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src/contents/drylab.tsx deleted 100644 → 0
View file @ 9143d0b0
import { useEffect } from "react";
import { useLocation } from "react-router-dom";
import { openFromOtherPage } from "../components/Buttons";
export function Drylab() {
const location = useLocation();
useEffect(() => {
const params = new URLSearchParams(location.search);
const collapseId = params.get('collapseId');
const tabId = params.get('tab');
// Scroll to the section specified by collapseId
if (collapseId) {
const collapseElement = document.getElementById(collapseId);
if (collapseElement) {
const elementTop = collapseElement.getBoundingClientRect().top + window.pageYOffset;
const offset = window.innerHeight / 2 - collapseElement.offsetHeight / 2;
const scrollPosition = elementTop - offset;
window.scrollTo({
top: scrollPosition,
behavior: 'smooth',
});
}
}
// Open the tab specified by tabId
if (tabId) {
openFromOtherPage(tabId)({ currentTarget: document.getElementById(tabId)! });
}
}, [location.search]);
return (
<>
<div className="row">
<div className="col">
</div>
</div>
<div className="row">
</div>
</>
);
}
\ No newline at end of file
src/contents/engineering.tsx
View file @ 3fd292fd
import { ButtonOneEngineering, openIt } from "../components/Buttons";
import { LoremShort } from "../components/loremipsum";
import { useEffect } from "react";
import { useLocation } from "react-router-dom";
import { ButtonOneEngineering } from "../components/Buttons";
import { openElement } from "../utils/openElement";
import { H2, H3, H4, H5, PhilipH3 } from "../components/Headings";
import { useTabNavigation } from "../utils/TabNavigation";
import { Collapsible } from "../components/Collapsible";
import { useNavigation } from "../utils";
import { TabScrollLink } from "../components/Link";
import { InfoBox } from "../components/Boxes";
import { DownloadLink } from "../components/Buttons";
import { Section } from "../components/sections";
import EngTrfsources from "../sources/eng-trf-sources";
import EngRepsources from "../sources/eng-reporter-sources";
import EngPEsystems from "../sources/eng-pe-sources";
import EngPegsources from "../sources/eng-peg-sources";
import EngNicksources from "../sources/eng-nickases-sources";
import EngDelsources from "../sources/eng-delivery-sources";
import { TwoLinePDF, PDF } from "../components/Pdfs";
import { OneFigure, TwoFigureRow } from "../components/Figures";
export function Engineering() {
const location = useLocation();
useEffect(() => {
const params = new URLSearchParams(location.search);
const collapseId = params.get('collapseId');
const tabId = params.get('tab');
// Open the tab specified by tabId
if (tabId) {
// Hide all tabs
const tabs = document.querySelectorAll('.enginneeringtab');
tabs.forEach((tab) => {
(tab as HTMLElement).style.display = 'none';
});
// Show the selected tab
const selectedTab = document.getElementById(tabId);
if (selectedTab) {
selectedTab.style.display = 'block';
}
}
// Scroll to the section specified by collapseId after opening the tab
if (collapseId) {
const collapseElement = document.getElementById(collapseId);
if (collapseElement) {
const elementTop = collapseElement.getBoundingClientRect().top + window.pageYOffset;
const offset = window.innerHeight / 2 - collapseElement.offsetHeight / 2;
const scrollPosition = elementTop - offset;
window.scrollTo({
top: scrollPosition,
behavior: 'smooth',
});
}
}
}, [location.search]);
useTabNavigation();
const {goToPagesAndOpenTab} = useNavigation ();
const {goToPageAndScroll} = useNavigation();
const {goToPageWithTabAndScroll} = useNavigation();
return (
<>
<div className="row mt-4">
<div className="col">
<br/> <br/> <br/>
<div id="tab-our-cycle" className="enginneeringtab" style={{display: "block"}}>
<section id="Our Cycle">
<h3>Our Cycle</h3>
<p><LoremShort></LoremShort></p>
<br/> <br/>
<div id="tab-our-cycle" className="enginneeringtab" style={{display: "none"}}>
<section > <br id="obenengineering"/>
<div className="eng-box box" >
<H2 text="Our cycle" id="our-cycle-header"></H2>
<p>
In the course of our project, innovative thoughts were taken up, rejected, elaborated, discussed and tested. On this page we present a selection of ideas that occupied us the most in the last month. Our trains of thought are represented in form of iterations of engineering cycles. Each engineering cycle is comprised of four steps:
</p>
<ul>
<li>
<b>Design</b>: In this step the motivation or problem, that the individual iteration addresses, is mentioned. A theoretical approach addressing the motivation is explained.
</li>
<li>
<b>Build</b>: The practical examination of the theoretical plan described in the design section is executed, e. g. by planning an experiment, by creating a construct in silico, by cloning.
</li>
<li>
<b>Test</b>: The initial design is tested, either by conducting an experiment, testing a design in silico, e. g. by modeling, refuting ideas based on new input or background research or discussing approaches with an expert.
</li>
<li>
<b>Learn</b>: In this last section, test results are discussed and insights gained in testing the design are formulated.
</li>
</ul>
</div>
<br/>
<div className="row">
<div className="col">
</div>
<div className="col button-left">
<div className="right"><ButtonOneEngineering label="Next" open="proof-of-concept"/></div>
<div className="right"><ButtonOneEngineering label="Next" open="reporter" scrollToId="reporter-header"/></div>
</div>
</div>
</section>
</div>
<div id="tab-proof-of-concept" className="enginneeringtab" style={{display: "none"}}>
<section id="Proof of Concept" >
<div className="bg-lb box" >
<h3>Proof of Concept</h3>
<p><LoremShort></LoremShort></p>
<div className="enginneeringtab" id="tab-reporter" style={{display: "none"}}>
<section id="reporter sec" >
<div className="eng-box box" >
<H2 id="reporter-header" text="Prime Editing Reporter"></H2>
<p>Prime editing is a is a very precise and safe method. However, depending on the genomic locus targeted, the editing efficiency can be very low. The Cystic Fibrosis causing CFTR F508del mutation is, as <a onClick={() => goToPagesAndOpenTab('mattijsinv', '/human-practices')}> Mattijs Bulcaen </a> stated in our interview, one of, if not the most obvious application of prime editing, considering the large amount of people affected. The lack of publications addressing CFTR target implied, that the mutation might be particularly hard to edit. At low editing efficiency, successful edits are hard, if not impossible to distinguish from the background noise using conventional methods like sanger sequencing or qPCR. As a basis to effectively test our approach and screen for working pegRNAs, we needed a highly sensitive method of detection with as little noise as possible to optimize our prime editing approach for genomic CFTR targeting.</p>
</div>
<div className="box" >
<p id="rep1">
<H3 text="A Fluorescence Reporter" id="rep1head"/>
<H4 text="Design" id="design-head"/>
<p>
We reasoned that the easiest way of detecting DNA changes in a cell would be fluorescence. Our initial idea was to create pegRNAs targeting the coding sequence of a fluorescent protein, that would introduce a mutation resulting in a different emission, giving easily detectable feedback of correct editing. The original Aequorea victoria GFP protein differs from avGFP(Y66W), emitting light in a wavelength of around 509 nm (cyan), and avGFP(Y66H), emitting light in a wavelength of around 448 nm (blue) by only one amino acid substitution each.<TabScrollLink tab="tab-reporter" num="1" scrollId="desc-1"/> Prime editing could therefore be visualized by facilitating these substitutions with a prime editor.
</p>
<H4 text="Build" id="build-head"/>
<p>
To this end, the wild-type and edited versions of the avGFP were put in contrast and we started searching for potential pegRNAs for editing one into the other.
</p>
<figure>
<img src="https://static.igem.wiki/teams/5247/engineering-cycle/rep-it1.svg" alt="Illustration of fluorescence wavelength change reporter"/>
<figcaption><b>Figure 1: Illustration of a reporter system based on the introduction of a single amino acid substitution into GFP plasmids transformed into HEK293 cells changing the emission spectrum in a detectable way.</b> </figcaption>
</figure>
<H4 text="Test" id="test-head"/>
<p>
When trying to find protospacers for Cas9 and other possible <a onClick={() => goToPageWithTabAndScroll ({scrollToId: 'nickase-header', path: '/engineering', tabId: 'tab-nickase'})}>nickases</a>, we noticed, that the locus of the mutations is too far away from any SpuFz1 TAM sequences. Additionally, the applicability of insights gained through pegRNA optimization in this locus to CFTR editing would also be very limited due to the vast differences in the sequence of protospacer and surrounding genomic region. Additionally, we learned from our interview with <a onClick={() => goToPagesAndOpenTab('mattijsinv', '/human-practices')}>Mattijs Bulcaen</a> that the type of edit (insertion, substitution or deletion) significantly impacts editing efficiency. A mutation changing GFP to BFP would have to be a substitution instead of the three-nucleotide insertion needed to correct CFTR F508del.
</p>
<H4 text="Learn" id="learn-head"/>
<p>
From our observations we learned that a reporter system is only of use, if it can really mimic the genomic target of choice. The adjustments to be made to create a pegRNA targeting the genomic target from a pegRNA targeting the reporter should be as minor as possible. This includes a similar spacer and a similar edit to be made.
</p>
</p>
</div>
<div className="box" >
<p id="rep2">
<H3 text="Proof of Concept for PEAR" id="rep2head"/>
<H4 text="Design" id="design-head"/>
<p>
After extensive research we came across the prime editor activity reporter (PEAR) created by Simon et al. (2022)<TabScrollLink tab="tab-reporter" num="2" scrollId="desc-2"/>, which is the template our modified reporter plasmid is based on. The PEAR plasmid contains an eGFP coding sequence with an intron derived from the mouse Vim gene. If the intron is removed during RNA splicing, the two exons form a continuous open reading frame. By mutating the 5’ splicing signal, a target is created which, upon correct editing, leads to a gain-of-function. The resulting fluorescence can be imaged using confocal microscopy or quantified by means of flow cytometry. Notably, the area downstream of the 5’ splice signal is intronic, and thus can be edited without any impact on the coding sequence. Additionally, Simon et al. showed, that “efficiency of prime editing to modify PEAR plasmids is governed by the same factors as prime editing in genomic context”. We reasoned that this system might be flexible, and sensitive enough to build our optimizations strategies upon.
</p>
<H4 text="Build" id="build-head"/>
<p>
Since none of us had any experience in prime editing before our project, we wanted to test whether we can facilitate prime editing in the first place. To do this and also assess the functionality of the PEAR system, we set up a proof of concept using the PEAR 2in1 system. This plasmid includes not only the eGFP with and intron and disrupted 5’ splice site, but also a pegRNA expression cassette. The pegRNA is designed in a way that, in combination with a prime editing protein complex, corrects the disrupted splicing signal.
</p>
<H4 text="Test" id="test-head"/>
<p>
In the experiment, we transfected HEK293 cells (as recommended by <a onClick={() => goToPagesAndOpenTab('mattijsinv', '/human-practices')}>Mattijs Bulcaen</a>) with the <a onClick={() => goToPageWithTabAndScroll ({scrollToId: 'current-pe-systems', path: '/engineering', tabId: 'tab-pe-systems' })}>pCMV-PE2 prime editor</a> plasmid and the pDAS12489_PEAR-GFP_2in1_2.0 mentioned above. Our first proof of concept succeeded as we could see fluorescent cells 72 h after transfection. In contrast, negative controls with only one of the plasmids transfected did not show any fluorescence. However, the transfection efficiency in our initial test runs was quite low, as indicated by a technical positive control.
</p>
<figure>
<img src="https://static.igem.wiki/teams/5247/engineering-cycle/rep-it2.svg" alt="Illustration of the proof of concept using the PEAR2in1 system"/>
<figcaption><b>Figure 2:</b> Illustration of the proof of concept experiment. HEK293 cells transiently transformed with the pDas12189_PEAR-GFP_2in1_2.0 plasmid on the left side show fluorescence after transformation with a prime editor expression plasmid.</figcaption>
</figure>
<H4 text="Learn" id="learn-head"/>
<p>
This proved, that not only we were able to use prime editing in our model, but also that the PEAR reporter system can report successful prime editing. Though this was a very promising start, further steps had to be taken to enable context specific testing of prime editing. Firstly, the transfection efficiency had to be improved (see <a onClick={() => goToPageWithTabAndScroll ({scrollToId: 'transfection-header', path: '/engineering', tabId: 'transfection' })}> Transfection Optimization</a>). Secondly, the reporter had to be modified in a way that resembles the genomic CFTR target.
</p>
</p>
</div>
<div className="box" >
<p id="rep3">
<H3 text="Contextualization of PEAR" id="rep3head"/>
<H4 text="Design" id="design-head"/>
<p>
The original PEAR plasmid pDAS12124_PEAR-GFP-preedited that we bought from AddGene represents, as the name suggests, how the reporter should look like after successful editing and can thus be used as a positive control and for normalization. To alter the PEAR plasmid so that it mimics the mutated genomic CFTR target, we first analyzed the region surrounding CFTR F508del mutation. As the mutation is a three base pair deletion, we introduced the very same at the 5’ splicing signal. For this modification to reliably disrupt intron splicing and thus eGFP expression, we effectively removed the GT bases of the intronic 5’ splice donor site as well as the preceding, exonic G base of the 5’ flanking sequence. Secondly, we replaced the intronic region downstream of the four base pair 3’ flanking region with the respective sequence from the CFTR locus. This 27 bp substitute included a PAM sequence, an entire spacer as well as four additional base pairs in between present in the original gene sequence. Lastly, we introduced silent mutations upstream of the 5’ flanking sequence that lowered the GC content. This was to mimic the AT-rich region preceding the F508del mutation in the CFTR gene. This reveals one of the necessary shortcomings of this reporter: Edits upstream of the 5’ donor site are heavily restricted by the eGFP coding sequence.
</p>
<figure>
<img src="https://static.igem.wiki/teams/5247/engineering-cycle/rep-strategy-it3.svg" alt="Modification strategy for creation of the pPEAR_CFTR plasmid"/>
<figcaption><b>Figure 3:</b> Illustration of the modification strategy for the pPEAR_CFTR reporter. The pDAS12124_PEAR-GFP-preedited plasmid was modified by introducing a 3 bp deletion resembling the F508del mutation, inserting a 27 bp sequence from the genomic CFTR target including PAM and protospacer sequence as well as making silent edits to account for the AT-rich region upstream of the mutation.</figcaption>
</figure>
<H4 text="Build" id="build-head"/>
<p>
We constructed the reporter system by first analyzing the original plasmid to identify appropriate restriction sites. We then digested the plasmid backbone and cloned in a gene synthesis fragment ordered at IDT containing the edits via Gibson Assembly cloning. The correct cloning was validated first by colony PCR and then by sequencing the regions of the plasmid containing the cloning sites and our modifications.
</p>
<H4 text="Test" id="test-head"/>
<p>
We evaluated the functionality of our reporter system by co-transfecting our reporter construct with a pCMV-PE2 prime editor plasmid as well as a plasmid expressing pegRNA that targeted our reporter (see <a onClick={() => goToPagesAndOpenTab('pegrna', '/engineering')}> pegRNA engineering cycle</a>) into HEK293 cells. After 72 h we saw a significant number of cells showing fluorescence.
</p>
<p>
Additionally, for positive controls we transfected a technical control plasmid as well the unmodified pDAS12124_PEAR-GFP-preedited plasmid, which could be used to determine the transfection efficiency as well as normalize the editing efficiency. As negative controls, our modified plasmid, pCMV-PE2 and the pegRNA plasmid were transfected. The positive controls showed fluorescence, while the negative control did not.
</p>
<figure>
<img src="https://static.igem.wiki/teams/5247/engineering-cycle/rep-it3.svg" alt="Illustration of pegRNA testing using the pPEAR_CFTR system"/>
<figcaption><b>Figure 4:</b> Illustration of the pegRNA testing using the pPEAR_CFTR system. HEK293 cells transiently transformed with the pPEAR_CFTR plasmid (in the middle) show fluorescence after transformation with a prime editor and a reporter-specific pegRNA expression plasmid. The pDAS12124_PEAR-GFP-preedited is used as an internal positive control and for normalization.</figcaption>
</figure>
<H4 text="Learn" id="learn-head"/>
<p>
Our results demonstrate three things: Firstly, the original pDAS12124_PEAR-GFP-preedited plasmid leads to undisrupted expression of eGFP in the transfected cells. Secondly, the modifications that we made to create our own, context specific PEAR plasmid prevented proper expression of eGFP in transfected, unedited cells as planned and notably with no apparent noise. The last and most important insight gained was, that editing of the reporter plasmid using respective pegRNAs successfully restores eGFP expression, proving that our reporter works as intended.
</p>
<p>
<b>This achievement formed a convenient basis for the following optimization of prime editing in the CFTR F508del locus for us as well as other research groups.</b>
</p>
</p>
</div>
<div className="box" >
<p id="rep4">
<H3 text="Application in epithelial Cells" id="rep4head"/>
<H4 text="Design" id="design-head"/>
<p>
Although we could show that our PEAR reporter plasmid works in a HEK cell model, according to <a onClick={() => goToPagesAndOpenTab('ignatova', '/human-practices')}> Prof.Dr. Zoya Ignatova </a> insights gained here might still not entirely transfer to cells actively expressing CFTR. As recommended, we applied our reporter to a system closer to a therapeutic target <a onClick={() => goToPageAndScroll ('Cell Culture2H', '/materials-methods')}>CFBE41o-</a>. The cells are derived from bronchial epithelial cells of a Cystic Fibrosis patient and are homozygous for CFTR F508del.
</p>
<H4 text="Build" id="build-head"/>
<p>
For experimenting in CFBE41o- cells, the same reporter construct was used as for the HEK293 test. However, we used a different prime editor (pCMV-PE6c, see prime editing systems engineering cycle<a onClick={() => goToPagesAndOpenTab('pe-systems', '/engineering')}> prime editing systems circle </a>), and only pegRNAs were used, that proved the most efficient in preceding experiments (see <a onClick={() => goToPagesAndOpenTab('pegrna', '/engineering')}> pegRNA engineering cycle </a>).
</p>
<H4 text="Test" id="test-head"/>
<p>
Similar to the previous cycle, we evaluated the functionality of our reporter system by co-transfecting our reporter construct with a pCMV-PE6c prime editor plasmid as well as a plasmid expressing pegRNA that targeted our reporter this time into CFBE41o- cells. After 72 h we saw a significant number of cells showing fluorescence.
</p>
<p>
Like with the experiments in HEK cells, we transfected a technical control plasmid as well the unmodified pDAS12124_PEAR-GFP-preedited plasmid as positive controls and our modified plasmid, pCMV-PE6c and the pegRNA plasmid individually as negative controls. Again, the positive controls showed solid fluorescence, while the negative control did not.
</p>
<figure>
<img src="https://static.igem.wiki/teams/5247/engineering-cycle/rep-it4.svg" alt="Illustration of applying the pPEAR_CFTR system to lung epithelial cell lines"/>
<figcaption><b>Figure 5:</b> Illustration of the pegRNA testing using the pPEAR_CFTR system. CFBE41o- cells transiently transformed with the pPEAR_CFTR plasmid (in the middle) show fluorescence after transformation with a prime editor and a reporter-specific pegRNA expression plasmid. The pDAS12124_PEAR-GFP-preedited is used as an internal positive control and for normalization.</figcaption>
</figure>
<H4 text="Learn" id="learn-head"/>
<p>
This experiment confirms that our reporter can not only be used in cell lines distantly related to patient cells of interest, in our case HEK203 cells, but also works in cells actively expressing CFTR and carrying the mutation. The reporter still showed no noise.
</p>
</p>
</div>
<div className="box" >
<p id="rep5">
<H3 text="Application in Primary Cells" id="rep5head"/>
<H4 text="Design" id="design-head"/>
<p>
The model closest to application in actual patient cells are human derived primary cells. For our last test of our modified PEAR reporter, we thus chose to use <a onClick={() => goToPageAndScroll ('Cell Culture3H', '/materials-methods')}>human nasal epithelial cells</a> derived from members of our team.
</p>
<H4 text="Build" id="build-head"/>
<p>
For testing our reporter in the human nasal epithelial cells, the same constructs have been used as in the previous iteration with CFBE41o- cells.
</p>
<H4 text="Test" id="test-head"/>
<p>
The experimental setup for this experiment was a scaled down version of the previous cycle with the only altered variable being the cells transfected. In this case, we did not observe any fluorescence, neither in the tested cells, nor the technical or pDAS12124_PEAR-GFP-preedited positive controls.
</p>
<H4 text="Learn" id="learn-head"/>
<p>
In this last experiment, the negative technical positive control implies a failed transfection of the cells. Thus, this attempt did not allow to draw any conclusion regarding the function of our reporter in primary cells. The experiment is to be repeated in the future.
</p>
</p>
</div>
<div className="box" >
<p id="rep6">
<H3 text="Outlook" id="rep6head"/>
<p>
Our CFTR contextualized PEAR reporter proved to consistently allow detection of prime editing without notable noise, laying the foundation for optimization of existing and testing of new prime editing systems. Although very versatile in the context of targeting CFTR F508del with the <a onClick={() => goToPagesAndOpenTab('pegRNA-genau-collapsible', '/description')}>spacer of our choice</a>, a wider applicability to other genomic targets and other possible prime editor variants working differently than Cas9-based systems would be favorable. In the original PEAR plasmid however, modification of variable region is quite impractical. Also, as a part the eGFP is RCF[1000] but not RCF[10] BioBrick standard conform and hardly compatible with other parts like our <a onClick={() => goToPageWithTabAndScroll ({scrollToId: 'pe3', path: '/engineering', tabId: 'pe-systems'})}>PreCyse cassette</a>.
</p>
<H4 text="Design" id="design-head"/>
<p>
This is why, as an outlook and contribution for future iGEM teams, we aim to create a more modular and compatible part similar to our PreCyse Casette. For this we made use of the experience gained when cloning pegRNAs. An oligonucleotide-based golden gate cloning site in the region of interest surrounding the 5’ splice donor site allows for cheap and convenient modification of the sequence. The area between the TypeIIS restriction sites is designed as a dropout cassette coding for a fluorescence marker expressed in E. coli, that enables rapid screening for transformants containing correctly digested plasmid backbones.
</p>
{/* <H4 text="Build" id="build-head"/>
<p>
</p>
<H4 text="Test" id="test-head"/>
<p>
</p>
<H4 text="Learn" id="learn-head"/>
<p>
</p> */}
</p>
</div>
<Section title="References" id="references">
<EngRepsources/>
</Section>
<br/>
<div className="row ">
<div className="col">
<div className="left"><ButtonOneEngineering label="Previous" open="our-cycle"/></div>
<div className="left"><ButtonOneEngineering label="Previous" open="our-cycle" scrollToId="our-cycle-header"/></div>
</div>
<div className="col button-left">
<div className="right"><ButtonOneEngineering label="Next" open="pe-systems"/></div>
<div className="right"><ButtonOneEngineering label="Next" open="transfection" scrollToId="transfection-header"/></div>
</div>
</div>
</section>
</div>
<div className="enginneeringtab" id="tab-pe-systems" style={{display: "none"}}>
<section id="PE Systems" >
<div id="tab-transfection" className="enginneeringtab" style={{display: "none"}}>
<section >
<div className="eng-box box" >
<H2 id="transfection-header" text="Optimization of Transfection"></H2>
<p>
To test prime editors, a reliable model system is required. HEK293 cells are a human derived cell line and widely used in a variety of fields in biology<TabScrollLink tab="transfection" num="3" scrollId="desc-3"/>. Apart from easy handling and comparatively easy transfection, they have, as we found out in our exchange with <a onClick={() => goToPagesAndOpenTab('mattijsinv', '/human-practices')}>Mattijs Bulcaen</a>, one advantage over other models: They are naturally impaired in DNA repair mechanisms and therefore easier to edit. To properly compare editing efficiencies, a high transfection efficiency is of utmost importance. This engineering cycle focuses on our work in simulating prime editing using the PEAR reporter system<TabScrollLink tab="transfection" num="4" scrollId="desc-4"/> and optimizing transfection protocols.
</p>
</div>
<div className="box" >
<p id="trf1">
<H3 text="Test of Lipofectamine 2000" id="trf1head"/>
<H4 text="Design" id="text"/>
<p>
Before testing any of our mechanistic approaches, we had to examine whether we can facilitate and detect prime editing in the first place. During our research we eventually stumbled upon the PEAR reporter system (see <a onClick={() => goToPagesAndOpenTab('pegrna', '/engineering')}> pegRNA engineering cycle </a>). The PEAR 2in1 plasmid reporter includes a GFP that is to be edited for sensitive prime editing detection, and a pegRNA expression cassette with a pegRNA targeting the plasmid itself. Having found a system capable of detecting even small-scale prime editing, the next step was to find transfection conditions that would work. In the literature, Lipofectamine is described as a common transfection agent.
</p>
<p>
Transfection with Lipofectamine 2000 was performed in accordance with the Anzalone protocol. However, the result was characterized by insufficient transfection efficiency.
</p>
<H4 text="Build" id="text"/>
<p>
Anzalone et al. 2019<TabScrollLink tab="transfection" num="5" scrollId="desc-5"/> describe a transfection of prime-editing complexes with Lipofectamine 2000.
</p>
<H4 text="Test" id="text"/>
<p>
Transfection with Lipofectamine 2000 was performed in accordance with the Anzalone protocol. However, the result was characterized by insufficient transfection efficiency.
</p>
<H4 text="Learn" id="text"/>
<p>
The low efficiency of Lipofectamine 2000 indicates that the product is not optimally suited to the specific conditions under consideration. In contrast, Lipofectamine 3000 is described in the literature as potentially more efficient.
</p>
</p>
</div>
<div className="box" >
<p id="trf2">
<H3 text="Initial Test with Lipofectamine 3000" id="trf2head"/>
<H4 text="Design" id="text"/>
<p>
In light of the aforementioned findings, the decision was taken to test Lipofectamine 3000, given its reputation for greater efficiency. A new test design was devised, utilizing Lipofectamine 3000 with an equivalent quantity of DNA and modified transfection conditions.
</p>
<H4 text="Build" id="text"/>
<p>
In accordance with the established protocol, the recommended ratio of 1 µg DNA to 2 µl Lipofectamine 3000, as provided by ThermoFisher, was to be employed.
</p>
<H4 text="Test" id="text"/>
<p>
The objective of the experiment was to enhance the transfection efficiency of Lipofectamine 3000. The transfection protocol was conducted in accordance with the manufacturer's instructions (1 µg DNA, 2 µl Lipofectamine 3000 reagent).
</p>
<p>
The outcome revealed that despite the modification, the transfection efficiency remained inadequate, although a marginal improvement was discernible.
</p>
<H4 text="Learn" id="text"/>
<p>
Although a switch to Lipofectamine 3000 resulted in a marginal improvement, the efficiency fell short of expectations. This indicates that further optimization is required in terms of the amount of Lipofectamine and DNA, as well as the medium used.
</p>
</p>
</div>
<div className="box" >
<h3>PE Systems</h3>
<p><LoremShort></LoremShort></p>
<p id="trf3">
<H3 text="Optimization of DNA and Lipofectamine Volumes" id="trf3head"/>
<H4 text="Design" id="text"/>
<p>
In order to optimize the transfection process, a new optimization test was designed, which incorporated a variable design with regard to the quantity of Lipofectamine 3000 and DNA.
</p>
<H4 text="Build" id="text"/>
<p>
The protocol entailed the utilization of varying concentrations of Lipofectamine 3000, specifically 1 µl and 1.5 µl, with a DNA quantity of 1 µg or 0.5 µg. In this phase, we developed the transfection method with calcium chloride (CaCl<sub>2</sub>) as an alternative to conventional lipofectamine transfection. The aim was to test whether this more cost-effective method offers comparable transfection efficiency. Three different DNA concentrations were used to investigate the effect on transfection efficiency.
</p>
<H4 text="Test" id="text"/>
<p>
To enhance transfection efficiency, optimization tests were conducted, in which the quantities of Lipofectamine and DNA were varied. The objective of this iteration was to find the optimal ratio of Lipofectamine 3000 to DNA. To this end, 1 µl and 1.5 µl of Lipofectamine 3000 at a DNA concentration of either 1 µg or 0.5 µg were compared with each other. In the next step, the tests were carried out with the different DNA concentrations using the CaCl<sub>2</sub> transfection method. The transfection efficiencies were compared with those from the Lipofectamine transfection to determine whether the new method represents an improvement.
</p>
<H4 text="Learn" id="text"/>
<p>
The experiment demonstrated that a quantity of 1 µl Lipofectamine 3000 was sufficient for successful transfection, and that increasing the quantity does not result in a notable difference. Additionally, the findings indicated that an amount of 1 µg DNA exhibited a higher efficiency than an amount of 0.5 µg DNA. It can be reasoned that additional factors may have contributed to the previously observed decline in transfection efficiency. One potential explanation is that the cells may have been in an excessively high passage level. It became clear from the tests that CaCl<sub>2</sub> transfection did not deliver better results than Lipofectamine transfection. On the contrary, the efficiency was significantly lower, although the method is less expensive. This led to the realisation that the CaCl<sub>2</sub> technique in this form was not a suitable alternative for our specific requirements.
</p>
<p>
It can be reasonably deduced that the aforementioned factors may have contributed to the observed decline in transfection efficiency.
</p>
</p>
</div>
<div className="box" >
<p id="trf4">
<H3 text="Validation of optimized Protocol" id="trf4head"/>
<H4 text="Design" id="text"/>
<p>
The results obtained were used to develop an optimized protocol that takes into account both the concentration of Lipofectamine and the amount of DNA.
</p>
<H4 text="Build" id="text"/>
<p>
In subsequent research, a DNA quantity of 1 µg and a defined quantity of 1 µl of Lipofectamine 3000 will be utilized.
</p>
<H4 text="Test" id="text"/>
<p>
Following a series of optimizations, the proof of concept was conducted once more to confirm the efficacy of the optimized protocol. The objective was to perform the transfection with the final, optimized protocol. This protocol involved the utilization of 1 µl Lipofectamine 3000, 1 µg DNA, 2 µl Reagent 3000 and Opti-MEM as a medium. The outcomes were encouraging, as the transfection efficiency was markedly enhanced.
</p>
<H4 text="Learn" id="text"/>
<p>
The utilization of an optimized quantity of 1 µl Lipofectamine 3000, a defined quantity of DNA and the suitable Opti-MEM medium resulted in a notable enhancement in transfection efficiency. This substantiates the assertion that the aforementioned conditions constitute an optimal foundation for the transfection of HEK cells with the prime editing complex.
</p>
</p>
</div>
<Section title="References" id="references">
<EngTrfsources/>
</Section>
<br/>
<div className="row ">
<div className="col">
<div className="left"><ButtonOneEngineering label="Previous" open="proof-of-concept"/></div>
<div className="left"><ButtonOneEngineering label="Previous" open="reporter" scrollToId="reporter-header"/></div>
</div>
<div className="col button-left">
<div className="right"><ButtonOneEngineering label="Next" open="nikase"/></div>
<div className="right"><ButtonOneEngineering label="Next" open="pe-systems" scrollToId="pe-systems-header"/></div>
</div>
</div>
</section>
</div>
<div className="enginneeringtab" id="tab-nikase" style={{display: "none"}}>
<section id="Nikase" >
<div id="tab-pe-systems" className="enginneeringtab" style={{display: "none"}}>
<section id="PE Systems sec" >
<div className="eng-box box" >
<H2 id="pe-systems-header" text="Prime Editing Systems"></H2>
<p>Different versions of the original prime editing system have been developed since its initial introduction.
Deciding on what system to use for the application in therapeutic human gene editing, especially concerning the correction of
F508del, was the goal of this engineering cycle.</p>
<p>
Since we aim to develop a therapy delivered to the human body, we wanted to obtain high editing efficiency while risking as
little off-targets as possible and also reducing the size for improved packability.
</p>
<InfoBox title="Existing Prime Editing Systems" id="current-pe-systems">
<details>
<summary>
</summary>
<div className='row align-items-center'>
<div className='col'>
<p>
<b>PE1</b><TabScrollLink tab="tab-pe-systems" num="6" scrollId="desc-6"/>{/* ehem 1 */}, the first version of the Prime Editor features a Cas9(H840A), a Streptococcus pyogenes Cas9 (SpCas9, hereafter just referred to as Cas9) mutant that only cuts the non-target strand of the DNA template<TabScrollLink tab="tab-pe-systems" num="7" scrollId="desc-7"/>{/* ehem 2 */}, and a wildtype reverse transcriptase from the Moloney Murine Leukaemia Virus (M-MLV RT) connected by a serine and glycine rich flexible linker.
</p>
<p>
<b>PE2</b><TabScrollLink tab="tab-pe-systems" num="6" scrollId="desc-6"/>{/* ehem 1 */} improves on this concept by incorporating an improved RT with five mutations improving affinity to the template RNA, enzyme processivity and thermostability (D200N/L603W/T330P/T306K/W313F). This version of the prime editor showed varying improvement of editing efficiency over all tested loci and edits with no apparent downsides. Building on the PE2 system, a smaller version of the M-MLV RT was introduced by Gao et al. (2022)<TabScrollLink tab="tab-pe-systems" num="8" scrollId="desc-8"/>{/* ehem3 */}. The RT was truncated by 621 bp through deletion of the RNaseH domain, which originally degrades the RNA template, but is not needed for prime editing. The codon optimized version of this truncated RT prime editor (in literature usually called PE2∆RNaseH) was named <b>PE<sup>CO</sup>-Mini</b> in the paper and will be addressed as such here.
</p>
</div>
<div className='col-4'>
<figure>
<img src="https://static.igem.wiki/teams/5247/engineering-cycle/pe2-open.svg" alt="PE2 Prime Editor"/>
<figcaption><b>Figure 1: Illustration of PE2 Prime Editor</b> </figcaption>
</figure>
</div>
</div>
<div className='row align-items-center'>
<div className='col'>
<p>
The <b>PE3</b><TabScrollLink tab="tab-pe-systems" num="6" scrollId="desc-6"/>{/* ehem 1 */} system, described in the same paper as PE1 and PE2, introduces the use of a second single guide RNA besides the pegRNA which leads to a nick in the strand opposite to the edited strand. This is supposed to improve integration of edits by directing cellular DNA repair systems to use the edited strand as a template for resolving base mismatches. Nicks positioned 3‘ of the edit about 40–90 base pairs from the pegRNA-induced nick were able to further increase editing efficiencies about threefold when compared to PE2, but with a higher range of on-target indels , meaning random Insertions and/or Deletions that appear after faulty repair of double strand breaks in the DNA. PE3b, where the protospacer for the nicking sgRNA lies within the edited regions, decreased the indel rate greatly compared to PE3.
</p>
</div>
<div className='col-4'>
<img src="https://static.igem.wiki/teams/5247/engineering-cycle/pe3-open.svg" alt="PE3 Prime Editor"/>
</div>
</div>
<div className='row align-items-center'>
<div className='col'>
<p>
<b>PE4</b> and <b>PE5</b><TabScrollLink tab="tab-pe-systems" num="8" scrollId="desc-8"/> {/* ehem 4 */}expand the PE2 and PE3 systems, respectively, by co-expressing a dominant negative MLH1 protein (MLH1(Δ754–756), hereafter referred to as MLH1dn). The MLH1 protein plays a crucial role in the mismatch repair (MMR) mechanism of the human cell<TabScrollLink tab="tab-pe-systems" num="9" scrollId="desc-9"/>{/* ehem 5 */} by recruiting other repair proteins and facilitating catalytic function. The mutant still recruits other factors but is impaired in its endonuclease function, disrupting function of the entire repair mechanism. This leads to an average 7.7-fold and 2.0-fold increase in editing efficiency, respectively, compared to PE2 and PE3. This is possibly due to slower repair of mismatches and thus more time for the proteins encoded by LIG1 and FEN1 genes to excise the non-edited 5’ flap and ligate the nick in the edited strand. Additionally, MLH1dn co-expression slightly reduced on-target indels as well as unintended editing outcomes in PE3 systems and did not lead to higher off-target indel rates or overall mutation rates.
</p>
<p>
With <b>PEmax</b><TabScrollLink tab="tab-pe-systems" num="8" scrollId="desc-8"/>{/* ehem 4 */}, the structure of PE2 is further enhanced by using human codon-optimized RT, a new linker containing a bipartite SV40 nuclear localization sequence (NLS)<TabScrollLink tab="tab-pe-systems" num="11" scrollId="desc-11"/>, an additional C-terminal c-Myc NLS<TabScrollLink tab="tab-pe-systems" num="12" scrollId="desc-12"/> and R221K N394K mutations in SpCas9 previously shown to improve Cas9 nuclease activity<TabScrollLink tab="tab-pe-systems" num="13" scrollId="desc-13"/>. These changes led to moderate improvements in editing efficiency compared to previous editor architectures.
</p>
</div>
<div className='col-4'>
<img src="https://static.igem.wiki/teams/5247/engineering-cycle/pe4-open-new.svg" alt="PE4 Prime Editor"/>
<img src="https://static.igem.wiki/teams/5247/engineering-cycle/pe5-open-new.svg" alt="PE5 Prime Editor"/>
</div>
</div>
<div className='row align-items-center'>
<div className='col'>
<p>
<b>PE6</b><TabScrollLink tab="tab-pe-systems" num="14" scrollId="desc-14"/> was made by improving the reverse transcriptase domain of the prime editor using Phage-Assisted Continuous Evolution (PACE). Multiple RT mutants (PE6a-d), derived from RTs of Escherichia coli Ec48 retron, Schizosaccharomyces pombe Tf1 retrotransposon and Moloney Murine Leukaemia Virus, were identified to increase editing efficiency over and/or were smaller than the M-MLV RT used in previous PE systems. Especially <b>PE6c</b> (evolved Tf1 RT) and <b>PE6d</b> (evolved M-MLV RT) showed significantly higher editing efficiencies than even PEmax depending on the targeted loci, with PE6d showing benefits especially in loci forming more complex secondary structures. Recent advancements in prime editing targeting the CFTR F508Δ mutation showed that PE6c was the most promising for editing in this loci<TabScrollLink tab="tab-pe-systems" num="15" scrollId="desc-15"/>. Improvements of nCas9 on the other hand (PE6e-g) were only marginal and highly site specific. All PE6 systems use nicking gRNAs (PE3) by default, but do not co-express MLH1dn.
</p>
</div>
<div className='col-4'>
<img src="https://static.igem.wiki/teams/5247/engineering-cycle/pe6c-open.svg" alt="PE6c Prime Editor"/>
</div>
</div>
<div className='row align-items-center'>
<div className='col'>
<p>
<b>PE7</b><TabScrollLink tab="tab-pe-systems" num="16" scrollId="desc-16"/> adds an additional RNA binding domain to the Prime Editor. The domain is derived from the La Protein (La(1-194)), an endogenous eukaryotic protein involved RNA metabolism and known for its role in binding polyuridine (polyU) tails at the 3’ ends of nascent transcripts, thus protecting them from exonuclease activity. PE7 showed considerable improvements over PEmax at different loci and different types of edits when used with the PE2 strategy (no nicking gRNAs, no MLH1dn co-expression). Notably, PE7 did perform worse when used with engineered pegRNAs than with regular ones (see pegRNA design).
</p>
</div>
<div className='col-4'>
<img src="https://static.igem.wiki/teams/5247/engineering-cycle/pe7-open.svg" alt="PE7 Prime Editor"/>
</div>
</div>
</details>
</InfoBox>
</div>
<div className="box" >
<p id="pe1">
<PhilipH3 id="pe1head"><span>PE2 and PE<sup>CO</sup>-Mini</span></PhilipH3>
<H4 text="Design" id="text"/>
<p>
For our initial approach, we wanted to start from the beginning and use the PE2 prime editing system. Since our goal of stripping the size of the prime editor was a big factor from the beginning, we did a researched into that direction and found a truncated version of M-MLV RT, PE<sup>CO</sup>-Mini. We then ordered the plasmids for both PE2 and PE<sup>CO</sup>-Mini. Since the PE<sup>CO</sup>-Mini plasmid had a different promotor than pCMV-PE2, we decided to clone the PE<sup>CO</sup>-Mini RT into the pCMV-PE2 vector to allow for direct comparison.
</p>
<H4 text="Build" id="text"/>
<p>
We designed primers for the amplification of PE<sup>CO</sup>-Mini RT and cloned it into pCMV-PE2 via double digestion and Gibson assembly.
</p>
<H4 text="Test" id="text"/>
<p>
To compare the prime editing performances of M-MLV RT (PE2) and PE<sup>CO</sup>-Mini RT, both were tested using a 2in1 prime editing reporter plasmid system<TabScrollLink tab="tab-pe-systems" num="17" scrollId="desc-17"/> (see <a onClick={() => goToPageWithTabAndScroll({scrollToId: 'Proof of Concept', path: '/engineering', tabId: 'tab-transfection' })}>Proof of Concept</a>) in HEK293 cells. Contrary to the findings of Gao et al., here the PE<sup>CO</sup>-Mini prime editor performed a lot worse than the PE2 prime editor.
</p>
<H4 text="Learn" id="text"/>
<p>
Since we knew, that for a successful therapy targeting the F508del mutation a very high prime editing efficiency was of utmost importance, we decided against using PE<sup>CO</sup>-Mini as the basis for our approach and that we have to look for other alternatives.
</p>
</p>
</div>
<div className="box" >
<p id="pe2">
<H3 text="PE6c" id="pe2head"/>
<H4 text="Design" id="text"/>
<p>
During our initial talk with <a onClick={() => goToPagesAndOpenTab('mattijsinv', '/human-practices')}>Mattijs Bulcaen</a>, he recommended a talk of <a onClick={() => goToPagesAndOpenTab('liu', '/human-practices')}>David Liu</a> at an online conference, where he presented unpublished data about his laboratory working on prime editing for F508del correction. We investigated it and through this came across the PE6 generation of prime editors. Seeing that the Liu Laboratory eventually decided on using the PE6c system, we adopted the findings.
</p>
<H4 text="Build" id="text"/>
<p>
We got the plasmid carrying the PE6c prime editor. Except for the RT and a few improving mutations in the Cas9 enzyme, it has the same architecture as PE2, which made comparison quite easy.
</p>
<H4 text="Test" id="text"/>
<p>
We tested PE6c against PE2 using the same reporter system as mentioned above for PE<sup>CO</sup>-Mini. PE6c, as expected from the literature, proved way more efficient in prime editing.
</p>
<H4 text="Learn" id="text"/>
<p>
The data from literature as well as our own experiments confirmed that PE6c architecture is superior to PE2 even without using nicking gRNAs that help suppress mismatch repair. This led us to the decision to use the PE6c reverse transcriptase and parts of the overall architecture for our subsequent tests.
</p>
</p>
</div>
<div className="box" >
<h3>Nikase</h3>
<p><LoremShort></LoremShort></p>
<p id="pe3">
<H3 text="PreCyse Casette" id="pe3head"/>
<H4 text="Design" id="text"/>
<p>
In the later stages of our project, the Liu laboratory published their own findings regarding CFTR F508del targeting with prime editing<TabScrollLink tab="tab-pe-systems" num="15" scrollId="desc-15"/>. The data showed that the editing efficiency of PE2 based systems, even when using PE6c reverse transcriptase, might not be sufficient for application in a therapy. Also, the plasmids of current prime editors did not include restriction sites that would have allowed replacing components like the nickase to test alternatives. This is why, in a cherry-picking manner, we combined the PE6c architecture prime editor with the most promising aspects of other prime editors, creating the PreCyse cassette.
</p>
<p>
Our decision on what components of existing prime editors we wanted to use was mainly driven by two factors: efficiency and precision. In prime editing, these two are often opposing forces, which means advancements improving efficiency often also increase the risk of off-targets mutations and on-target undesired editing. For this reason, we decided against using nicking gRNAs. Although they have been proven to reliably improve editing efficiency, they increase the risk and possible scope of off-target cleavage and mutations. Additionally, if <b>PE3b</b> is not applicable, there is a chance for double strand breaks to occur, which diminishes the safety advantage of prime editing over other common CRISPR-based methods. Co-expression of MLH1dn can improve editing efficiency in the same way as nicking gRNAs do, by helping to evade of the cellular mismatch repair mechanisms. The use of MLH1dn is especially impactful, when nicking gRNAs are not used, which is perfect in our case. Recently, the La poly(U)-binding motif has been shown to enhance prime editing efficiency, presumably through protection of the 3’ poly(U) tail of the pegRNA from RNases. The motif is also comparatively small, which aligns with the overall goal to create a compact prime editing tool. This is why PreCyse Casettes have been designed to include the La RNA binding motif fusion and the dominant negative MLH1 protein.
</p>
<H4 text="Build" id="text"/>
<p>
The PreCyse cassette comes in three versions: PreCyseA, the most basic version, comprises of a T7 promoter and an open reading frame, which includes NLS and one typeIIS restriction enzyme cloning site for a nickase and a reverse transcriptase each. For possible future additions like e. g. selection markers, a BamHI restriction site at the end of the coding sequence allows for easy in-frame Gibson cloning. Building on this basis, PreCyseB expands PreCyseA by the La Poly(U)-binding motif. PreCyseC additionally introduces the co-expressed MLH1dn. The cassettes were ordered in three individual parts to be put together with a pCMV-PE6c backbone via Gibson Cloning in different configurations to create the three variants. In the plasmid the cassette is expressed under a CMV promoter and followed by a polyadenylation signal. The PreCyse Casettes themselves can be used as a BioBrick RFC<TabScrollLink tab="tab-pe-systems" num="16" scrollId="desc-16"/> standard compatible composite part can thus be freely combined with other parts. The nickase and RT slots can be used for inserting any basic or composite part compatible with the Type IIS RCF[1000] standard for fusion proteins. The PreCyse Casette is meant to be a contribution to the iGEM community and a base for other teams to join us and researchers around the world to innovate in the exciting field of prime editing.
</p>
<div className="casettecontainer">
<div className="casettebox">
<H5 text="PreCyseA" id="PCA"/>
<img src="https://static.igem.wiki/teams/5247/engineering-cycle/precysea-casette.svg" alt="image 1" />
</div>
<div className="casettebox">
<H5 text="PreCyseB" id="PCB"/>
<img src="https://static.igem.wiki/teams/5247/engineering-cycle/precyseb-casette.svg" alt="image 2" />
</div>
<div className="casettebox">
<H5 text="PreCyseC" id="PCC"/>
<img src="https://static.igem.wiki/teams/5247/engineering-cycle/precysec-casette.svg" alt="image 3" />
</div>
</div>
{/* <div className="row align-items-center">
< div className='col align-items-center'>
<H5 text="PreCyseA" id="PreCyseA"/>
<img src="https://static.igem.wiki/teams/5247/engineering-cycle/precysea-casette.svg" alt="PreCyseA modular PE casette" style={{height: "80pt", width: "auto"}}/>
</div>
<div className='col align-items-center'>
<H5 text="PreCyseB" id="PreCyseB"/>
<img src="https://static.igem.wiki/teams/5247/engineering-cycle/precyseb-casette.svg" alt="PreCyseB modular PE casetter" style={{height: "80pt", width: "auto"}}/>
</div>
</div>
<div className='row align-items-center'>
<H5 text="PreCyseC" id="PreCyseC"/>
<img src="https://static.igem.wiki/teams/5247/engineering-cycle/precysec-casette.svg" alt="PreCyseC modular PE casette" style={{height: "80pt", width: "auto"}}/>
</div> */}
</p>
</div>
<Section title="References" id="references">
<EngPEsystems/>
</Section>
<br/>
<div className="row ">
<div className="col">
<div className="left"><ButtonOneEngineering label="Previous" open="pe-systems"/></div>
<div className="left"><ButtonOneEngineering label="Previous" open="transfection" scrollToId="transfection-header"/></div>
</div>
<div className="col button-left">
<div className="right"><ButtonOneEngineering label="Next" open="pegrna"/></div>
<div className="right"><ButtonOneEngineering label="Next" open="pegrna" scrollToId="pegrna-header"/></div>
</div>
</div>
</section>
</div>
<div className="enginneeringtab" id="tab-pegrna" style={{display: "none"}}>
<section id="pegRNA" >
<section id="pegRNA sec" >
<div className="eng-box box" >
<H2 id="pegrna-header" text="pegRNA"></H2>
<p>The <a onClick={() => goToPagesAndOpenTab('pegrna', '/engineering')}> pegRNA </a> is of paramount importance for function and efficiency of prime editors, as it plays a role in every step of the prime editing mechanism. It is therefore equally important to optimize the pegRNA than it is to have an optimized prime editor. Hence this engineering cycle explains our process of optimizing the pegRNAs for our genomic target, CFTR F508del. Given that different areas of the pegRNA have different functionalities, the following iteration cycles will demonstrate how improvements and optimizations have been made to these various functional domains in relation to the CFTR context. This was achieved through research, the correspondence with of experts and experiments.</p>
<div className="casettecontainer">
<div className="pegrnabox" style={{height: 'auto', width: '50%'}}>
<img src="https://static.igem.wiki/teams/5247/engineering-cycle/pegrna-overview.svg" alt="Illustration of the key components in our pegRNAs, including spacer, gRNA scaffold, primer binding sequence, reverse transcriptase template, tevopreQ1 3' stem loop motif and templates for main and silent edits."/>
<figcaption><b>Figure 1:</b> Illustration of the key components in our pegRNAs, including spacer, gRNA scaffold, primer binding sequence
(PBS), reverse transcriptase template (RTT), tevopreQ1 3' stem loop motif and templates for main and silent edits. </figcaption>
</div>
</div>
</div>
<div className="box" >
<p id="peg1">
<H3 text="Initial pegRNA Design and Silent Edits" id="peg1head"/>
<p>
The first iteration of our engineering cycle, we designed our first set of pegRNAs targeting the
<a onClick={() => goToPageWithTabAndScroll ({scrollToId: 'reporter-header', path: '/engineering',
tabId: 'reporter' })}>modified pPEAR_CFTR reporter</a>. We also focused on the incorporation of
silent edits.
</p>
<H4 text="Design" id="design-head"/>
<p>
Following an interview with <a onClick={() => goToPagesAndOpenTab('JPpegRNA', '/human-practices')}>
Jan-Phillipp Gerhard</a>, we came across the concept of silent edits. Silent edits refer to single-base
alterations of the nucleotide sequence that do not change the encoded amino acid. Jan-Phillipp pointed out
that introducing silent edits in addition to the intended edit offers two major advantages.
</p>
<p>
Firstly, silent edits can increase the likelihood of flap incorporation during the prime editing process,
especially in the context of MMR (Mismatch Repair) in the cell. Without silent edits, the cell is more likely
to detect the mismatches that only occur at the desired mutation site, leading to a higher chance of the
wild-type flap being reinserted. By introducing silent edits, multiple mismatches are present which this
increases the probability of the synthesized flap being incorporated.
</p>
<p>
Secondly, silent edits can prevent re-binding of the prime editing complex to the target region after
successful editing. This is be achieved by introducing silent edits to the regions making up PAM sequence
and/or protospacer. PAM or protospacer disruption make the editing process more secure. This is because it
reduces the likelihood of editing the target region repeatedly, which would increase the probability of
on-target undesired editing outcomes. He suggested that swapping cytosine or guanine bases for these silent
edits can be particularly effective in improving prime editing efficiency.
</p>
<H4 text="Build" id="build-head"/>
<p>
We designed several pegRNAs, both with and without silent edits. To assist with this, we used the pegFinder
software<TabScrollLink tab="tab-pegrna" num="18" scrollId="desc-18"/>, which generated possible variations of
pegRNAs based on the sequence of the reporter plasmid. We selected the optimal pegRNA as suggested by the
software, and then tested it in two forms: one unmodified and one with silent edits. For the unmodified
variant, we included a single silent edit that introduced a PAM disrupt in terms of our biosafety measures.
For the modified variant, we introduced three silent edits in total, adding two more to the initial edit.
</p>
<p>
Once we had designed these variants, we ordered them in their individual components and cloned them into a
pU6-peg-GG-acceptor backbone using Golden Gate cloning according to the protocol from Anzalone et al.
2019<TabScrollLink tab="tab-pegrna" num="19" scrollId="desc-19"/>. We then screened the assembled pegRNAs to ensure that the individual components had the correct orientation and then cloned them into the pU6-GG-pegRNA-acceptor plasmid so that they were ready to be tested.
</p>
<H4 text="Test" id="test-head"/>
<p>
These two variants were then tested against each other using our <a onClick={() => goToPageWithTabAndScroll ({scrollToId: 'rep3', path: '/engineering', tabId: 'reporter' })}>pPEAR_CFTR reporter plasmid system</a> and a <a onClick={() => goToPageWithTabAndScroll ({scrollToId: 'scroll target id', path: '/page', tabId: 'tabid' })}>PE2 prime editor</a>. The test of the pegRNAs was conducted by co-transfecting the reporter system, the pegRNA plasmids and the PE2 plasmids into HEK293 cells.
</p>
<H4 text="Learn" id="learn-head"/>
<p>
The results showed that the editing efficiency of the variant without silent edits was superior to the variant with silent edits, which considering our input was not expected. But as we have learned in the interview with Jan-Phillipp Gerhard, these silent edits are especially effective in avoiding mismatch repair (MMR) inside human cells. Form <a onClick={() => goToPagesAndOpenTab('mattijsinv', '/human-practices')}> Mattijs Bulcaen </a> we learned, that HEK293 cells are deficient in this very mechanism. From this we deduced that we had to test the silent edits in lung ephital cells to get a valid result.
</p>
</p>
</div>
<div className="box" >
<h3>pegRNA</h3>
<p><LoremShort></LoremShort></p>
<p id="peg2">
<H3 text="Screening of pegRNA variants" id="peg2head"/>
<p>
In this second iteration, we focused on further optimizing our pegRNA by incorporating a stem loop and experimenting with different lengths of the PBS (Primer Binding Site) and RTT (Reverse Transcriptase Template). These modifications were inspired by a combination of literature research and expert interviews. After evaluating the performance of the pegRNAs using flow cytometry, we selected the three most effective candidates.
</p>
<H4 text="Design" id="design-head"/>
<p>
Based on literature reviews and our interview with <a onClick={() => goToPagesAndOpenTab('mattijsinv', '/human-practices')}> Mattijs Bulcaen </a>, we decided to modify our pegRNA by adding a stem loop to enhance its stability. Specifically, Mattijs recommended using the tevopreQ1 stem loop, a small structural motif that increases the pegRNA's resistance to RNases. This stem loop was added to the 3' end of the pegRNA, positioned after the PBS.
</p>
<p>
Additionally, during a webinar with B. Sc. Jordan Doman<TabScrollLink tab="tab-pegrna" num="20" scrollId="desc-20"/>, we learned that it is important to test various lengths of PBS and RTT, as there is no universally optimal length for all applications. Instead, the ideal lengths are application specific. Following this advice, we designed six different pegRNA variants with combinations of two different PBS lengths (16 and 17 nucleotides) and three different RTT lengths (27, 30, and 33 nucleotides).
</p>
<p>
We chose the PBS lengths of 16 and 17 nucleotides based on an earlier recommendation from Jan-Phillipp Gerhard, who emphasised that the annealing temperature of the PBS should match the environmental conditions relevant to the intended application. In our case, since we are exploring a potential therapeutic approach, it is important that the annealing temperature of the PBS is close to the body temperature of 37 °C, which is the case for these lengths. The RTT lengths were selected based on suggestions from the pegFinder software. As with our previous insights, we designed all six variants both with and without silent edits for a wider comparison of the silent edits, making it 12 variants in total.
</p>
<H4 text="Build" id="build-head"/>
<p>
Once we had designed these variants, we ordered them in their individual components and cloned them together using Golden Gate cloning. This was a much more resource-efficient and sustainable option, as only the PBS and/or RTT lengths differed. Thus, there was a constant pegRNA part, consisting of spacer and scaffold, and a variable part, consisting of PBS, RTT and stem loop. We then cloned these variants into the pU6-GG-pegRNA-acceptor plasmid and confirmed the correct orientation and successful cloning of all constructs through screening.
</p>
<H4 text="Test" id="test-head"/>
<p>
We tested these twelve pegRNA variants against each other and the two previous variants without the trevopreQ1 stem loop, again within the PE2 system, using our reporter system, to assess their editing efficiency. The experimental setup was similar to the cycle before.
</p>
<H4 text="Learn" id="learn-head"/>
<p>
From this round of testing, we found out that our engineered pegRNA variants pegRNA04, 05, 07 and 08 exhibited the highest levels of efficiency and stability, while the pegRNA12 showed the lowest level of editing efficiency. Therefore, we reasoned to go with these four pegRNA variants as well as pegRNA12 as a negative example for follow-up experiments.
</p>
</p>
</div>
<div className="box" >
<p id="peg3">
<H3 text="Application lung epithelial cell lines" id="peg3head"/>
<p>
HEK cells are an easy to handle and easy to edit cell model. However, they are not particularly similar to the cells that would actually be useful targets for a gene therapy. In our context, two key differences are especially grave: HEK cells, as mentioned above, are impaired in mismatch repair, making them easier to edit, and they do not naturally express CFTR.
</p>
<H4 text="Design" id="design-head"/>
<p>
In this third iteration, we wanted to investigatee the applicability of a pegRNA optimized in a model closer to therapeutic application. In our case we used in <a onClick={() => goToPageAndScroll ('Cell Culture2H', '/materials-methods')}>CFBE41o- epithelial cells lines</a> homozygous for the CFTR F508del mutation.
</p>
<H4 text="Build" id="build-head"/>
<p>
For this test, we used one of the pegRNAs (pegRNA04) that showed the highest efficiencies in previous optimization steps. Since we expected only low editing efficiencies compared to HEK cells for reasons mentioned above, we used the <a onClick={() => goToPageWithTabAndScroll ({scrollToId: 'pe2', path: '/engineering', tabId: 'pe-systems' })}>PE6c prime editor</a>. It had proven to be most effective in HEK cells in our <a onClick={() => goToPagesAndOpenTab('pe-systems', '/engineering')}> pe systems engineering cycle </a> and should ensure detectability of possible editing.
</p>
<H4 text="Test" id="test-head"/>
<p>
We co-transfected the CFBE41o- with our modified <a onClick={() => goToPagesAndOpenTab('reporter', '/engineering')}> reporter system </a>, the plasmid expressing pegRNA04 as well as pCMV-PE6c. As a result, we observed fluorescence, indicating successful editing of the reporter plasmid. The negative controls transfected with only one of the plasmids each showed no fluorescence, routing out other factors.
</p>
<H4 text="Learn" id="learn-head"/>
<p>
Thanks to this experiment we knew, that our pegRNAs work not only in HEK, but also in epithelial cells that express CFTR F508del.
</p>
</p>
</div>
<div className="box" >
<p id="peg4">
<H3 text="Application to genomic CFTR targeting" id="peg4head"/>
<p>
In this fourth iteration, we aimed to transfer our findings in optimizing the pegRNAs, generated in previous iterations, to the genomic CFTR context. To this end we modified our pegRNAs to be used in the CFTR gene editing process.
</p>
<H4 text="Design" id="design-head"/>
<p>
Using the pegFinder software and our acquired expertise in creating pegRNAs, we designed the new variants specifically tailored to the genomic CFTR region. These pegRNAs included the same combinations of PBS and RTT lengths as the ones we created for our reporter plasmid. Notably, scaffold, spacer, PBS and a part of the RTT did not have to be changed from the reporter targeting to genome targeting pegRNAs. Of the created pegRNAs, we wanted to focus on testing the most effective four variants found in the previous cycles and also a variant designated comparatively ineffective to test for consistency of our reporter system.
</p>
<H4 text="Build" id="build-head"/>
<p>
The newly designed pegRNAs were ordered as separate components, identical to the process used for the pegRNAs targeting the reporter system. Each RNA had both a constant and variable region, which we assembled using Golden Gate cloning. Afterwards we confirmed the correctness and completeness of the cloning into the pU6-peg-GG-acceptor plasmid through colony PCR screening. Unfortunately to this point, we were not able to produce positive clones.
</p>
<H4 text="Test" id="test-head"/>
<p>
The next step is to test the correction of CFTR F508del using these pegRNAs in the CFBE41o- epithelial cells. Additionally, we also want to test the pegRNAs in primary cells derived from friend of the team and Cystic Fibrosis patient <a onClick={() => goToPagesAndOpenTab('maxfirst', '/human-practices')}> Max </a>, testing whether our approaches are applicable not only in model systems, but also work in patient cells. To validate the editing efficiency of our designed pegRNAs were going to co-transfect a plasmid carrying an eYFP variant which is sensitive to chloride and iodide ion concentrations<TabScrollLink tab="tab-pegrna" num="21" scrollId="desc-21"/><TabScrollLink tab="tab-pegrna" num="22" scrollId="desc-22"/>. The intensity of the fluorescence correlates with these ion concentrations, which in turn reflects the functionality of the CFTR channel. This enables us to evaluate the editing efficiency of the different pegRNA variants on a phenotypic level. After 72 hours, we are going to perform a final analysis using flow cytometry to quantify the results and determine the editing efficiency of each pegRNA. Secondly, we wanted to detect the editing on a genomic level by facilitating a qPCR with a primer specific only to the corrected F508del locus.
</p>
<H4 text="Learn" id="learn-head"/>
<p>
With this experiment we hope to achieve two things: Firstly, we want to examine whether optimizations of pegRNAs designed for our reporter system actually transfer to the genomic CFTR targeting. Secondly and most importantly, we want to find out whether we actually created an effective gene editing strategy for the genomic context of CFTR, thereby providing a foundation for a future gene therapy with high efficiency and precision when used with the right prime editor.
</p>
</p>
</div>
<div className="box" >
<p id="peg5">
<H3 text="Outlook" id="peg5head"/>
<p>
In this final iteration, we focus on the outlook for future modifications and optimizations of our pegRNA design. These concepts are meant to further improve both the stability and editing efficiency through additional research and the implementation of new design strategies.
</p>
<H4 text="Design" id="design-head"/>
<p>
As we continued to refine our approach, further literature research was conducted, and new design ideas considered. The overarching goal remained to enhance both the stability and editing efficiency of the pegRNAs. One concept we are already exploring involves the incorporation of 3’ and 5’ UTRs (Untranslated Regions)<TabScrollLink tab="tab-pegrna" num="23" scrollId="desc-23"/>. These elements, typically found in mRNA, could be added to the pegRNA to increase its stability.
</p>
<p>
Another promising idea is the use of circular RNA (circRNA)<TabScrollLink tab="tab-pegrna" num="24" scrollId="desc-24"/>, which could provide additional stability by maintaining the closed-loop structure of the pegRNA. This would prevent degradation and increase the longevity of the pegRNA in the cell.
</p>
<p>
Additionally, further nucleotide modifications could be explored, such as experimenting with alternative silent edits to see if this leads to improved editing efficiency. We also nucleotide substitutions in the scaffold region to enhance RNA-binding affinity to the protein complex could be of use.
</p>
<H4 text="Build" id="build-head"/>
<p>
To implement these new design features, the individual components, such as UTRs, would need to be cloned into the existing pegRNAs. If we pursue alternative silent edits, the pegRNA sequences would need to be redesigned, ordered, and re-cloned. The circular RNA would also require a new assembly method to achieve the desired structure. However, the fundamental workflow would remain consistent with the processes used in previous iterations.
</p>
<H4 text="Test" id="test-head"/>
<p>
To maintain consistency and comparability, the same testing protocols used for the previous pegRNA screening would be applied. This includes co-transfection in the appropriate cell lines, fluorescence-based readouts for editing efficiency, and flow cytometry analysis. By keeping the experimental conditions the same, we can ensure that the effects of the new modifications can be accurately assessed and compared to previous results.
</p>
<H4 text="Learn" id="learn-head"/>
<p>
From these tests, we would aim to derive new insights not only specific to our particular context but also for pegRNA design as a whole. These future modifications could also yield valuable information on how to further improve the overall efficiency and stability of pegRNAs, contributing to the broader field of gene editing.
</p>
</p>
</div>
<Section title="References" id="references">
<EngPegsources/>
</Section>
<br/>
<div className="row ">
<div className="col">
<div className="left"><ButtonOneEngineering label="Previous" open="nikase"/></div>
<div className="left"><ButtonOneEngineering label="Previous" open="pe-systems" scrollToId="pe-systems-header"/></div>
</div>
<div className="col button-left">
<div className="right"><ButtonOneEngineering label="Next" open="delivery"/></div>
<div className="right"><ButtonOneEngineering label="Next" open="nickase" scrollToId="nickase-header"/></div>
</div>
</div>
</section>
</div>
<div className="enginneeringtab" id="tab-delivery" style={{display: "none"}}>
<section id="Delivery" >
<div className="enginneeringtab" id="tab-nickase" style={{display: "none"}}>
<section id="Nickase sec" >
<div className="eng-box box" >
<H2 id="nickase-header" text="Alternative Nickases"></H2>
<p>The Cas9 nickase is the key component of most current prime editing system. It is needed for localizing the genomic target and cutting a single DNA strand. The complex's size and RNA stability issues limit its efficiency. To overcome these challenges, we explored smaller endonucleases like CasX and Fanzor, which not only reduce the size of the complex but also offer structural advantages such as a reversed guide RNA architecture. We theorize that this unique configuration protects the RNA from degradation and improves editing precision by reducing the risk of unwanted genomic alterations by scaffold readthrough, making CasX and SpuFz1 promising alternatives to Cas9-based systems for prime editing.</p>
</div>
<div className="box" >
<h3>Delivery</h3>
<p id="del1">
<h3>del1</h3>
<LoremShort></LoremShort>
<p id="nic1">
<H3 text="SpuFz1 Zink Finger Mutation" id="nic1head"/>
<H4 text="Design" id="text"/>
<p>
In our quest to identify smaller endonucleases suitable for creating nickases, we focused on a newly characterized family of eukaryotic endonucleases known as Fanzor proteins first described in June 2023<TabScrollLink tab="tab-nickase" num="25" scrollId="desc-25"/>, with SpuFz1 (Fig. 1) being a standout candidate due to its smaller size compared to Cas9 (SpuFz1 consists of 638 amino acids<TabScrollLink tab="tab-nickase" num="25" scrollId="desc-25"/>, whereas Cas9 has a size of 1368 amino acids<TabScrollLink tab="tab-nickase" num="26" scrollId="desc-26"/>). We selected SpuFz1 not only because of its smaller size, but also due to structural advantages, such as the reversed positioning of the spacer, which provides better protection from RNase degradation and improves editing precision.
</p>
<p>
The Cas9 endonuclease contains two active domains, each responsible for cutting one of the two DNA strands. Cas9 uses the RuvC and HNH domains, with each domain making a cut on a different strand of the target DNA<TabScrollLink tab="tab-nickase" num="27" scrollId="desc-27"/>. To create a nickase from Cas9, scientists deactivate one of these active domains, typically the HNH domain, so that the enzyme only cuts one strand instead of both, producing a single-strand break rather than a double-strand break<TabScrollLink tab="tab-nickase" num="27" scrollId="desc-27"/>.
</p>
<p>
Based on the function of this prototypical Cas9 nickase, we assumed that SpuFz1 would operate similarly, with two active centers—RuvC and TNB—each cutting one DNA strand. Following this logic, we hypothesized that by deactivating the TNB domain, which contains a zinc finger motif (Fig. 2) crucial for DNA coordination, we could convert SpuFz1 into a nickase. To test this, we aimed to replace the cysteine residues involved in zinc ion coordination within the TNB domain with alanine, thereby impairing its DNA-binding ability and producing a SpuFz1 nickase that cuts only one strand. At that time, we believed both domains in SpuFz1 were directly responsible for DNA cleavage, and our strategy was based on this assumption.
</p>
<TwoFigureRow
pic1="https://static.igem.wiki/teams/5247/engineering-cycle/spufz-wt-3d-model.webp"
description="Schematic illustration of SpuFz1 (PDB code: 8GKH) visualized in ChimeraX"
alt1="Schematic illustration of SpuFz1 (PDB code: 8GKH) visualized in ChimeraX"
num={1}
num2={2}
description2="Close up of the zinc finger motif of SpuFz1 (PDB code: 8GKH) visualized in ChimeraX - in the middle of the image the zinc ion of the motif can be seen, which is coordinated by 4 surrounding cysteine residues"
pic2="https://static.igem.wiki/teams/5247/engineering-cycle/spufz-wt-3d-model-zinc-finger.webp"
/>
<H4 text="Build" id="text"/>
<p>
Using the protein visualization software ChimeraX, we carefully examined the structure of SpuFz1 to identify the key cysteine residues responsible for coordinating the zinc ion. With this insight, we designed our nickase candidates by modifying the wild-type sequence, specifically substituting these cysteines with alanine, to disrupt the zinc ion coordination and potentially alter the protein's function.
</p>
<H4 text="Test" id="text"/>
<p>
First, we discussed our approach with <a onClick={() => goToPagesAndOpenTab('hammerkai', '/human-practices')}> Kai Schülke </a>, a PhD student from the Hammer Group at Bielefeld University, which specializes in enzyme engineering. He confirmed that our plan to focus on specific mutation candidates was appropriate given the constraints of our project. He emphasized that we lacked the time and resources to conduct large-scale, quantitative studies on a wide range of mutations. Instead, he supported our decision to target specific candidates that could be thoroughly tested within the scope of our project.
</p>
<p>
Additionally, we carefully considered the potential effectiveness of our modified SpuFz1 nickase in a Prime Editing scenario, specifically targeting the F508del mutation in Cystic Fibrosis. During this detailed analysis, we identified a critical challenge: the TAM sequence required for SpuFz1 binding was located too far from the target mutation site. This distance could limit the efficiency of the Prime Editor, raising concerns about its overall effectiveness for this particular mutation.
</p>
<InfoBox title="TAM sequences" id="current-pe-systems">
<details>
<summary>
A TAM sequence is the equivalent to a PAM sequence for OMEGA systems.
</summary>
<p>
A <b>TAM sequence</b> (Targeted Activity Modification sequence) is a short DNA sequence, typically only a few bases long, that provides a binding site for the nickase within the Prime Editing complex. This sequence is crucial because it allows the nickase to bind to the DNA and make a precise single-strand cut. For the Prime Editing complex to correct a mutation at a specific location guided by the pegRNA, a TAM sequence must be located near that target site. While the pegRNA directs the editing machinery to the region where the correction will occur, the TAM sequence enables the nickase to physically interact with the DNA and initiate the cut. Therefore, both the pegRNA and the TAM sequence are essential for efficient and accurate editing: the pegRNA specifies the site of the correction, and the TAM sequence facilitates the nickase's binding and action. For instance, SpuFz1 recognizes the TAM sequence <b>5'-CATA-3'</b>, and CasX binds to <b>5'-TTCN-3'</b>.
</p>
</details>
</InfoBox>
<H4 text="Learn" id="text"/>
<p>
Through this iteration, we learned that targeted mutagenesis is a promising approach for generating our mutant nickases. We also recognized the importance of carefully selecting the appropriate PAM or TAM sequences for our chosen endonucleases. Specifically, we realized that the TAM sequence for SpuFz1 might be too far from our target mutation, prompting us to explore other endonucleases within the Fanzor family that could serve as better candidates for nickase development. Additionally, this process highlighted the critical role of expert consultation in refining our strategies and ensuring the feasibility of our approach.
</p>
</p>
<div className="box" >
<p id="nic2">
<H3 text="Fusion Protein from GtFz1 & SpuFz1" id="nic2head"/>
<H4 text="Design" id="design-head"/>
<p>
In our ongoing exploration of Fanzor proteins, we identified another potential candidate, GtFz1, which had a suitable TAM sequence for our target application of correcting the F508del mutation in Cystic Fibrosis. However, GtFz1 showed low cutting efficiency in the tests reported in the literature<TabScrollLink tab="tab-nickase" num="25" scrollId="desc-25"/>. To address this, we devised a strategy to combine the favorable TAM-binding properties of GtFz1 with the higher cutting efficiency of SpuFz1. Specifically, we planned to engineer a fusion protein by replacing the TAM-binding domain of SpuFz1 with that of GtFz1. This approach aims to create an endonuclease that retains the strong TAM-binding ability of GtFz1 while utilizing the robust cutting efficiency of SpuFz1, optimizing it for our Prime Editing application.
</p>
<p>
Given that we were swapping entire domains rather than just single amino acids, we realized that the fusion protein might not retain the ideal TAM-binding efficiency or cutting efficiency of the original proteins. Our strategy was to create a fusion protein that could bind to the TAM site and perform DNA cutting to a certain extent, albeit weakly. We planned to use directed evolution techniques, such as Phage Assisted Continuous Evolution (PACE), to enhance these functionalities over time. This approach relies on having a starting point with some degree of the desired activity, which can then be incrementally improved through evolution.
</p>
<H4 text="Build" id="build-head"/>
<p>
The build phase involved designing this fusion protein by integrating the TAM-binding region from GtFz1 into the SpuFz1 protein structure. We engineered the sequence to include this hybrid configuration, intending to test its functionality as a nickase after introducing the zinc finger mutation, which we had hypothesized would inactivate one of the DNA-cutting domains.
</p>
<H4 text="Test" id="test-head"/>
<p>
To validate our approach, we conducted two key interviews. First, we consulted with <a onClick={() => goToPagesAndOpenTab('hammer', '/human-practices')}> Prof. Dr. Hammer </a> from Bielefeld University, who highlighted the possibility that the zinc finger domain might be structurally significant and cautioned that mutating it could destabilize the protein. He recommended that we explore whether there were any known enzymes with similar mechanisms where analogous mutations had successfully converted endonucleases into nickases. This approach, he suggested, might offer a more reliable pathway.
</p>
<p>
Next, we spoke with <a onClick={() => goToPagesAndOpenTab('svenja', '/human-practices')}> Svenja Finke </a>, a Postdoctoral Fellow at the Harvard Institute and an expert in directed enzyme evolution, including PACE. We reached out to her specifically because we anticipated that our fusion protein might require optimization to achieve strong TAM-binding and cutting efficiency. Svenja informed us that while PACE could theoretically optimize our fusion protein, the process was too complex and time-consuming for the scope of our project. As a result, we decided to reconsider this method and look for simpler, more feasible alternatives.
</p>
<H4 text="Learn" id="learn-head"/>
<p>
From this iteration, we learned several important lessons. First, we decided to abandon the fusion protein approach. Feedback from Svenja’s interview highlighted that this strategy was too complex, time-consuming, and involved significant uncertainty regarding its effectiveness. Given the long testing times and the inherent risks, we concluded that this approach was not viable within our project’s constraints. Initially, we considered moving away from SpuFz1 due to the TAM sequence being too far from the ΔF508 mutation. However, with ongoing improvements in reverse transcriptases within Prime Editing systems, which allow for greater distances between the mutation site and the TAM sequence, we refocused our efforts on SpuFz1, considering it a viable candidate for further development.
</p>
<p>
Secondly, we realized the importance of verifying whether the zinc finger mutation we proposed is structurally feasible and whether it might compromise protein stability. This insight further emphasized the need to carefully assess our design choices before proceeding to extensive testing.
</p>
<p><LoremShort></LoremShort></p>
<p><LoremShort></LoremShort></p>
</p>
</div>
<div className="box" >
<p id="Modeling of Mutant Structural Integrity">
<H3 text="nic3" id="nic3head"/>
<H4 text="Design" id="design-head"/>
<p>
In the previous iteration, we consulted <a onClick={() => goToPagesAndOpenTab('hammer', '/human-practices')}> Prof. Dr. Hammer </a>, who suggested that the zinc finger domain in the SpuFz1 protein might play a critical structural role. Based on this feedback, the goal of this iteration was to investigate whether mutating the zinc finger would destabilize the protein and compromise its function. Specifically, we aimed to determine if altering this domain would still be a viable strategy for generating a SpuFz1-based nickase without losing structural integrity.
</p>
<H4 text="Build" id="build-head"/>
<p>
Before, we identified the specific amino acids responsible for coordinating the zinc ion within the zinc finger domain. Using the software Geneious, we proceeded to design DNA sequences by substituting these key amino acids with ones that would impair their ability to coordinate the zinc ion. These designed sequences corresponded to our potential mutation candidates, which we prepared for further structural analysis.
</p>
<H4 text="Test" id="test-head"/>
<p>
We used AlphaFold to model the 3D structures of our zinc finger mutation candidates. After generating these models, we used ChimeraX to perform a structural overlay comparison between the native SpuFz1 protein and the mutated versions (Fig. 3). This comparison revealed significant differences, particularly in the TNB domain, indicating that the zinc finger plays a crucial structural role (Fig. 4).
</p>
<TwoFigureRow
pic1="https://static.igem.wiki/teams/5247/engineering-cycle/spufz-wt-vs-zf-nikase.webp"
pic2="https://static.igem.wiki/teams/5247/engineering-cycle/spufz-wt-vs-zf-nikase-zinc-finger.webp"
num={3}
num2={4}
alt1="Structural overlay of wildtype SpuFz1 (color: Lilac) (PDB code: 8GKH) and modeled zinc-finger mutation candidate (color: orange) visualized in ChimeraX – the yellow circle shows the location of the zinc-finger. A structural deviation of both proteins locally is evident"
description="Structural overlay of wildtype SpuFz1 (color: Lilac) (PDB code: 8GKH) and modeled zinc-finger mutation candidate (color: orange) visualized in ChimeraX – the yellow circle shows the location of the zinc-finger. A structural deviation of both proteins locally is evident"
description2="Close-up of the zinc finger motif of the structural overlay - the zinc finger appears to be structurally significant: there are strong structural differences locally"
/>
<H4 text="Learn" id="learn-head"/>
<p>
From this analysis, we concluded that the zinc finger mutation is not a suitable candidate for generating a nickase, as altering this domain would likely compromise the structural integrity of SpuFz1. Prof. Hammer suggested that instead of focusing on SpuFz1, we explore other endonucleases with similar mechanisms. His recommendation was to identify endonucleases that are structurally comparable to SpuFz1 and analyze the strategies used to convert these into nickases. We would then apply these same strategies to our selected endonucleases, adapting them for our purposes.
</p>
</p>
</div>
<div className="box" >
<p id="nic4">
<H3 text="nCas12 analog Mutations" id="nic4head"/>
<H4 text="Design" id="design-head"/>
<p>
After concluding that the zinc finger mutation approach was not suitable for converting SpuFz1 into a nickase, we revisited our understanding of its cutting mechanism. Initially, we believed that SpuFz1, similar to Cas9, contained two active centers that each cut one of the DNA strands, and that by deactivating one of these centers, we could generate a nickase that only cuts one strand. However, through further research, we discovered that this assumption was incorrect. SpuFz1 actually functions with a different cutting mechanism: the RuvC domain is responsible for cutting the non-target strand, while the TNB domain does not directly cut the DNA. Instead, it assists the process by guiding the target strand into the RuvC domain for sequential cleavage<TabScrollLink tab="tab-nickase" num="29" scrollId="desc-29"/>. This discovery shifted our focus from simply deactivating an active site to better understanding how the sequential cleavage works in order to inform future mutation strategies.
</p>
<p>
In addition to these insights, we noticed a significant phylogenetic relationship between Fanzor endonucleases, like SpuFz1, and Cas12 endonucleases<TabScrollLink tab="tab-nickase" num="25" scrollId="desc-25"/>. This connection was crucial, as Cas12 proteins have a similar cutting mechanism to Fanzor proteins, utilizing a single active site for cleavage while coordinating both DNA strands. More importantly, we identified a precedent in the literature where a Cas12a endonuclease was successfully converted into a nickase by substituting a single amino acid in the TNB domain<TabScrollLink tab="tab-nickase" num="30" scrollId="desc-30"/> (Fig. 5 and 6). This provided us with a clear model strategy to follow, as this targeted mutation allowed the endonuclease to selectively cut only one DNA strand, effectively converting it into a nickase.
</p>
<TwoFigureRow
pic1="https://static.igem.wiki/teams/5247/engineering-cycle/cas12-nikase.webp"
pic2="https://static.igem.wiki/teams/5247/engineering-cycle/cas12-nikase-close-up.webp"
num={5}
num2={6}
alt1="Schematic representation of Cas12a (PDB code: 8SFH) visualized in ChimeraX - the yellow circle highlights the position of arginine (R) (1226th amino acid in the primary structure) which, when replaced by an alanine (A), converts the Cas12a endonuclease into an nCas12a nickase"
description="Schematic representation of Cas12a (PDB code: 8SFH) visualized in ChimeraX - the yellow circle highlights the position of arginine (R) (1226th amino acid in the primary structure) which, when replaced by an alanine (A), converts the Cas12a endonuclease into an nCas12a nickase"
description2="Close-up of Cas12a (PDB code: 8SFH) - arginine (R) (1226th amino acid in the primary structure) is colored purple"
/>
<p>
Based on these findings, two key decisions emerged. First, recognizing the structural and mechanistic similarities between Fanzor and Cas12 endonucleases, we decided to explore CasX—a smaller Cas12-related endonuclease—as an additional candidate in our project. CasX shares many of the advantages of SpuFz1, such as a more compact structure compared to Cas9, making it ideal for applications requiring smaller editing systems. Secondly, we resolved to adapt the mutation strategy used to convert Cas12a into a nickase for both CasX and SpuFz1. By applying these learnings, we aimed to generate effective nickases from these endonucleases to further optimize the Prime Editing complex.
</p>
<InfoBox title="The rationale behind designing SpuFz1 and CasX Nickases" id="how-to-create-nickases-online-fast">
<details>
<summary>
The mutation strategy aimed to convert the endonucleases SpuFz1 and CasX into nickases by targeting specific positively charged amino acids, similar to R1226 in mutated to create a AsCas12a nickase, to disrupt their double-strand cleavage function while retaining single-strand cutting capability.
</summary>
<p>
In our project, we set out to engineer the endonucleases SpuFz1 and CasX into nickases, a process that required a more targeted approach than random mutagenesis due to the time and financial constraints of our project. Random mutagenesis, while a possible strategy, would have required an extensive scope, making it difficult to achieve meaningful results within our timeframe. As a result, we aimed to identify specific mutational candidates that would allow for a more focused and efficient approach.
</p>
<p>
One strategy we explored was finding an endonuclease with structural and mechanistic similarities to SpuFz1 and CasX, for which a successful precedent existed in converting an endonuclease into a nickase. After studying the phylogenetic relationships of SpuFz1 and CasX, we identified AsCas12a, an endonuclease with a similar sequential DNA cleavage mechanism. Importantly, there was already a known example where AsCas12a had been engineered into a nickase through a single mutation—specifically, the mutation of arginine 1226. This provided a strong foundation for us to develop a similar strategy for SpuFz1 and CasX.
</p>
<p>
We hypothesized that the role of arginine 1226 in the sequential cleavage mechanism of AsCas12a was to coordinate the DNA strands during the cutting process. AsCas12a performs a sequential cut, where the RuvC domain first cleaves the non-target strand, and the TNB (NUC) domain helps guide the target strand into the RuvC domain for cleavage (Fig. 7). We suspected that arginine 1226 could play a key role in this process by coordinating the DNA due to its long, positively charged side chain. If removing or mutating this arginine disrupts the sequential cut, it would suggest that the arginine helps guide the second strand into the RuvC domain.
</p>
<p>
Structurally, we observed that arginine 1226 protrudes from the NUC domain of AsCas12a and is oriented toward the RuvC domain (Fig. 8). This positioning led us to hypothesize that the arginine helps coordinate the DNA strand as it moves into the RuvC domain for cutting. Based on this observation, we speculated that the mutation of arginine 1226 disrupts this coordination, preventing the full double-strand cut and effectively converting AsCas12a into a nickase.
</p>
<TwoFigureRow
pic1="https://static.igem.wiki/teams/5247/engineering-cycle/ascas12a-nuc-domain.webp"
pic2="https://static.igem.wiki/teams/5247/engineering-cycle/ascas12a-nuc-domain-close-up.webp"
num={7}
num2={8}
description2="Close-up of NUC domain (colored purple) of AsCas12a(PDB code: 8SFH) - the arginines (R) (1226th amino acid in the primary structure) is colored yellow. Its positively charged side chain is oriented towards the RuvC domain, as well as the DNA strand fixated in the RuvC domain"
description="AsCas12a (PDB code: 8SFH) visualized in ChimeraX. The NUC domain (TNB) is colored purple and is attached to the RuvC domain. The DNA strand is colored orange"
alt1="AsCas12a (PDB code: 8SFH) visualized in ChimeraX. The NUC domain (TNB) is colored purple and is attached to the RuvC domain. The DNA strand is colored orange"
/>
<p>
We then applied this structural insight to SpuFz1 and CasX, searching for positively charged amino acids with long side chains, similar to arginine 1226, that were positioned at the interface between the NUC and RuvC domains. We specifically looked for amino acids that protruded from the NUC domain and oriented toward the RuvC domain, mirroring the structural role of arginine 1226 in AsCas12a. These amino acids became our mutational targets, allowing us to design a strategy to convert SpuFz1 and CasX into nickases by disrupting their ability to make double-strand cuts, while preserving their functionality for single-strand cuts. The amino acids we identified in SpuFz1 are the 564th and the 568th arginine located in its NUC domain. For CasX we identified the 904th arginine as a promising candidate.
</p>
</details>
</InfoBox>
<H4 text="Build" id="build-head"/>
<p>
We structurally analyzed CasX and SpuFz1, as well as the known AsCas12a nickase, using Chimera. Our objective was to understand why the specific amino acid substitution converted AsCas12a into a nickase. We then identified analogous amino acids in SpuFz1 (Fig. 7 and Fig. 8) and CasX (Fig. 9 and Fig. 10) that might play a similar role, allowing us to design new mutation candidates for our project. After designing these new mutation candidates, we modeled them using AlphaFold to predict their 3D structures and assess their potential effectiveness.
</p>
<TwoFigureRow
pic1="https://static.igem.wiki/teams/5247/engineering-cycle/casx-nikase.webp"
pic2="https://static.igem.wiki/teams/5247/engineering-cycle/casx-nikase-close-up.webp"
num={9}
num2={10}
description2="Close-up of PlmCasX (PDB code: 7WAZ) - arginine (R) (904th amino acid in the primary structure) and glutamine (Q) (907th amino acid in the primary structure) are purple in color"
alt1="Schematic representation of PlmCasX (PDB code: 7WAZ) in ChimeraX - the yellow circle highlights the position of arginine (R) (904th amino acid in the primary structure) and glutamine (Q) (907th amino acid in the primary structure), which, when replaced by an alanine (A), convert the endonuclease into a nickase, according to our hypothesis"
description="Schematic representation of PlmCasX (PDB code: 7WAZ) in ChimeraX - the yellow circle highlights the position of arginine (R) (904th amino acid in the primary structure) and glutamine (Q) (907th amino acid in the primary structure), which, when replaced by an alanine (A), convert the endonuclease into a nickase, according to our hypothesis"
/>
<TwoFigureRow
pic1="https://static.igem.wiki/teams/5247/engineering-cycle/spufz-nikase.webp"
pic2="https://static.igem.wiki/teams/5247/engineering-cycle/spufz-nikase-close-up.webp"
num={11}
num2={12}
description2="Close-up of SpuFz1 (PDB code: 8GKH) - the two arginines (R) (564th and 568th amino acid in the primary structure) are purple in color"
alt1="Schematic representation of SpuFz1 (PDB code: 8GKH) in ChimeraX - the yellow circle highlights the position of the two arginines (R) (564th and 568th amino acid in the primary structure), which, when replaced by an alanine (A), transform the endonuclease into a nickase according to our hypothesis"
description="Schematic representation of SpuFz1 (PDB code: 8GKH) in ChimeraX - the yellow circle highlights the position of the two arginines (R) (564th and 568th amino acid in the primary structure), which, when replaced by an alanine (A), transform the endonuclease into a nickase according to our hypothesis"
/>
<H4 text="Test" id="test-head"/>
<p>
To validate our approach, we conducted an interview with <a onClick={() => goToPagesAndOpenTab('saito', '/human-practices')}> Makoto Saito </a>, the lead author of the main Fanzor paper. Given his expertise, there was no better person to consult on this topic. We presented our project and our strategy for creating nickases, and he found our approach to be very plausible. He confirmed that the zinc finger mutation is likely structurally critical and agreed that our new strategy, based on the precedent with AsCas12a, was more promising. This conversation gave us confidence that we were on a good track.
</p>
<H4 text="Learn" id="learn-head"/>
<p>
From this iteration, we gained several key insights. First, our initial understanding of the cutting mechanism used by SpuFz1—based on the assumption that it contained two active centers, like Cas9, each cutting a DNA strand—was incorrect. We discovered that SpuFz1 operates differently, with the RuvC domain cutting the non-target strand and the TNB domain assisting by guiding the target strand into the RuvC domain for sequential cleavage. This shift in understanding allowed us to refine our approach, moving away from deactivating an active site to focusing on the sequential cutting mechanism.
</p>
<p>
Additionally, we found that Fanzor endonucleases, like SpuFz1, share a significant phylogenetic relationship with Cas12 endonucleases, which have a similar single-site cutting mechanism. This connection, along with the precedent of converting Cas12a into a nickase through the substitution of a single amino acid in the TNB domain, provided us with a clear strategy for converting SpuFz1 and CasX into nickases. The similarity in cutting mechanisms between Fanzor and Cas12 proteins reinforced the viability of this approach.
</p>
<p>
This iteration led us to incorporate CasX, a smaller Cas12-related endonuclease, into our project. CasX offers the same advantages as SpuFz1, such as a compact structure, making it ideal for applications that require smaller editing systems. Additionally, we adapted the mutation strategy used to convert Cas12a into a nickase for both CasX and SpuFz1, guiding our future work in optimizing the Prime Editing complex.
</p>
</p>
</div>
<div className="box" >
<p id="nic5">
<H3 text="Ongoing: In Vitro Cleavage Assays" id="nic5head"/>
<H4 text="Design" id="design-head"/>
<p>
In this iteration, our focus shifted to testing whether our mutation candidates had successfully converted the endonucleases into functional nickases. To do this, we adapted an existing assay that had been originally designed to determine whether mutated endonucleases exhibited nickase activity<TabScrollLink tab="tab-nickase" num="25" scrollId="desc-25"/>. We tailored this assay to fit our specific needs, allowing us to accurately assess the properties of our mutated proteins in the lab. The key question was whether the mutations had rendered the proteins dysfunctional, left them as endonucleases, or successfully converted them into nickases.
</p>
<H4 text="Build" id="build-head"/>
<p>
We started off by amplification of our nickase candidates, ordered as gene syntheses, to add restriction sites. We then facilitated restriction cloning of the amplificates into an E. coli protein expression vector provided by the laboratory of or PI Kristian Müller. We subsequently transformed E. coli with the gene fragments of our nickase candidates for CasX and SpuFz. However, the transformant cells did not grow, leading us to suspect that the plasmid backbone we received maybe impaired in some way. Given the timeline, we were not able to complete the testing of our nickase candidates. Our current steps involve troubleshooting regarding the restriction cloning and continuing with protein expression and purification once the issue is resolved.
</p>
<H4 text="Test" id="test-head"/>
<p>
The next phase of our plan, once we overcome the current issues with cloning and successfully overexpress our nickase candidates, would involve conducting an in vitro plasmid cleavage assay (Fig. 13). In this assay, the purified nickases would be combined with their respective guide RNAs and a supercoiled test plasmid. The guide RNAs would direct the nickases to the target sequence on the plasmid. Depending on the results, the plasmid would remain supercoiled if untouched, become relaxed if nicked, or be linearized if cut by an endonuclease. To analyze these outcomes, we would perform gel electrophoresis, where the different conformations of the plasmid (supercoiled, relaxed, or linearized) would migrate differently through the gel. Supercoiled plasmids would migrate the furthest, relaxed plasmids would move the slowest, and linearized plasmids would fall between these two. As controls, we would have used the plasmid in its uncut form, nicked by nCas9 and digested using a restriction enzyme.
</p>
<H4 text="Learn" id="learn-head"/>
<p>
If we could have proceeded with the nickase assays, the readout would allow us to determine whether the tested proteins function as nickases, endonucleases, or remain inactive.
</p>
<div className="row align-items-center">
<OneFigure
pic1="https://static.igem.wiki/teams/5247/engineering-cycle/nickase-assay.webp"
num={13}
description="Theoretical gel electrophoresis results for our nickase assay. Lanes 1 and 8 represent molecular weight ladders, which provide size markers for the plasmid fragments. Lane 2 contains the untreated reporter plasmid, which remains supercoiled and travels the farthest through the gel. Lane 3 serves as a positive control, containing nCas9, gRNA, and the reporter plasmid. The nCas9 nickase nicks the plasmid, relaxing its structure, and as a result, the relaxed circular plasmid moves slower than the supercoiled form. Lane 4 acts as a negative control, containing a restriction enzyme and the reporter plasmid. The enzyme fully cuts the plasmid, linearizing it, and this linear form moves slower than the supercoiled plasmid but faster than the relaxed circular form. Lane 5 includes CasX and the reporter plasmid without gRNA, meaning no cleavage occurs, leaving the plasmid in its supercoiled state, which migrates similarly to the untreated plasmid in lane 2. Lane 6 contains CasX, gRNA, and the reporter plasmid, resulting in full cleavage and plasmid linearization, causing it to migrate similarly to the linear plasmid in lane 4. Finally, lane 7 includes our nickase candidate (either CasX or SpuFz1), gRNA, and the reporter plasmid. Ideally, the candidate would nick the plasmid, resulting in a relaxed circular form that moves similarly to the nicked plasmid in lane 3."
alt1="Theoretical gel electrophoresis results for our nickase assay. Lanes 1 and 8 represent molecular weight ladders, which provide size markers for the plasmid fragments. Lane 2 contains the untreated reporter plasmid, which remains supercoiled and travels the farthest through the gel. Lane 3 serves as a positive control, containing nCas9, gRNA, and the reporter plasmid. The nCas9 nickase nicks the plasmid, relaxing its structure, and as a result, the relaxed circular plasmid moves slower than the supercoiled form. Lane 4 acts as a negative control, containing a restriction enzyme and the reporter plasmid. The enzyme fully cuts the plasmid, linearizing it, and this linear form moves slower than the supercoiled plasmid but faster than the relaxed circular form. Lane 5 includes CasX and the reporter plasmid without gRNA, meaning no cleavage occurs, leaving the plasmid in its supercoiled state, which migrates similarly to the untreated plasmid in lane 2. Lane 6 contains CasX, gRNA, and the reporter plasmid, resulting in full cleavage and plasmid linearization, causing it to migrate similarly to the linear plasmid in lane 4. Finally, lane 7 includes our nickase candidate (either CasX or SpuFz1), gRNA, and the reporter plasmid. Ideally, the candidate would nick the plasmid, resulting in a relaxed circular form that moves similarly to the nicked plasmid in lane 3."
/>
</div>
</p>
</div>
<div className="box" >
<p id="nic6">
<H3 text="Ongoing: SpuFz1 expression in Yeast" id="nic6head"/>
<H4 text="Design" id="design-head"/>
<p>
When talking to <a onClick={() => goToPagesAndOpenTab('saito', '/human-practices')}> Makoto Saito </a>, he told us that expressing SpuFz1 in E. coli did not work for him. He advised us to instead establish a yeast expression system. Since none of us had any experience with using yeast for protein production, we reached out to <a onClick={() => goToPagesAndOpenTab('nberelsmann', '/human-practices')}> Nils Berelsmann </a> at the faculty of chemistry at our university. He was able to provide us with a yeast expression strain as well as the corresponding expression vector. Because we were strongly restricted by time at that point, we then asked <a onClick={() => goToPagesAndOpenTab('hakan', '/human-practices')}> Hakan Soytürk </a> from the biological faculty for help and he offered to facilitate transformation and selection of positive Yeast transformants for us.
</p>
<H4 text="Build" id="build-head"/>
<p>
The workflow for cloning SpuFz1 amplificates into the pPIC9K yeast vector was similar to the cloning into the E. coli expression vector. After multiple attempts of transformations and colony PCRs we found positive clones for two of the nickase variants. Hakan transformed them into the yeast strain for us, but no colonies formed, indicating unsuccessful transformation. Due to time constraints, we were not able to repeat the cloning. However, testing our SpuFz1 and also CasX nickase candidates and eventually using them for prime editing.
</p>
</p>
</div>
<div className='row align-items-center'>
<div className='col'>
<TwoLinePDF link='https://static.igem.wiki/teams/5247/pdfs/cloning-strategy-spufz1.pdf' name="cloning-strategy-spufz1.pdf"/>
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<div className='seperator-2 col-2'>
</div>
<div className='col'>
<TwoLinePDF link='https://static.igem.wiki/teams/5247/pdfs/cloning-strategy-plmcasx.pdf' name="cloning-strategy-plmcasx.pdf"/>
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</div>
<div className='row align-items-center'>
<div className='col'>
<PDF link='https://static.igem.wiki/teams/5247/pdfs/cloning-strategy-cas9.pdf' name="cloning-strategy-cas9.pdf'"/>
</div>
</div>
</div>
<Section title="References" id="references">
<EngNicksources/>
</Section>
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</section>
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<section id="References" >
<h3>References</h3>
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<section id="Delivery sec" >
<div className="eng-box box" >
<H2 id="delivery-header" text="Delivery"></H2>
<p>The design path of our lipid nanoparticle (LNP) for mRNA delivery underwent multiple cycles of research and discussion, marked by important decision points and learnings along the way. By ongoing further improvement, we designed our lungs-specific LNP called AirBuddy with improved stability aspects, becoming more precise in the delivery of our therapeutic cargo LNP by LNP.</p>
<div className="row align-items-center">
<div className="col">
<img src="https://static.igem.wiki/teams/5247/delivery/airbuddy.webp" className="center" style={{maxHeight: "80pt", margin: "auto"}}/>
</div>
</div>
</div>
<div className="box" >
<p id="del1">
<H3 text="Iteration 1 - AVVs vs LNPs" id="del1head" />
<p>Initially, this project part started with a discussion with <a onClick={() => goToPagesAndOpenTab('kristian', '/human-practices')}> Prof. Dr. Krisitan Müller</a>, PI of our team with expertise in Adeno-associated viruses (AAVs), focusing on whether to pursue LNPs or AAVs for mRNA delivery. The deciding factor leaned towards LNPs, as they offered a significant advantages including less immunogenic potential<TabScrollLink tab="tab-delivery" num="31" scrollId="desc-31"/> and bigger loading capacity<TabScrollLink tab="tab-delivery" num="32" scrollId="desc-32"/>. LNPs loading capacity depends on various factors, but in general they offer a bigger cargo size compared to 4.7 kb for AVVs<TabScrollLink tab="tab-delivery" num="33" scrollId="desc-33"/>. This allows the delivery of bigger mRNA constructs compared to AAVs, which is needed for our Prime Editing construct.</p>
<p><a onClick={() => goToPagesAndOpenTab('weber', '/human-practices')}>Prof. Wolf-Michael Weber and Dr. Jörg Große-Onnebrink</a> from the UKM in Münster were our first point of contact for the development of our LNP for CFTR treatment. Moreover, <a onClick={() => goToPagesAndOpenTab('radukic', '/human-practices')}>Dr. Marco Radukic </a>form Bielefeld University provided us with a very useful cargo, namely minicircle DNA carrying the EYFP gene from <a href="https://www.plasmidfactory.com/custom-dna/minicircle-dna/" title="PlasmidFactory" >PlasmidFactory</a> as a positive control for our experiments. He also helped us establish protocols for LNP synthesis and LNP transfection in our lab.</p>
</p>
</div>
<div className="box" >
<p id="del2">
<H3 text="Interation 2 - Cayman LNP" id="del2head" />
In the first experimental phase, LNPs from <strong>Cayman Chemical LNP Exploration Kit (LNP-102)</strong> consisting of SM-102, 1,2-DSPC, Cholesterol, and DMG-PEG(2000)<TabScrollLink tab="tab-delivery" num="34" scrollId="desc-34"/> were tested with mRNA encoding fluorescent protein to evaluate their transfection efficiency. However, the results indicated good non-lung-specific transfection efficiency, which was a critical factor for the project. This led the team to reconsider their choice of this LNP.
</p>
<OneFigure
pic1="https://static.igem.wiki/teams/5247/delivery/cayman-lnp-freigestellt.webp"
alt1="Cayman LNP"
num={1}
description="Schematic view of LNP-102 from Cayman Chemical."
/>
</div>
<div className="box" >
<p id="del3">
<H3 text="Interation 3 - Corden LNP" id="del3head" />
In the next phase, we chose to use a new LNP formulation, namely the <strong>LNP Starter Kit #2</strong><TabScrollLink tab="tab-delivery" num="35" scrollId="desc-35"/> of <a onClick={() => goToPagesAndOpenTab('corden', '/human-practices')}>Corden Pharma</a>, because it offered several advantages over the initial option. The key benefit of this new LNP lies in the use of DOTAP, a cationic lipid that enhances interaction with negatively charged cell membranes in the lungs, improving cellular uptake efficiency. While SM-102 in the Cayman LNP-102 is effective for systemic delivery, it lacks the same specificity for lung tissue. Additionally, Corden Pharma’s plant-based BotaniChol® prevents animal-sourced contamination and helps address the global lipid shortage for vaccine production. mPEG-2000-DSPE provides superior stability and reduces immune system activation over time, making it particularly suitable for pulmonary delivery. This made the new formulation a better choice for safely and effectively targeting lung tissue, especially in delivering therapies for CFTR-related diseases. During this time, the team encountered a paper on capsaicin-chitosan nanoparticles, which explored its use in targeted delivery and higher transfection efficiency. However, after further investigation and consultation of <a onClick={() => goToPagesAndOpenTab('kolonkofirst', '/human-practices')}>Dr. Katharina Kolonko</a>, it was determined that capsaicin was not suitable in our way of pulmonary application.
</p>
<OneFigure
pic1="https://static.igem.wiki/teams/5247/delivery/corden-lnp-freigestellt.webp"
alt1="Corden LNP"
num={2}
description="Schematic view of LNP #2 from Corden Pharma with DOTAP as cationic lipid, DSPC as phospholipid and mPEG-2000-DSPE as PEG lipid."
/>
</div>
<div className="box" >
<p id="del4">
<H3 text="Interation 4 - Spray-dried SORT LNP called AirBuddy" id="del4head" />
The next design iteration incorporated the insights from Wang's LNP work for building upon SORT principles to make the nanoparticles lung-specific<TabScrollLink tab="tab-delivery" num="36" scrollId="desc-36"/>. The main components include DMG-PEG 2000, cholesterol, DOPE and DOTAP as phospholipids and cationic lipids such as 4A3-SC8. In our LNP development, we carefully considered the use of PEG. While PEG can improve stability, it can also reduce cellular uptake and induce immune responses, necessitating a balanced approach to its inclusion<TabScrollLink tab="tab-delivery" num="37" scrollId="desc-37"/>.
<Collapsible id="Col1" open={false} title="Ambivalence of PEG and our implementation">
<p>
<H4 text="What is PEG and why is it important for LNPs?" id="text" />
Polyethylene glycol (PEG) is an essential component in the formulation of LNPs, which are widely used in drug delivery systems, particularly for mRNA-based therapies like vaccines. PEG-lipids are hybrid molecules consisting of a hydrophilic PEG chain attached to a hydrophobic lipid anchor. This unique structure enables PEG-lipids to interact effectively with both aqueous environments and lipid structures, such as cell membranes and lipid nanoparticles themselves.
<p>PEGylation—attaching PEG to lipids—provides numerous benefits. It increases the stability of LNPs by forming a protective outer layer, preventing aggregation, extending circulation time in the bloodstream, and reducing immune system detection. These advantages are critical in ensuring that the LNPs reach their target cells and deliver the therapeutic payload effectively. </p>
<H4 text="Why is PEG relevant for LNPs in mRNA delivery?" id="text" />
PEG improves the pharmacokinetics of LNPs by extending their systemic circulation time, which is crucial for therapies like mRNA vaccines, where the nanoparticles must remain in the bloodstream long enough to reach their target cells. Additionally, PEG-lipids can reduce the size of LNPs, enhancing their ability to penetrate cell membranes and deliver the therapeutic material efficiently. However, a balance must be struck. Increasing PEG content can lead to smaller, more stable particles, but it may also reduce intracellular delivery and protein expression. Therefore, while PEG boosts circulation and stability, too much can hinder therapeutic effectiveness.
<H4 text="Cytotoxicity and mPEG-2000-DSPE" id="text" />
One challenge with PEGylation is the potential for immune responses, such as the <i>accelerated blood clearance</i> (ABC) phenomenon, where repeated exposure to PEGylated particles leads to faster clearance by the immune system. There are also risks of hypersensitivity reactions like <i>complement activation-related pseudoallergy</i> (CARPA). Thus, selecting the right PEG-lipid type is essential to mitigate these risks.
<p>We collaborated with <a onClick={() => goToPagesAndOpenTab('corden', '/human-practices')}>Corden Pharma</a>, a specialist in LNP technologies, to address these concerns. Based on their recommendations, we opted for <strong>mPEG-2000-DSPE</strong> as our PEG-lipid of choice. This variant minimizes cytotoxicity while providing excellent stability and circulation time. It has also proven effective in reducing immune-related side effects while preserving the integrity and performance of our nanoparticles. </p>
<H4 text="DMG-PEG2000 vs mPEG-2000-DSPE" id="text" />
While mPEG-2000-DSPE has traditionally been used for stabilizing LNPs and enhancing delivery efficiency, we decided to incorporate DMG-PEG2000 into our SORT LNP-based AirBuddy due to its superior properties. DMG-PEG2000 offers better biodegradability and enhanced stability in pulmonary applications. Unlike mPEG-2000-DSPE, which tends to accumulate in the body and may lead to immune activation over time, DMG-PEG2000 is known for its faster clearance and reduced potential for long-term toxicity. For lung-specific delivery, where stability and safety are critical, DMG-PEG2000 ensures that the nanoparticles remain stable long enough to deliver the therapeutic material effectively, but also degrade at a rate that minimizes unwanted immune responses. This makes DMG-PEG2000 a more suitable choice for therapies targeting CFTR-related diseases, where precise and safe delivery to the lungs is essential for treatment success.
<p>Details about the biosafety aspects of our LNP can be read <a onClick={() => goToPageAndScroll ('sort-lnp-and-cytotoxicity', '/safety')}> here </a>. </p>
<H4 text="Conclusion" id="text" />
We use DMG-PEG2000 in our SORT LNP-based AirBuddy because of its superior biodegradability, enhanced stability, and reduced risk of immune system activation. By building on insights from experts and incorporating principles from Wang’s LNP work, we’ve tailored our nanoparticles for lung-specific delivery. This choice ensures that our formulations remain stable long enough to deliver the therapeutic payload effectively while minimizing potential long-term toxicity. This balance is crucial for pulmonary applications, where DMG-PEG2000 outperforms alternatives like mPEG-2000-DSPE, making it the ideal choice for treating CFTR-related lung diseases.
</p>
</Collapsible>
<p>DMG-PEG2000 of the SORT LNP offers better biodegradability and enhanced stability in pulmonary applications - it is known for its faster clearance and reduced potential for long-term toxicity. To ensure we addressed this issue, cytotoxicity tests were performed in addition to the determination of physicochemical properties in cooperation with the <a onClick={() => goToPagesAndOpenTab('biophysik', '/human-practices')}>Physical and Biophysical Chemistry working group of Bielefeld University</a> to characterize the LNPs. More details about the composition of the SORT LNPs and function of the components can be read below.</p>
<OneFigure
pic1="https://static.igem.wiki/teams/5247/delivery/airbuddy-svg.svg"
alt1="AirBuddy"
num={3}
description="Schematic view of our lung-specific SORT LNP called AirBuddy carrying chitosan-protected pegRNA and mRNA for the assembly of our prime editing technology named PrimeGuide."
/>
<Collapsible id="Col2" open={false} title="Composition of our SORT LNP called Airbuddy // LNP Handbook Cooperation">
<H4 text="Components of AirBuddy" id="text" />
<H5 text="Ionizable Lipid" id="text" />
<p>The primary ingredient, 4A3-SC8 or MC3, are ionizable cationic lipids that forms the core of the LNP. Ionizable cationic lipids become positively charged in acidic environments, such as when a pH change occurs for example in acidic buffers or in the endosome. This allows them to bind to negatively charged nucleic acids and form protective capsules around it. In the endosome these lipids facilitate endosomal escape through electrostatic interactions between the LNPs and the endosomal or cellular membranes.</p>
<H5 text="Helper Lipids" id="text" />
<p>DOTAP (Dioleoyltrimethyl-ammonium propane) is a cationic lipid that makes up 50 % of the total molar lipid ratio. It plays a crucial role in binding to the negatively charged surface of lung epithelial cells. This enhances transfection efficiency and helps make the LNP formulation more lung-specific, improving targeted delivery. The neutral helper lipid DOPE (Dioleoylphosphatidylethanolamine) enhances endosomal escape by fusing with the endosomal membrane and improves transfection efficiency.</p>
<H5 text="Sterol" id="text" />
<p>Cholesterol, is an important cationic lipid, providing structural stability, fluidity and permeability to the LNPs, thereby improving their overall transfection efficiency. </p>
<H5 text="PEGylated Lipids" id="text" />
<p>DMG-PEG (Dimyristoylglycerin-polyethyleneglycol) is an important component by improving the LNP stability and preventing aggregation of the LNPs. </p>
<H4 text="Production Methods" id="text" />
<H5 text="LNP Assembly" id="text" />
<p>Our LNP can be formulated using various methods depending on the scale of production, including pipette mixing, vortex mixing, or microfluidic mixing. For cargo protection the mRNA can be mixed with chitosan for stability improvement before adding with the LNP components. After mixing the lipids with mRNA in carefully controlled ratios, the mixture is typically dialyzed to remove organic solvents like ethanol and citrate buffer. The choice of lipid composition and preparation method influences the tissue-targeting capabilities of the LNPs, allowing for selective delivery to organs like the liver, lungs, or spleen. For more detailed information on formulation methods and lipid selection, refer to our LNP Handbook deigned in <a onClick={() => goToPageAndScroll ('handbook', '/human-practices')}> cooperation with iGEM Team Linköping </a> and others.</p>
<p>Click this Button to gain the LNP Handbook</p>
<DownloadLink url="https://static.igem.wiki/teams/5387/liposomes-handbook.pdf" fileName="liposomes-handbook.pdf" />
<H5 text="Spray drying Procedure" id="text" />
<p>By combining these components with the spray drying method from <a onClick={() => goToPageAndScroll ('rnhale', '/human-practices')}> RNhale </a> we offer a versatile and efficient method for delivering RNA therapeutics to the lung, paving the way for gene therapy, especially our Prime Guide. The effective delivery of the prime editing complex is a crucial point in our project. </p>
<H5 text="Storage" id="text" />
<p>The final LNP solution can be stored at 4 °C for a few days. It is recommended to use the formulated LNPs as soon as possible to maintain consistent results. Storage at RT is not recommended. Storage at freezing temperatures is also not recommended unless optimized cryoprotectants are used.</p>
</Collapsible>
<p>The final innovation for our LNP to become <strong>AirBuddy</strong> came through consultation with Benjamin Winkeljann from <a onClick={() => goToPagesAndOpenTab('rnhale', '/human-practices')}> RNhale</a>, when the use of spray-drying techniques was discussed. Spray-drying the LNPs, instead of using traditional methods, helps improve stability and eco-friendliness of the product<TabScrollLink tab="tab-delivery" num="38" scrollId="desc-38"/>. Our samples are set for transfer to RNhale for spray-drying, with scheduling aligned to resume promptly after the wiki freeze. Meanwhile, the discussion with <a onClick={() => goToPagesAndOpenTab('moorlach', '/human-practices')}>Benjamin Moorlach</a>, chitosan expert working at FH Bielefeld, provided new ideas for improvement by <strong>complexing the cargo with chitosan</strong> to improve the stability of the cargo during spray drying and nebulization. The positive effect of chitosan-complexing for opimized LNP delivery could be confirmed in our lab. In conclusion, we created a stable LNP for efficient delivery of RNA therapeutics to the lungs since the successful delivery of the prime editing complex via inhalation is key to our project. </p>
<OneFigure
pic1="https://static.igem.wiki/teams/5247/delivery/big-plan-inhalation-teil-del.webp"
alt1="Flow DEL"
description="Application strategy - AirBuddy is inhaled by the patient, enabling uptake of PrimeGuide RNA in lung epithelial cells via endocytosis. "
num={4}
/>
</p>
</div>
<div className="box" >
<p id="del5">
<H3 text="Outlook" id="del5head" />
Ultimately, through continuous cycles of experimentation, feedback, and optimization, our LNP formulation called AirBuddy was designed using SORT LNPs incorporating chitosan-complexation of the cargo and processing via spray-drying, achieving lung-specificity and improved stability suited for inhalation strategies. We also want to state that for our LNP is further room for improvement. Intensive research led us to the realization that, among other modifications, <strong>antibody conjugation</strong> as a surface modification of our LNP for cell type-specific administration, more specifically club cells<TabScrollLink tab="tab-delivery" num="39" scrollId="desc-39"/> and ionocytes<TabScrollLink tab="tab-delivery" num="40" scrollId="desc-40"/> as most CFTR-expressing lung epithelial cells, would round off our most important aspect of precision.
</p>
</div>
<Section title="References" id="references">
<EngDelsources/>
</Section>
<br/>
<div className="row ">
<div className="row">
<div className="col">
<div className="left"><ButtonOneEngineering label="Previous" open="delivery"/></div>
<div className="left"><ButtonOneEngineering label="Previous" open="nickase" scrollToId="nickase-header"/></div>
</div>
<div className="col button-left">
</div>
</div>
</section>
......@@ -203,7 +1171,7 @@ export function EngineeringCycleTab(){
<g
id="g25"
transform="translate(7.500978,3.0504898)">
<a typeof="button" className="svg-button" onClick={openIt({it: "designing"})}>
<a typeof="button" className="svg-button" onClick={openElement({elementToOpen: "designing", classToHide: "cycletab"})}>
<path
className="cls-7"
d="m 42.05,115.59 c 27.77,9.19 57.83,-4.79 69.76,-32.23"
......@@ -221,7 +1189,7 @@ export function EngineeringCycleTab(){
id="polygon25"
style={{fill:"#850f78",fillOpacity:"1",strokeWidth:"6",strokeDasharray:"none"}}
transform="matrix(2.9806259,0,0,2.9806259,-225.43722,-156.45123)" />
<a typeof="button" className="svg-button" onClick={openIt({it: "learning"})}>
<a typeof="button" className="svg-button" onClick={openElement({elementToOpen: "learning", classToHide: "cycletab"})}>
<path
className="cls-12"
d="M 114,78.55 C 123.45,50.86 110,20.75 82.66,8.6"
......@@ -229,7 +1197,7 @@ export function EngineeringCycleTab(){
style={{strokeWidth:"15",strokeDasharray:"none",stroke:"#850f78",strokeOpacity:"1"}} />
<text
style={{fontSize:"17.3333px",lineHeight:"0",fontFamily:"Arial",fill:"#000000",fillOpacity:"1",stroke:"none",strokeWidth:"15",strokeLinecap:"round",strokeLinejoin:"round",strokeDasharray:"none",strokeOpacity:"1",paintOrder:"fill markers stroke"}}
style={{fontSize:"17.3333px",lineHeight:"0",fontFamily:"Arial",fill:"var(--offblack)",fillOpacity:"1",stroke:"none",strokeWidth:"15",strokeLinecap:"round",strokeLinejoin:"round",strokeDasharray:"none",strokeOpacity:"1",paintOrder:"fill markers stroke"}}
id="text31"
transform="translate(5.6902194,-0.11551883)"><textPath
xlinkHref="#path25"
......@@ -248,7 +1216,7 @@ export function EngineeringCycleTab(){
style={{fill:"#a0a7f3",fillOpacity:"1",strokeWidth:"6",strokeDasharray:"none"}}
inkscape:transform-center-x="3.6173751"
inkscape:transform-center-y="5.1978852" />
<a typeof="button" className="svg-button" onClick={openIt({it: "testing"})}>
<a typeof="button" className="svg-button" onClick={openElement({elementToOpen: "testing", classToHide: "cycletab"})}>
<path
className="cls-9"
d="M 78.599111,7.5468264 C 43.820346,-2.6177588 13.956746,14.286046 4.2106281,46.368749"
......@@ -256,7 +1224,7 @@ export function EngineeringCycleTab(){
style={{stroke:"#a0a7f3",strokeWidth:"15",strokeDasharray:"none",strokeOpacity:"1"}} />
<text
style={{fontSize:"17.3333px",lineHeight:"0",fontFamily:"Arial",fill:"#000000",fillOpacity:"1",stroke:"none",strokeWidth:"6",strokeLinecap:"round",strokeLinejoin:"round",strokeDasharray:"none",strokeOpacity:"1",paintOrder:"fill markers stroke"}}
style={{fontSize:"17.3333px",lineHeight:"0",fontFamily:"Arial",fill:"var(--offblack)",fillOpacity:"1",stroke:"none",strokeWidth:"6",strokeLinecap:"round",strokeLinejoin:"round",strokeDasharray:"none",strokeOpacity:"1",paintOrder:"fill markers stroke"}}
id="text28"
transform="translate(-0.03023506,-5.9602336)"><textPath
......@@ -273,7 +1241,7 @@ export function EngineeringCycleTab(){
id="polygon25-6"
style={{fill:"#f57d22",fillOpacity:"1",strokeWidth:"6",strokeDasharray:"none"}}
transform="matrix(-2.9650314,0.30449893,-0.30449893,-2.9650314,364.84067,249.28249)" />
<a typeof="button" className="svg-button" onClick={openIt({it: "building"})}>
<a typeof="button" className="svg-button" onClick={openElement({elementToOpen: "building", classToHide: "cycletab"})}>
<path
className="cls-11"
d="M 2.6659753,50.953505 C -2.0956694,72.727915 10.936866,102.94273 36.656234,113.62834"
......@@ -281,7 +1249,7 @@ export function EngineeringCycleTab(){
style={{strokeWidth:"15",strokeDasharray:"none",stroke:"#f57d22",strokeOpacity:"1"}} />
<text
style={{fontSize:"17.3333px",lineHeight:"0",fontFamily:"Arial",fill:"#000000",fillOpacity:"1",stroke:"none",strokeWidth:"15",strokeLinecap:"round",strokeLinejoin:"round",strokeDasharray:"none",strokeOpacity:"1",paintOrder:"fill markers stroke"}}
style={{fontSize:"17.3333px",lineHeight:"0",fontFamily:"Arial",fill:"var(--offblack)",fillOpacity:"1",stroke:"none",strokeWidth:"15",strokeLinecap:"round",strokeLinejoin:"round",strokeDasharray:"none",strokeOpacity:"1",paintOrder:"fill markers stroke"}}
id="text32"
transform="translate(-5.7110315,1.7453243)"><textPath
xlinkHref="#path24"
......@@ -294,10 +1262,10 @@ export function EngineeringCycleTab(){
id="polygon22"
style={{strokeWidth:"6",strokeDasharray:"none",fill:"#f4cc1e",fillOpacity:"1"}}
transform="matrix(2.8248588,0,0,2.8248588,-67.797781,-207.96977)" />
<a typeof="button" className="svg-button" onClick={openIt({it: "designing"})}>
<a typeof="button" className="svg-button" onClick={openElement({elementToOpen: "designing", classToHide: "cycletab"})}>
<text
style={{fontSize:"17.3333px",lineHeight:"0",fontFamily:"Arial",fill:"#000000",fillOpacity:"1",stroke:"none",strokeWidth:"15",strokeLinecap:"round",strokeLinejoin:"round",strokeDasharray:"none",strokeOpacity:"1",paintOrder:"fill markers stroke"}}
style={{fontSize:"17.3333px",lineHeight:"0",fontFamily:"Arial",fill:"var(--offblack)",fillOpacity:"1",stroke:"none",strokeWidth:"15",strokeLinecap:"round",strokeLinejoin:"round",strokeDasharray:"none",strokeOpacity:"1",paintOrder:"fill markers stroke"}}
id="text29"
transform="translate(8.4052921,8.8553334)"><textPath
xlinkHref="#path22"
......@@ -305,7 +1273,7 @@ export function EngineeringCycleTab(){
id="tspan29" /></textPath></text> </a>
</g>
</g>
<a typeof="button" className="svg-button" onClick={openIt({it: "overview"})}>
<a typeof="button" className="svg-button" onClick={openElement({elementToOpen: "overview", classToHide: "cycletab"})}>
<circle
style={{opacity:"0.85",fill:"#e2dad7",fillOpacity:"1",strokeWidth:"15",strokeLinecap:"round",strokeLinejoin:"round",paintOrder:"fill markers stroke"}}
id="path1"
......@@ -313,7 +1281,7 @@ export function EngineeringCycleTab(){
cy="63.214005"
r="20" />
<text
style={{fontSize:"8px",lineHeight:"0",fontFamily:"Arial",opacity:"0.85",fill:"#000000",fillOpacity:"1",strokeWidth:"15",strokeLinecap:"round",strokeLinejoin:"round",strokeDasharray:"none",paintOrder:"fill markers stroke"}}
style={{fontSize:"8px",lineHeight:"0",fontFamily:"Arial",opacity:"0.85",fill:"var(--offblack)",fillOpacity:"1",strokeWidth:"15",strokeLinecap:"round",strokeLinejoin:"round",strokeDasharray:"none",paintOrder:"fill markers stroke"}}
x="50.929825"
y="66.676674"
id="text1">
......
src/contents/ethics.tsx deleted 100644 → 0
View file @ 9143d0b0
import { useEffect } from "react";
import { useLocation } from "react-router-dom";
import { openFromOtherPage } from "../components/Buttons";
export function Ethics() {
const location = useLocation();
useEffect(() => {
const params = new URLSearchParams(location.search);
const collapseId = params.get('collapseId');
const tabId = params.get('tab');
// Scroll to the section specified by collapseId
if (collapseId) {
const collapseElement = document.getElementById(collapseId);
if (collapseElement) {
const elementTop = collapseElement.getBoundingClientRect().top + window.pageYOffset;
const offset = window.innerHeight / 2 - collapseElement.offsetHeight / 2;
const scrollPosition = elementTop - offset;
window.scrollTo({
top: scrollPosition,
behavior: 'smooth',
});
}
}
// Open the tab specified by tabId
if (tabId) {
openFromOtherPage(tabId)({ currentTarget: document.getElementById(tabId)! });
}
}, [location.search]);
return (
<>
<div className="row mt-4">
<div className="col-lg-8">
</div>
</div>
</>
);
}
\ No newline at end of file
src/contents/example.css
View file @ 3fd292fd
:root {
/* our colours*/
--text-primary: #850F78;
--mediumpurple: #bc15aa;
/*--purple: #B85BD1; */
--accen-secondary: #F57D22;
--accent-primary: #F4CC1E;
--lightyellow: #fae99e;
--lightblue: #A0A7F3 ;
--verylightblue: #ebecfd;
--offblack: #32232C ;
--cebitecgray: #8295A4;
/*--offwhite: #e9dff1; */
--ourbeige: #FFF6F2;
--darkerbeige: #e2dad7;
--background: #FFF6F2;
/*igem colours*/
--igemdarkgreen: #006530;
--igemmediumgreen: #019968;
--igemlightgreen: #99cb9a;
--info-border-color: var(--mediumpurple);
--vp-ct: var(--text-primary);
--info-border-color: var(--accent-primary);
--info-bg-color: var(--lightyellow);
--info-title-color: var(--text-primary);
--info-code-bg-color: var(--lightyellow);
--note-border-color: var(--text-primary);
--note-bg-color: var(--darkoffwhite);
--note-title-color: var(--text-primary);
--note-code-bg-color: var(--darkoffwhite);
--tip-border-color: var(--text-primary);
--tip-bg-color: var(--darkoffwhite);
--tip-title-color: var(--text-primary);
--tip-code-bg-color: var(--darkoffwhite);
--warning-border-color: var(--accen-secondary);
--warning-bg-color: var(--lightorange);
--warning-title-color: var(--text-primary);
--warning-code-bg-color: var(--lightorange);
}
.example-docu{
background-color: var(--igemlightgreen);
width: fit-content;
......@@ -60,10 +19,10 @@
}
.st196{fill:none;stroke:black;stroke-miterlimit:10;}
.st196{fill:none;stroke:var(--offblack);stroke-miterlimit:10;}
.st455{font-size:auto;}
/* .d-none{
/* .{
background-color: red;
} */
......
src/contents/example.tsx
View file @ 3fd292fd
import TestSVG from "../components/testsvganimation";
import { BlueInfoBox, BulbBox, InfoBox, NoteBox, QaBox, WarnBox } from "../components/Boxes";
import { BFHMoreButton, ButtonOne } from "../components/Buttons";
import Collapsible from "../components/Collapsible";
import PieChart, { HowOftenTreatmentatients, MoreInfoOnTherapyBoth, OpenToGeneTherapyatients } from "../components/Graph";
import H1, { H2, H3, Hhighlight, Hhopp, Hsmoke, Hspoiler, Hwave } from "../components/Headings";
import { LoremMedium, LoremShort } from "../components/Loremipsum";
import SimpleSlider from "../components/Slider";
import React from 'react';
import { Bar, Doughnut, PolarArea } from 'react-chartjs-2';
import { Chart as ChartJS, Tooltip, Legend, BarElement, CategoryScale, LinearScale, Title, RadialLinearScale } from 'chart.js';
/* import ProteinViewer from '../components/Fanzorviewer.tsx'; */
import { useTabNavigation } from "../utils/TabNavigation";
ChartJS.register(
CategoryScale,
RadialLinearScale,
LinearScale,
BarElement,
Title,
Tooltip,
Legend
);
export function Example() {
useTabNavigation();
return (
<>
{/* <div className="container">
<h1>Protein Structure Viewer</h1>
<ProteinViewer/>
</div> */}
<h1> Here you can see what we can use</h1>
<h2>Collapisbles</h2>
<div className="row">
<div className="col">
<details>
<summary>Expand me</summary>
<LoremMedium></LoremMedium>
</details>
</div>
<div className="col">
<Collapsible title="Title" id="collapsible"> <LoremMedium></LoremMedium></Collapsible>
</div>
<HowOftenTreatmentatients/>
<OpenToGeneTherapyatients/>
<MoreInfoOnTherapyBoth/>
</div>
<h2>Boxes</h2>
<div className="row">
<div className="col">
<InfoBox title="InfoBox" id="boxid"><LoremShort></LoremShort></InfoBox>
<BlueInfoBox title="BlueInfoBox"><LoremShort></LoremShort></BlueInfoBox>
<NoteBox title="NoteBox" id="notebox"><LoremShort></LoremShort></NoteBox>
</div>
<div className="col">
<WarnBox title="WarnBox"><LoremShort></LoremShort></WarnBox>
<BulbBox title="BulbBox"><LoremShort></LoremShort></BulbBox>
<QaBox
q="A question or sentence."
a="An answer or sentence"/>
</div>
</div>
<h2>Headings</h2>
<div className="row">
<div className="col">
<H1 text="H1" id="text" />
<H2 text="H2" id="text" />
<H3 text="H3" id="text" />
</div>
<div className="col">
<Hsmoke text="Hsmoke" id="text" />
<Hhopp text="Hhopp" id="text" />
<Hhighlight> Highlight</Hhighlight>
<Hspoiler> Hspoiler </Hspoiler>
<Hwave text="Hwave" id="text" />
<a href="#" className="underline--magical">PreCyse</a>
</div>
</div>
<h2>Buttons</h2>
<div className="row">
<div className="col">
<TestSVG></TestSVG>
{/* <h3 className="example">Exercises</h3>
<div className="col">
<ButtonOne text="The Public" open="pubs" openclass="subcycletab"></ButtonOne>
</div>
</div>
<div className="col">
<BFHMoreButton it="id of part to be opened" />
</div>
<div className="col">
<button className="tablinks Patient hp-more-button" > Something </button>
</div>
</div>
<h2>Graphs</h2>
<div className="row">
<div className="col">
<PieChart></PieChart>
<DoughnutChart></DoughnutChart>
</div>
<div className="col">
<BarChartTwoSets></BarChartTwoSets>
<BarChart></BarChart>
</div>
<div className="col">
<BarChartVertical></BarChartVertical>
<PolarChart></PolarChart>
</div>
</div>
<h2>Boxes</h2>
<div className="row">
<div className="col">
</div>
<div className="col">
</div>
</div>
<div className="col">
<h3 className="example">Exercises</h3>
<i><h6>By Your name</h6></i>
<div className="example-exercise">
<p> All components should be in this file. The extra css has to be put into exapmle.css. Turn to Liliana if you need or want a scss file, too.</p>
......@@ -42,28 +158,6 @@ export function Example() {
</div>
</div>
<hr/>
<div className="col exercise">
<div className="row align-items-center">
<div className="col"><h4>Timeline BFH</h4></div>
<div className="col-1 "><div className="example-easy-tag">Easy</div></div>
</div>
<i><h6>By </h6></i>
<div className="example-exercise">
<p> Add a dummy timeline item</p>
</div>
<div className="timeline-container">
<TimelineItem
date='How to SynBio'
tag='Workshop Session I.'
color='var(--text-primary)'
csstag="Workshop"
>
Design genetic constructs and re-write the genomic code, and plan experiments using AI. Learn how to effectively build genetic circuit systems for implementation in your iGEM project.
</TimelineItem>
</div>
</div>
<hr/>
<div className="col">
<div className="row align-items-center">
<div className="col"><h4>Picture Slider</h4></div>
......@@ -74,16 +168,16 @@ export function Example() {
<p> Add a dummy sponsor to this slider.</p>
<SimpleSlider>
<a className="sponsor-container" href="https://bts-ev.de/">
<img className="img-sponsor" src="https://static.igem.wiki/teams/5247/sponsors/bts.png"/>
<img className="img-sponsor side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/bts.png"/>
</a>
<a className="sponsor-container" href="https://www.uni-bielefeld.de/fakultaeten/technische-fakultaet/arbeitsgruppen/multiscale-bioengineering/campusbrauerei/">
<img className="img-sponsor" src="https://static.igem.wiki/teams/5247/sponsors/campus-brauerei-hinterlegt.jpeg"/>
<img className="img-sponsor side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/campus-brauerei-hinterlegt.jpeg"/>
</a>
<a className="sponsor-container" href="www.idtdna.com">
<img className="img-sponsor" src="https://static.igem.wiki/teams/5247/sponsors/idt-logo.png"></img>
<img className="img-sponsor side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/idt-logo.png"></img>
</a>
<a className="sponsor-container" href="https://www.cebitec.uni-bielefeld.de/">
<img className="img-sponsor" src="https://static.igem.wiki/teams/5247/sponsors/cebitec-farbe.png"/>
<img className="img-sponsor side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/cebitec-farbe.png"/>
</a>
</SimpleSlider>
</div>
......@@ -168,8 +262,8 @@ export function Example() {
<p> See DocuBox component. </p>
</div>
</div>
<hr/> */}
{/* <div className="col exercise">
<hr/>
<div className="col exercise">
<div className="row align-items-center">
<div className="col"><h4> </h4></div>
<div className="col-1 "><div className="example-easy-tag"></div></div>
......@@ -179,11 +273,11 @@ export function Example() {
<p> </p>
</div>
</div>
<hr/> */}
<hr/>
</div>
</>
);
}
......@@ -201,4 +295,217 @@ export function Example() {
</>
)
}
\ No newline at end of file
const BarChartTwoSets: React.FC = () => {
const labels = ['January', 'February', 'March', 'April', 'May', 'June', 'July'];
const data = {
labels,
datasets: [
{
label: 'Dataset 1',
data: [
0.004858000000000001,
0.0008859999999999997,
0.7264179999999972,
0.2384159999999995,
0.003703,
0.2384159999999995,
0.003703
],
backgroundColor: 'rgba(255, 99, 132, 0.5)',
},
{
label: 'Dataset 2',
data: [
0.004858000000000001,
0.0008859999999999997,
0.7264179999999972,
0.2384159999999995,
0.003703,
0.2384159999999995,
0.003703
],
backgroundColor: 'rgba(53, 162, 235, 0.5)',
},
],
};
const options = {
responsive: true,
plugins: {
legend: {
position: 'top' as const,
},
title: {
display: true,
text: 'BarChartTwoSets',
},
},
};
return (
<div className="bar-chart-container">
<Bar options={options} data={data} />
</div>
);
};
const BarChart: React.FC = () => {
const labels = ['January', 'February', 'March', 'April', 'May', 'June', 'July'];
const data = {
labels,
datasets: [
{
label: 'Dataset 1',
data: [
0.004858000000000001,
0.0008859999999999997,
0.7264179999999972,
0.2384159999999995,
0.003703,
0.2384159999999995,
0.003703
],
backgroundColor: 'rgba(255, 99, 132, 0.5)',
},
],
};
const options = {
responsive: true,
plugins: {
legend: {
position: 'top' as const,
},
title: {
display: true,
text: 'BarChartOneSet',
},
},
};
return (
<div className="bar-chart-container">
<Bar options={options} data={data} />
</div>
);
};
const BarChartVertical: React.FC = () => {
const labels = ['January', 'February', 'March', 'April', 'May', 'June', 'July'];
const data = {
labels,
datasets: [{
axis: 'y',
label: 'My First Dataset',
data: [65, 59, 80, 81, 56, 55, 40],
fill: false,
backgroundColor: [
'rgba(255, 99, 132, 0.2)',
'rgba(255, 159, 64, 0.2)',
'rgba(255, 205, 86, 0.2)',
'rgba(75, 192, 192, 0.2)',
'rgba(54, 162, 235, 0.2)',
'rgba(153, 102, 255, 0.2)',
'rgba(201, 203, 207, 0.2)'
],
borderColor: [
'rgb(255, 99, 132)',
'rgb(255, 159, 64)',
'rgb(255, 205, 86)',
'rgb(75, 192, 192)',
'rgb(54, 162, 235)',
'rgb(153, 102, 255)',
'rgb(201, 203, 207)'
],
borderWidth: 1
}
],
};
const options = {
indexAxis: 'y' as const,
responsive: true,
plugins: {
legend: {
position: 'top' as const,
},
title: {
display: true,
text: 'Vertical Bar Chart',
},
},
};
return (
<div className="bar-chart-container">
<Bar options={options} data={data} />
</div>
);
};
const DoughnutChart: React.FC = () => {
const data = {
labels: ['Red', 'Blue', 'Yellow', 'Green', 'Purple', 'Orange'],
datasets: [
{
label: '# of Votes',
data: [12, 19, 3, 5, 2, 3],
backgroundColor: [
'rgba(255, 99, 132, 0.2)',
'rgba(54, 162, 235, 0.2)',
'rgba(255, 206, 86, 0.2)',
'rgba(75, 192, 192, 0.2)',
'rgba(153, 102, 255, 0.2)',
'rgba(255, 159, 64, 0.2)',
],
borderColor: [
'rgba(255, 99, 132, 1)',
'rgba(54, 162, 235, 1)',
'rgba(255, 206, 86, 1)',
'rgba(75, 192, 192, 1)',
'rgba(153, 102, 255, 1)',
'rgba(255, 159, 64, 1)',
],
borderWidth: 1,
},
],
};
return (
<div className="bar-chart-container">
<Doughnut data={data} />
</div>
);
};
const PolarChart: React.FC = () => {
const data = {
labels: ['Red', 'Blue', 'Yellow', 'Green', 'Purple', 'Orange'],
datasets: [
{
label: '# of Votes',
data: [12, 19, 3, 5, 2, 3],
backgroundColor: [
'rgba(255, 99, 132, 0.5)',
'rgba(54, 162, 235, 0.5)',
'rgba(255, 206, 86, 0.5)',
'rgba(75, 192, 192, 0.5)',
'rgba(153, 102, 255, 0.5)',
'rgba(255, 159, 64, 0.5)',
],
borderWidth: 1,
},
],
};
return (
<div className="bar-chart-container">
<PolarArea data={data} />;
</div>
);
};
\ No newline at end of file
src/contents/experiments.tsx deleted 100644 → 0
View file @ 9143d0b0
import { useEffect } from "react";
import { useLocation } from "react-router-dom";
import { openFromOtherPage } from "../components/Buttons";
export function Experiments() {
const location = useLocation();
useEffect(() => {
const params = new URLSearchParams(location.search);
const collapseId = params.get('collapseId');
const tabId = params.get('tab');
// Scroll to the section specified by collapseId
if (collapseId) {
const collapseElement = document.getElementById(collapseId);
if (collapseElement) {
const elementTop = collapseElement.getBoundingClientRect().top + window.pageYOffset;
const offset = window.innerHeight / 2 - collapseElement.offsetHeight / 2;
const scrollPosition = elementTop - offset;
window.scrollTo({
top: scrollPosition,
behavior: 'smooth',
});
}
}
// Open the tab specified by tabId
if (tabId) {
openFromOtherPage(tabId)({ currentTarget: document.getElementById(tabId)! });
}
}, [location.search]);
return (
<>
<div className="row mt-4">
<div className="col-lg-8">
</div>
</div>
</>
);
}
src/contents/human-practices.tsx deleted 100644 → 0
View file @ 9143d0b0
/*
- seitwärts
- mit Farben und Text anzeigen wozu die gehören
- bei show more unten drunter Tabs öffnen
- wenn man auf die Person klickt soll es Infos über die Person anzeigen
- kann mn die Karten nach Links und nach rechts ausweiten zb für Mehr infos für die Person?
- Filter mit HalbkreisDing als Tabsteuerung | Einteilung nach Bereich (Academia, Insustry, ..., und auch nach Delivery und Mechanism)
- DNA Strang als Timeline?
*/
/* <br/>
<h3>Name</h3>
<hr/>
<div className="row">
<div className="col-2">
<div className="t-tag Tag">
Beruf
</div>
</div>
<div className="col">Original language: German</div>
</div>
<div className="row">
<div className="col">
<BlockQuoteB text="Quote" cite="."></BlockQuoteB>
</div>
<div className="col-3">
<img className="middle sechpro" src="https://static.igem.wiki/teams/5247/placeholders/placehilderperson.jpeg"/>
</div>
</div>
<h4>Aim of contact</h4>
<p></p>
<h4>Insights</h4>
<p></p>
<h4>Clarification</h4>
<p></p>
<h4>Implementation</h4>
<p></p> */
/*
<h4>Aim of contact</h4>
<p></p>
<h4>Insights</h4>
<p></p>
<h4>Clarification</h4>
<p></p>
<h4>Implementation</h4>
<p></p>
*/
import { TimeHori } from "../components/HorizontalTimeline";
import { BFHStyleTabs, ButtonRowTabs } from "../components/Tabs";
import { ButtonOne, TabButtonRow, openFromOtherPage, openTab } from "../components/Buttons";
import { BlockQuoteB } from "../components/Quotes";
import { Box, Tab } from "@mui/material";
import {TabContext, TabList, TabPanel} from '@mui/lab';
import React, { useEffect } from "react";
import { useLocation } from "react-router-dom";
let timelinebuttonrowdata = [
{
buttonname: "All",
node: <TimeHori tab="" ></TimeHori>,
cssname: "First"
},
{
node: <TimeHori tab="Patient" ></TimeHori>,
buttonname: "Patients",
cssname: "Patient"
},
{
node: <TimeHori tab="Medical Professional" ></TimeHori>,
buttonname: "Medical Professionals",
cssname: "Medical"
},
{
node: <TimeHori tab="Academia" ></TimeHori>,
buttonname: "Academia",
cssname: "Academia"
},
{
node: <TimeHori tab="Industry" ></TimeHori>,
buttonname: "Industry",
cssname: "Industry"
}
]
let timelinepersontabs =[
{
node: <>
<br/>
<h3>Max Beckmann</h3>
<h5>First official interview</h5>
<hr/>
<div className="row">
<div className="col-2">
<div className="t-tag Patient">
Patient
</div>
</div>
<div className="col-3">Original language: German</div>
<div className="col"><button>Go to interview</button></div>
</div>
<div className="row">
<div className="col">
<BlockQuoteB text="Quote" cite="."></BlockQuoteB>
</div>
<div className="col-3">
<img className="middle sechpro" src="https://static.igem.wiki/teams/5247/placeholders/placehilderperson.jpeg"/>
</div>
</div>
<h4>Aim of contact</h4>
<p></p>
<h4>Insights</h4>
<p></p>
<h4>Clarification</h4>
<p></p>
<h4>Implementation</h4>
<p></p>
</>,
cssname: "InvMax1"
},
{
node: <>
<br/>
<h3>Max Beckmann</h3>
<h5>Feedback Interview</h5>
<hr/>
<div className="row">
<div className="col-2">
<div className="t-tag Patient">
Patient
</div>
</div>
<div className="col-3">Original language: German</div>
<div className="col"><button>Go to interview</button></div>
</div>
<div className="row">
<div className="col">
<BlockQuoteB text="Quote" cite="."></BlockQuoteB>
</div>
<div className="col-3">
<img className="middle sechpro" src="https://static.igem.wiki/teams/5247/placeholders/placehilderperson.jpeg"/>
</div>
</div>
<h4>Aim of contact</h4>
<p></p>
<h4>Insights</h4>
<p></p>
<h4>Clarification</h4>
<p></p>
<h4>Implementation</h4>
<p></p>
</>,
cssname: "InvMax2"
},
{
node: <>
<br/>
<h3>Dr. Eva-Maria Berens</h3>
<hr/>
<div className="row">
<div className="col-2">
<div className="t-tag Academia">
Ethics Committee
</div>
</div>
<div className="col-3">Original language: German</div>
<div className="col"><button>Go to interview</button></div>
</div>
<div className="row">
<div className="col">
<BlockQuoteB text="Quote" cite="Dr. Eva-Maria Berens"></BlockQuoteB>
</div>
<div className="col-3">
<img className="middle sechpro" src="https://static.igem.wiki/teams/5247/placeholders/placehilderperson.jpeg"/>
</div>
</div>
<h4>Aim of contact</h4>
<p>The aim of the interview was to get an answer to the question of whether we need an ethics vote for our project or not and to obtain guidelines for dealing with patient cells regarding ethical issues and data protection. </p>
<h4>Insights</h4>
<p>The discussion was very informative in terms of how we should approach this topic and focused primarily on the important factors that need to be considered when planning the handling of patient cells. These include which legal principles need to be observed, data protection, ethical considerations and, above all, detailed and specific information for the donor. It also made us look at the situation from many different angles and consider the risks of worst-case scenarios. Overall, this interview was very useful to us, and we were able to use the information we gained to develop a kind of guideline that allowed us to approach this sensitive topic, which was new to us, with a certain degree of confidence. </p>
{/* <h4>Clarification</h4>
<p></p> */}
<h4>Implementation</h4>
<p>Based on the knowledge we have gained, we have drawn up guidelines for our handling of the cells. We used this guide when handling the patient cells, to ensure they were handled in an ethically correct manner. </p>
</>,
cssname: "Berens"
},
{
node: <>
<br/>
<h3>Prof. Dr. Erhard Wischmeyer und Dr. Oliver Dräger </h3>
<h4>Research Group Cellular Neurophysiology, Bielefeld University</h4>
<hr/>
<div className="row">
<div className="col-2">
<div className="t-tag Academia">
Research Group
</div>
</div>
<div className="col-3">Original language: German</div>
<div className="col"><button>Got to Interview with Prof. Wischmeyer</button></div>
</div>
<div className="row">
<div className="col">
<BlockQuoteB text="Quote" cite="."></BlockQuoteB>
</div>
<div className="col-3">
<img className="middle sechpro" src="https://static.igem.wiki/teams/5247/placeholders/placehilderperson.jpeg"/>
</div>
</div>
<img className="img-right img-half" src="https://static.igem.wiki/teams/5247/photos/for-wiki-texts/hp-patch-clamp/20240625-184032.jpg"/>
<div>
<h4>Aim of contact</h4>
<p>As part of our project, we aimed to demonstrate the functionality of the CFTR ion channel, after restoring
it through our optimized Prime Editing, by using Patch-Clamp measurements. To ensure the optimal use of the
Patch-Clamp and to gain an insight into electrophysiology, we asked experts from the medical faculty at
Bielefeld University to critically examine our measurement planning. Prof. Dr. Erhard Wischmeyer, an
experienced scientist in this field who has worked at the Max Planck Institute for Biophysical Chemistry
in Göttingen, the development site of the Patch-Clamp technique<a href="desc-1"><sup>1</sup></a>, and currently leads the Cellular
Neurophysiology working group at Bielefeld University, seemed to be an ideal interviewee. His
knowledge and experience promised valuable insights and advice for conducting and optimizing our
experiments. </p>
</div>
<br/>
<div className="row">
<div className="col2punkt5">
<img src="https://static.igem.wiki/teams/5247/photos/for-wiki-texts/hp-patch-clamp/bild-patch-clamp-isi-oliver.jpeg" />
</div>
<div className="col">
<h4>Insights</h4>
<p>Prof. Dr. Wischmeyer taught us about the workflow of the Patch-Clamp technique. He highlighted the need
for specialized electrodes and glass pipettes that must form a smooth surface devoid of the extracellular
matrix (ECM). Additionally, he pointed out that measuring CFTR conductivity with the Patch-Clamp technique
poses a technical challenge due to the low currents involved<a href="desc-2"><sup>2</sup></a>. He recommended using expression vectors
for overexpressing the CFTR gene in HEK cells instead of epithelial cells from a nasal swab to achieve
better results. Since Patch-Clamp measurements require a very sensitive testing environment, even
challenging for the most experienced scientists, Prof. Dr. Wischmeyer invited us to conduct the
measurements together with members of his group.
</p>
<p>In addition to the Patch-Clamp technique, Prof. Dr. Wischmeyer informed us about E-cis measurements as a
current electrophysiological measurement method alongside the Patch-Clamp technique. This method allows
the measurement of the membrane potential above and below a monolayer of confluent cells<a href="desc-3"><sup>3</sup></a>. Consequently,
it enables precise measurement of conductivity dependent on CFTR expression. </p>
</div>
</div>
<h4>Implementation</h4>
<div className="row align-items-center">
<div className="col">
<p>We decided to use HEK293T cells lines from Mattijs Bulcaen from KU Leuven [Link] which do overexpress the
correct CFTR and those which express CFTR with F508del for the Patch-Clamp measurements. To conduct the
Patch-Clamp experiments, we contacted the Cellular Neurophysiology group to perform the necessary
measurements. It was a pleasure to work together with Dr. Oliver Dräger, who is working as a post-doc for
the Cellular Neurophysiology working group at Bielefeld University. He taught us about the Patch-Clamp
method and spent his valuable time supporting our project by guiding our Patch-Clamp measurements. </p>
<p>In summary, through the interview with Prof. Dr. Wischmeyer and the collaboration with his employee
Oliver Dräger, we gained valuable insights and optimized our approach to effectively investigate and
measure the functionality of the CFTR ion channel, thereby determining the efficiency of our Prime
Editing strategy. </p>
</div>
<div className="col-5">
<img src="https://static.igem.wiki/teams/5247/photos/for-wiki-texts/hp-patch-clamp/bild-interssierte-wissenschaftler-oho.jpeg" style={{maxHeight: "300px"}}/>
</div>
</div>
<h4>References</h4>
{/*<!-- Citation num 1--> */}
<li typeof="schema:ScolarlyArticle" role="doc-biblioentry" property="schema:citation" id="bielefeld-cebitec/human-practices#desc-1">
<span property="schema:author" typeof="schema:Person">
<span property="schema:Name"> Roth, F.</span>;
<span property="schema:Name"> Draguhn, A.</span>
</span>
<span property="schema:name">&nbsp;Die Entwicklung der Patch-Clamp-Technik. </span>
<i property="schema:publisher" typeof="schema:Organization"> Springer eBooks</i>
<b property="issueNumber" typeof="PublicationIssue"> </b>,&nbsp;
<span property="schema:pageBegin"> 1</span>-<span property="schema:pageEnd">14</span>
(<time property="schema:datePublished" datatype="xsd:gYear" dateTime=" 2023">2023</time>).
<a className="doi" href="https://doi.org/10.1007/978-3-662-66053-9_1"> doi: 10.1007/978-3-662-66053-9_1</a>
</li>
{/*<!-- Citation num 2--> */}
<li typeof="schema:ScolarlyArticle" role="doc-biblioentry" property="schema:citation" id="desc-2">
<span property="schema:author" typeof="schema:Person">
<span property="schema:Name"> Mete, V.</span>
</span>
<span property="schema:name">&nbsp;Entwicklung und Validierung neuer nicht-invasiver Diagnosesysteme für Mucociliary Clearance Disorders (MCCD). </span>
<i property="schema:publisher" typeof="schema:Organization"> Dissertation, Westfälische Wilhelms-Universität Münster</i>
<b property="issueNumber" typeof="PublicationIssue"> </b>,&nbsp;
<span property="schema:pageBegin"> </span>
(<time property="schema:datePublished" datatype="xsd:gYear" dateTime=" 2023">2023</time>).
<a className="doi" href="https://doi.org/10.17879/98958441905"> doi: 10.17879/98958441905</a>
</li>
{/*<!-- Citation num 3--> */}
<li typeof="schema:ScolarlyArticle" role="doc-biblioentry" property="schema:citation" id="desc-3">
<span property="schema:author" typeof="schema:Person">
<span property="schema:Name"> Giaever, I.</span>;
<span property="schema:Name"> Keese, C.</span>
</span>
<span property="schema:name">&nbsp;A morphological biosensor for mammalian cells. </span>
<i property="schema:publisher" typeof="schema:Organization"> Nature</i>
<b property="issueNumber" typeof="PublicationIssue"> 366</b>,&nbsp;
<span property="schema:pageBegin"> 591</span>-<span property="schema:pageEnd">592</span>
(<time property="schema:datePublished" datatype="xsd:gYear" dateTime=" 1993">1993</time>).
<a className="doi" href="https://doi.org/10.1038/366591a0"> doi: 10.1038/366591a0</a>
</li>
</>,
cssname: "InvWischmeyer"
},
{
node: <>
<br/>
<h3>Nicole Friedlein</h3>
<hr/>
<div className="row">
<div className="col-2">
<div className="t-tag Ethics">
Phd
</div>
</div>
<div className="col-3">Original language: German</div>
<div className="col"><button>Go to interview</button></div>
</div>
<div className="row">
<div className="col">
<BlockQuoteB text="Quote" cite="."></BlockQuoteB>
</div>
<div className="col-3">
<img className="middle sechpro" src="https://static.igem.wiki/teams/5247/placeholders/placehilderperson.jpeg"/>
</div>
</div>
<h4>Aim of contact</h4>
<p></p>
<h4>Insights</h4>
<p></p>
<h4>Clarification</h4>
<p></p>
<h4>Implementation</h4>
<p></p>
</>,
cssname: "InvFriedlein"
},
{
node: <>
<br/>
<h3>Rnahale</h3>
<hr/>
<div className="row">
<div className="col-2">
<div className="t-tag Industry">
Beruf
</div>
</div>
<div className="col">Original language: German</div>
</div>
<div className="row">
<div className="col">
<BlockQuoteB text="Quote" cite="."></BlockQuoteB>
</div>
<div className="col-3">
<img className="middle sechpro" src="https://static.igem.wiki/teams/5247/placeholders/placehilderperson.jpeg"/>
</div>
</div>
<h4>Aim of contact</h4>
<p></p>
<h4>Insights</h4>
<p></p>
<h4>Clarification</h4>
<p></p>
<h4>Implementation</h4>
<p></p>
</>,
cssname: "InvRNale"
},
{
node: <>
<br/>
<h3>Mattijs Bulcaen</h3>
<hr/>
<div className="row">
<div className="col-5">
<div className="t-tag Academia">
Phd. Student (Molecular Virology and Gene Therapy)
</div>
</div>
<div className="col-3">Original language: English</div>
<div className="col"><button>Go to interview</button></div>
</div>
<div className="row">
<div className="col">
<BlockQuoteB text="Quote" cite="."></BlockQuoteB>
</div>
<div className="col-3">
<img className="middle sechpro" src="https://static.igem.wiki/teams/5247/placeholders/placehilderperson.jpeg"/>
</div>
</div>
<h4>Aim of contact</h4>
<p></p>
<h4>Insights</h4>
<p></p>
<h4>Clarification</h4>
<p></p>
<h4>Implementation</h4>
<p></p>
</>,
cssname: "InvBulcaen1"
},
{
node: <>
<br/>
<h3>Mattijs Bulcaen</h3>
<h5>Feedback Interview</h5>
<hr/>
<div className="row">
<div className="col-5">
<div className="t-tag Academia">
Phd. Student (Molecular Virology and Gene Therapy)
</div>
</div>
<div className="col">Original language: English</div>
</div>
<div className="row">
<div className="col">
<BlockQuoteB text="Quote" cite="."></BlockQuoteB>
</div>
<div className="col-3">
<img className="middle sechpro" src="https://static.igem.wiki/teams/5247/placeholders/placehilderperson.jpeg"/>
</div>
</div>
<h4>Aim of contact</h4>
<p></p>
<h4>Insights</h4>
<p></p>
<h4>Clarification</h4>
<p></p>
<h4>Implementation</h4>
<p></p>
</>,
cssname: "InvBulcaen2"
},
{
node: <>
<br/>
<h3>Julia</h3>
<hr/>
<div className="row">
<div className="col-2">
<div className="t-tag Patient">
Parent
</div>
</div>
<div className="col-3">Original language: German</div>
<div className="col"><button>Go to interview</button></div>
</div>
<div className="row">
<div className="col">
<BlockQuoteB text="Quote" cite="."></BlockQuoteB>
</div>
<div className="col-3">
<img className="middle sechpro" src="https://static.igem.wiki/teams/5247/placeholders/placehilderperson.jpeg"/>
</div>
</div>
<h4>Aim of contact</h4>
<p></p>
<h4>Insights</h4>
<p></p>
<h4>Clarification</h4>
<p></p>
<h4>Implementation</h4>
<p></p>
</>,
cssname: "InvJulia"
},
{
node: <>
<br/>
<h3>Joshua Bauder</h3>
<hr/>
<div className="row">
<div className="col-1">
<div className="t-tag Patient">
Parent
</div>
</div>
<div className="col-3">Original language: English</div>
<div className="col"><button>Go to interview</button></div>
</div>
<div className="row">
<div className="col">
<BlockQuoteB text="Quote" cite="."></BlockQuoteB>
</div>
<div className="col-3">
<img className="middle sechpro" src="https://static.igem.wiki/teams/5247/placeholders/placehilderperson.jpeg"/>
</div>
</div>
<h4>Aim of contact</h4>
<p></p>
<h4>Insights</h4>
<p></p>
<h4>Clarification</h4>
<p></p>
<h4>Implementation</h4>
<p></p>
</>,
cssname: "InvJoshua"
},
{
node: <>
<br/>
<h3>Katrin Westhoff</h3>
<h5>Initial Interview</h5>
<hr/>
<div className="row">
<div className="col-2">
<div className="t-tag Medical">
Physiotherapist
</div>
</div>
<div className="col-3">Original language: German</div>
<div className="col"><button>See interview</button></div>
</div>
<div className="row">
<div className="col">
<BlockQuoteB text="The more we know, the more opportunities we have." cite="Katrin Westhoff"></BlockQuoteB>
</div>
<div className="col-3">
<img className="middle sechpro" src="https://static.igem.wiki/teams/5247/placeholders/placehilderperson.jpeg"/>
</div>
</div>
<h4>Aim of contact</h4>
<p>The objective of the contact was to gain in-depth insights into the treatment and care of children with cystic fibrosis. The therapist's expertise was intended to help develop a better understanding of the challenges and necessary measures in the treatment of this chronic disease. In addition, the aim was to ascertain how the therapy is implemented in everyday life and which specific approaches and methods are particularly effective. </p>
<h4>Insights</h4>
<p>The interview yielded valuable insights into the regular implementation of the therapy, the use of aids and the adaptation of exercises to the individual needs of the patients. It was notable that the therapy has improved considerably thanks to better medication and adapted exercises, with a concomitant increase in life expectancy for children affected by cystic fibrosis. Of particular interest was the emphasis on the importance of sport and exercise, which should not only be therapeutically effective, but also increase quality of life. </p>
{/* <h4>Clarification</h4>
<p></p> */}
<h4>Implementation</h4>
<p>The following statement by Katrin Westhoff had a particularly profound impact on our project: "The more we know, the more opportunities we have." We learned from the interview that the current medication is already helping many patients to a huge extent, but that there is still a significant opportunity for improvement. After all, successful gene therapy would markedly enhance the quality of life for those affected. The findings of this project will be disseminated to the relevant researchers in order to facilitate the rapid improvement of the quality of life of all cystic fibrosis patients, regardless of their mutation. </p>
<button>Jump to visit</button>
</>,
cssname: "InvWesthoff"
},
{
node: <>
<br/>
<h3>Katrin Westhoff</h3>
<h5>Visit</h5>
<hr/>
<div className="row">
<div className="col-2">
<div className="t-tag Medical">
Physiotherapist
</div>
</div>
<div className="col">Original language: German</div>
</div>
<div className="row">
<div className="col">
<BlockQuoteB text="Quote" cite="."></BlockQuoteB>
</div>
<div className="col-3">
<img className="middle sechpro" src="https://static.igem.wiki/teams/5247/placeholders/placehilderperson.jpeg"/>
</div>
</div>
<h4>Aim of contact</h4>
<p></p>
<h4>Insights</h4>
<p></p>
<h4>Clarification</h4>
<p></p>
<h4>Implementation</h4>
<p></p>
</>,
cssname: "BesWesthoff"
},
{
node: <>
<br/>
<h3>Cristian-Gabriel Olariu</h3>
<hr/>
<div className="row">
<div className="col-2">
<div className="t-tag Medical">
Pediatrician
</div>
</div>
<div className="col">Original language: German</div>
</div>
<div className="row">
<div className="col">
<BlockQuoteB text="Quote" cite="."></BlockQuoteB>
</div>
<div className="col-3">
<img className="middle sechpro" src="https://static.igem.wiki/teams/5247/placeholders/placehilderperson.jpeg"/>
</div>
</div>
<h4>Aim of contact</h4>
<p></p>
<h4>Insights</h4>
<p></p>
<h4>Clarification</h4>
<p></p>
<h4>Implementation</h4>
<p></p>
</>,
cssname: "InvOlariu"
},
{
node: <>
<br/>
<h3>Dr. Katharina Kolonko</h3>
<hr/>
<div className="row">
<div className="col-2">
<div className="t-tag Academia">
Beruf
</div>
</div>
<div className="col">Original language: German</div>
</div>
<div className="row">
<div className="col">
<BlockQuoteB text="Quote" cite="."></BlockQuoteB>
</div>
<div className="col-3">
<img className="middle sechpro" src="https://static.igem.wiki/teams/5247/placeholders/placehilderperson.jpeg"/>
</div>
</div>
<h4>Aim of contact</h4>
<p></p>
<h4>Insights</h4>
<p></p>
<h4>Clarification</h4>
<p></p>
<h4>Implementation</h4>
<p></p>
</>,
cssname: "InvKolonko"
},
{
node: <>
<br/>
<h3>Prof. Dr. Christoph Weber</h3>
<hr/>
<div className="row">
<div className="col-2">
<div className="t-tag Academia">
Beruf
</div>
</div>
<div className="col">Original language: German</div>
</div>
<div className="row">
<div className="col">
<BlockQuoteB text="Quote" cite="."></BlockQuoteB>
</div>
<div className="col-3">
<img className="middle sechpro" src="https://static.igem.wiki/teams/5247/placeholders/placehilderperson.jpeg"/>
</div>
</div>
<h4>Aim of contact</h4>
<p></p>
<h4>Insights</h4>
<p></p>
<h4>Clarification</h4>
<p></p>
<h4>Implementation</h4>
<p></p>
</>,
cssname: "InvWeber"
},
{
node: <>
<br/>
<h3>Prof. Dr. Stefan Hammer</h3>
<hr/>
<div className="row">
<div className="col-2">
<div className="t-tag Academia">
Beruf
</div>
</div>
<div className="col">Original language: German</div>
</div>
<div className="row">
<div className="col">
<BlockQuoteB text="Quote" cite="."></BlockQuoteB>
</div>
<div className="col-3">
<img className="middle sechpro" src="https://static.igem.wiki/teams/5247/placeholders/placehilderperson.jpeg"/>
</div>
</div>
<h4>Aim of contact</h4>
<p></p>
<h4>Insights</h4>
<p></p>
<h4>Clarification</h4>
<p></p>
<h4>Implementation</h4>
<p></p>
</>,
cssname: "InvHammer"
},
]
// die height für className="timeline row align-items-center" muss angepasst werden, damit die Boxen höher sein können
export function HumanPractices() {
openTab({cityName: "All", cla: "timelinecardtabs"});
const location = useLocation();
useEffect(() => {
const params = new URLSearchParams(location.search);
const collapseId = params.get('collapseId');
const tabId = params.get('tab');
// Scroll to the section specified by collapseId
if (collapseId) {
const collapseElement = document.getElementById(collapseId);
if (collapseElement) {
const elementTop = collapseElement.getBoundingClientRect().top + window.pageYOffset;
const offset = window.innerHeight / 2 - collapseElement.offsetHeight / 2;
const scrollPosition = elementTop - offset;
window.scrollTo({
top: scrollPosition,
behavior: 'smooth',
});
}
}
// Open the tab specified by tabId
if (tabId) {
openFromOtherPage(tabId)({ currentTarget: document.getElementById(tabId)! });
}
}, [location.search]);
return (
<div className="row mt-4">
<BlockQuoteB
text="Human Practices is the study of how your work affects the world, and how the world affects your work."
cite="- Peter Carr, Director of Judging"
/>
<HPOverview/>
<HPTimeline/>
{/* <HPTabsTwo/> */}
<section id="Reflection" >
<div className="center">
<h3 className="col personalstyleone">Reflection Framework</h3>
</div>
</section>
{/*
<section id="Implementation" className="section">
<div className="center">
<h3 className="col personalstyleone">Implementation</h3>
</div>
</section> */}
</div>
);
}
export function HPTabs(){
const [value, setValue] = React.useState('1');
const handleChange = (_event: React.SyntheticEvent, newValue: string) => {
setValue(newValue);
};
return(
<Box sx={{ width: '100%', typography: 'body1' }}>
<TabContext value={value}>
<Box sx={{ borderBottom: 1, borderColor: 'divider' }}>
<TabList onChange={handleChange} aria-label="lab API tabs example">
<Tab label="Patient Needs" value="1" />
<Tab label="Scientific Challenges" value="2" />
<Tab label="Academic consideration" value="3" />
<Tab label="Ethical questions" value="4" />
<Tab label="Medical perspectives" value="5" />
</TabList>
</Box>
<TabPanel value="1"> </TabPanel>
<TabPanel value="2"> </TabPanel>
<TabPanel value="3"> </TabPanel>
<TabPanel value="4"> </TabPanel>
<TabPanel value="5"> </TabPanel>
</TabContext>
</Box>
)
}
/* <div className="methods-node">
<div>R<sup>3</sup></div>
</div>
<div className="methods-node">
<div>Human Practices Cycle</div>
</div>
<div className="methods-node">
<div>Stakeholder Framework</div>
</div>
<div className="methods-node">
<div>Feedback Cycle</div>
</div>
<div className="methods-node">
<div></div>
</div> */
export function HPTabsTwo(){
return(
<>
<div className="row align-items-center">
<div className="col"></div>
<div className="col-8">
<div className="methods-node">
<div>Project selection</div>
</div>
<div className="methods-node">
<div>Foundation</div>
</div>
<div className="methods-node">
<div>Diversification</div>
</div>
<div className="methods-node">
<div>Fine-tuning</div>
</div>
<div className="methods-node">
<div>Finalization</div>
</div>
</div>
<div className="col"></div>
</div>
<div className="row align-items-center">
<div className="col-3">
{/* <Box sx={{ borderBottom: 1, borderColor: 'divider' }}>
<TabList onChange={handleChange} aria-label="lab API tabs example">
<Tab label="Patient Needs" value="1" />
<Tab label="Scientific Challenges" value="2" />
<Tab label="Academic consideration" value="3" />
<Tab label="Ethical questions" value="4" />
<Tab label="Medical perspectives" value="5" />
</TabList>
</Box> */}
</div>
<div className="col">
{/* <TabPanel value="1"> 1 </TabPanel>
<TabPanel value="2"> 2 </TabPanel>
<TabPanel value="3"> 3 </TabPanel>
<TabPanel value="4"> 4 </TabPanel>
<TabPanel value="5"> 5 </TabPanel> */}
</div>
</div>
</>
)
}
{/*
*/}
/* <Tab label="Problem" value="1" />
<Tab label="Influence" value="5" />
<Tab label="Patient Needs" value="2" />
<Tab label="Scientific Challenges" value="3" />
<Tab label="Surveys" value="4" /> */
function HPOverview(){
return(
<section id="Overview" className="section">
<div className="center" >
<h3 className="col personalstyleone">Overview</h3>
</div>
<div className="row align-items-center" style={{marginTop: "5vh", marginBottom: "5vh"}}>
<div className="col">
<ButtonOne text="Inspiration" open="inspiration"></ButtonOne>
</div>
<div className="col">
<ButtonOne text="Methods" open="methods"></ButtonOne>
</div>
<div className="col">
<ButtonOne text="Values and goals" open="values"></ButtonOne>
</div>
<div className="col">
<ButtonOne text="Stakeholders" open="stakeholders"></ButtonOne>
</div>
</div>
<div className="col cycletab" id="inspiration" style={{display: "block"}}>Hallo </div>
<div className="col cycletab" id="values" style={{display: "none"}}>Hallo </div>
<div className="col cycletab" id="methods" style={{display: "none"}}>Hallo </div>
<div className="col cycletab" id="stakeholders" style={{position: "relative", height: "fit-content", display: "none"}}>
<MindMapTwo></MindMapTwo>
</div>
</section>
)
}
function HPTimeline(){
return(
<section id="Timeline" className="section">
<div className="center">
<h3 className="col personalstyleone">Timeline</h3>
</div>
<TabButtonRow data={timelinebuttonrowdata} classy="" opentype="timelinecardtabs" closing="timelinepersontabs" />
<ButtonRowTabs cla="timelinecardtabs" data={timelinebuttonrowdata}/>
<BFHStyleTabs cla="timelinepersontabs" data={timelinepersontabs}></BFHStyleTabs>
</section>
)
}
function MindMapTwo(){
return(
<div className="mindmap">
{/* <!--LEFT--> */}
<ol className="children children_leftbranch">
<li className="children_item">
<div className="node" >
<div id="L1" className="node_text"> Medical <br/> Professionals</div>
</div>
<ol className="children">
<li className="children_item">
<div className="node">
<div id="L1.1" className="node_text">Katrin</div>
</div>
</li>
<li className="children_item">
<div className="node">
<div id="L1.2" className="node_text">Cristian</div>
</div>
</li>
</ol>
</li>
<li className="children_item">
<div className="node">
<div id="L2" className="node_text">Industry</div>
</div>
<ol className="children">
<li className="children_item">
<div className="node">
<div className="node_text">L2.1</div>
</div>
</li>
<li className="children_item">
<div className="node">
<div className="node_text">L2.2</div>
</div>
</li>
<li className="children_item">
<div className="node">
<div className="node_text">L2.3</div>
</div>
</li>
</ol>
</li>
<li className="children_item">
<div className="node">
<div id="L3" className="node_text">iGem</div>
</div>
<ol className="children">
<li className="children_item">
<div className="node">
<div className="node_text">L2.1</div>
</div>
</li>
<li className="children_item">
<div className="node">
<div className="node_text">L2.2</div>
</div>
</li>
<li className="children_item">
<div className="node">
<div className="node_text">L2.3</div>
</div>
</li>
</ol>
</li>
</ol>
{/* <!--CENTER--> */}
<div className="node node_root">
<div className="node_text">Stakeholders</div>
</div>
{/* <!--RIGHT--> */}
<ol className="children children_rightbranch">
<li className="children_item">
<div className="node">
<div id="R1" className="node_text">Patients & <br/> next of kin </div>
</div>
<ol className="children">
<li className="children_item">
<div className="node">
<div id="R1.1" className="node_text">Max</div>
</div>
</li>
<li className="children_item">
<div className="node">
<div id="R1.2" className="node_text">Julia</div>
</div>
</li>
<li className="children_item">
<div className="node">
<div id="R1.3" className="node_text">Joshua</div>
</div>
</li>
</ol>
</li>
<li className="children_item">
<div className="node">
<div id="R2" className="node_text">Academia</div>
</div>
<ol className="children">
<li className="children_item">
<div className="node">
<div id="R2.1" className="node_text">Mattijs</div>
</div>
</li>
<li className="children_item">
<div className="node">
<div className="node_text">R2.2</div>
</div>
</li>
<li className="children_item">
<div className="node">
<div className="node_text">R2.3</div>
</div>
</li>
<li className="children_item">
<div className="node">
<div className="node_text">R2.4</div>
</div>
</li>
</ol>
</li>
<li className="children_item">
<div className="node">
<div id="R3" className="node_text">Society</div>
</div>
<ol className="children">
<li className="children_item">
<div className="node">
<div id="R3.1" className="node_text">Krankk.</div>
</div>
</li>
<li className="children_item">
<div className="node">
<div id="R3.2" className="node_text">Ethik</div>
</div>
</li>
<li className="children_item">
<div className="node">
<div className="node_text">R3.3</div>
</div>
</li>
</ol>
</li>
</ol>
</div>
)
}
export function idTabLine({beruf, tagend, lang}:{beruf: string, tagend: string, lang: string}){
let cl = "t-tag " + tagend
return(
<div className="row">
<div className="col-2">
<div className={cl}>
{beruf}
</div>
</div>
<div className="col"> Original language: {lang}</div>
</div>
)
}
\ No newline at end of file
src/contents/igem-bielefeld.tsx 0 → 100644
View file @ 3fd292fd
import { PDF } from "../components/Pdfs";
import { BlockQuoteB } from "../components/Quotes";
import { Section, Subesction } from "../components/sections";
import { useTabNavigation } from "../utils/TabNavigation";
export function igemBielefeld() {
useTabNavigation();
return (
<>
<Section title="Bielefeld University" id="Bielefeld University">
<img src="https://static.igem.wiki/teams/5247/photos/university/bielefeld-3381870.jpg"/>
<p>
Bielefeld University, established in 1969, is distinguished by its interdisciplinary approach to research and teaching at the highest academic level. The university is constituted of 14 faculties, which encompass a comprehensive range of subject areas, including the humanities, natural and technical sciences, social sciences, and education. In the forthcoming years, the recently established faculty of medicine will serve to further expand the university's interdisciplinary research opportunities. With an enrollment of approximately 25,000 students, including 2,000 international students, Bielefeld University offers 123 different degree programs. The campus is currently undergoing a significant expansion, with the objective of becoming one of the most modern in Europe. The new facilities will offer students an exceptional environment. Bielefeld University actively collaborates with over 300 partner institutions worldwide, fostering global academic exchange. Through its Erasmus+ program, the university enables students and faculties to engage in research and study abroad programs across Europe, Asia, America and beyond. While Bielefeld has traditionally been known for its strong sociology department, recent advances in robotics and biotechnology have brought these fields to the forefront of the university's cutting-edge research. As a result, the Centre for Biotechnology (CeBiTec) has become one of the university's most important institutes.
</p>
<img src="https://static.igem.wiki/teams/5247/photos/university/img-1989.jpeg"/>
<p>
The Centre for Biotechnology (CeBiTec) constitutes a central institution at Bielefeld University, dedicated to interdisciplinary research in the field of life sciences. It is one of the largest research facilities on campus, facilitating innovative projects that span several disciplines. The research areas of focus include large-scale genomics, big data bioinformatics and metabolic engineering of single-cell systems for bioproduction. CeBiTec unites research from a multitude of disciplines, including biotechnology, molecular biology, genome research, systems biology, biochemistry, bioinformatics, and computer science. Additionally, it plays a significant role in academic training and doctoral programmes, as well as serving as a hub for biotechnological initiatives. CeBiTec is situated within the state-of-the-art Laboratory Building G on the campus of Bielefeld University, which opened in 2007. Through ist focus on Cross-disciplinary collaboration, CeBiTec serves as a viral link between academic innovation and real-world applications, shaping the future of biotechnology at Bielefeld University and beyond.
</p>
</Section>
<Section title="History" id="History">
<div className="row">
<div className="col">
<img src="https://static.igem.wiki/teams/5247/sponsors/uni-bielefeld-dunkel.png" style={{ height:"100px"}}/>
</div>
<div className="col">
<img src="https://static.igem.wiki/teams/5247/sponsors/cebitec-logo-hinterlegt.png" style={{height:"100px"}}/>
</div>
</div>
<br/>
<p>The glorious history of the iGEM team Bielefeld began in 2010 and marked its debut on the global stage
of synthetic biology. Since then, the teams were composed of over 160 enthusiastic students from various disciplines, including biology, chemistry, and engineering.</p>
<p>Their initial project focused on the development of a biosensor for the measurement of spiciness in food, which resulted in the team being awarded a gold medal at the competition. Following this successful debut, the teams continued to evolve, both in size and expertise.
Over more than ten years, the iGEM Team Bielefeld-CeBiTec engaged in a multitude of projects, each pushing the boundaries of synthetic biology. The teams earned 13 gold medals and have achieved a ranking within the top 16 teams twice (2011, 2012). One of the most notable achievements was reached in 2013, when the iGEM Team Bielefeld won the European Jamboree, were the First Runner Up at the Giant Jamboree and secured several Track and Special Prizes. Their project “Ecolectricity” focused on creating a microbial fuel cell, by transforming E. coli into an electro-active bacterium and incorporating it into a fuel cell that provides an environmentally friendly alternative for generating electricity. Their innovative approach and careful execution impressed the jury and solidified the Bielefeld team's reputation in the iGEM community. In acknowledgment of the pivotal role played by the Center for Biotechnology (CeBiTec) in the team’s success since the beginning, the name of the team was officially changed to Bielefeld-CeBiTec in 2014. This change highlighted the close collaboration between the team and the research center, thereby underscoring the importance of institutional support in advancing scientific research and education related to the iGEM competition.</p>
<p>In recent years, the teams continued to pursue innovative avenues of enquiry. Their projects ranged from developing biological systems for medical applications to creating sustainable solutions for industrial and environmental challenges. For instance, in 2018, the project "nanoFactory" aimed to scavenge heavy metal ions using optimized E. coli cells to accumulate heavy metal ions inside the cytoplasm addressing the issue of increasing scarcity of metal resources due to global consumption of electronics.
In the previous year, the iGEM team Bielefeld-CeBiTec developed a platform for diagnosis and therapy of brain tumors called "ASTERISK". The team focusing on a modular genetic system to target gliomas by designing mRNA molecules that, upon detection of a tumor-specific mutation or amplification, lead to the translation of a toxic protein that selectively kills cancer cells without harming healthy cells. This innovative approach is a perfect example of how synthetic biology can be used to improve the specificity and efficacy of medical treatments, providing valuable tools to the iGEM community.</p>
<p>A significant aspect of iGEM Bielefeld-CeBiTec’s success has been their emphasis on collaboration and community engagement. The team regularly collaborates with other iGEM teams, institutions, companies, and scientists sharing knowledge and resources. They also engage with the local community through outreach programs and workshops. he history of iGEM Bielefeld is a story of motivation, dedication, and scientific curiosity. From their early beginnings to their current endeavors, the team has consistently demonstrated the power of synthetic biology to tackle complex global challenges. As they continue to participate in the iGEM competition, they remain to demonstrate innovation and collaboration within the scientific community.</p>
</Section>
<Section title="Steering Committee" id="Steering Committee">
<BlockQuoteB text="iGEM is the biggest opportunity for young researchers to cross their own boundaries." cite="Prof. Dr. Jörn Kalinowski, Principle Investigator of iGEM Bielefeld since 2010"/>
<Subesction title="What is a Steering Committee?" id="Steering Committee1">
<p>The Steering Committee plays a central role in the resumption and further development of iGEM activities at Bielefeld University. After a pause in 2022 due to financial constraints and changes in participation conditions, the Steering Committee was established to ensure Bielefeld's sustainable participation in future iGEM competitions.</p>
<p>
<div className="row">
<div className="col">
<img src="https://static.igem.wiki/teams/5247/pdfs/steering-commitee.webp" />
</div>
<div className="col">
<p>The Steering Committee consists of five renowned scientists from the Faculties of Biology and Technology: Dr. Petra Lutter, Prof. Dr. Jörn Kalinowski, Prof. Dr. Kristian Müller, Prof. Dr. Karsten Niehaus, and Prof. Dr. Jens Stoye. Each of these experts brings specific expertise crucial for the successful execution of iGEM projects. Petra Lutter has contributed to modeling in previous iGEM projects, while Jörn Kalinowski has been significantly involved in all past iGEM projects. Kristian Müller has been an experienced supporter of the iGEM competitions since their inception, and Karsten Niehaus, former head of the "Genome-based Systems Biology" master’s program, brings extensive knowledge of the scientific foundations of the projects. Jens Stoye, representing bioinformatics at Bielefeld University, contributes his expertise in this area. </p>
</div>
</div>
</p>
<p>The main goal of the Steering Committee is to ensure the successful implementation of future iGEM projects. This includes not only academic support but also organizational leadership, securing funding, and providing the necessary infrastructure. The experts in the Steering Committee are significantly involved in the strategic direction of the projects and offer a platform where ideas, resources, and knowledge are pooled to continue Bielefeld's tradition of successful iGEM participation. </p>
<p>
<div style={{marginBottom: "1rem"}}><PDF link="https://static.igem.wiki/teams/5247/pdfs/igem-broschure.pdf" name="igem-broschure.pdf" /></div>
</p>
<p>
<div className="row">
<div className="col">
<img src="https://static.igem.wiki/teams/5247/pdfs/steering-committee-1.webp"/>
</div>
<div className="col">
<p>In helping the new iGEM Bielefeld team advance their project, the Steering Committee played an indispensable role, particularly by promoting the iGEM principle of "Contribution" and fostering the interdisciplinary nature of the project. The committee emphasized the importance of creating tools, data, and methods that can benefit the global iGEM community. This mindset was reflected in the team’s project design, ensuring that their work not only met local goals but also provided meaningful contributions to future projects. </p>
<p>Moreover, the interdisciplinary nature of the iGEM project was strongly encouraged by the Steering Committee. With members from various scientific fields, the committee facilitated collaboration between disciplines such as biology, bioinformatics, and biotechnology. This interdisciplinary approach allowed the team to tackle complex challenges from multiple perspectives, integrating computational models with experimental biology to drive innovation. This guidance helped the iGEM Bielefeld team develop a more robust and impactful project, aligning with both the scientific goals of the competition and the collaborative spirit of the iGEM community. </p>
</div>
</div>
</p>
</Subesction>
</Section>
</>
);
}
\ No newline at end of file
src/contents/impressum.tsx
View file @ 3fd292fd
import { useEffect } from "react";
import { useLocation } from "react-router-dom";
import { openFromOtherPage } from "../components/Buttons";
export function Impressum() {
const location = useLocation();
useEffect(() => {
const params = new URLSearchParams(location.search);
const collapseId = params.get('collapseId');
const tabId = params.get('tab');
// Scroll to the section specified by collapseId
if (collapseId) {
const collapseElement = document.getElementById(collapseId);
if (collapseElement) {
const elementTop = collapseElement.getBoundingClientRect().top + window.pageYOffset;
const offset = window.innerHeight / 2 - collapseElement.offsetHeight / 2;
const scrollPosition = elementTop - offset;
window.scrollTo({
top: scrollPosition,
behavior: 'smooth',
});
}
}
// Open the tab specified by tabId
if (tabId) {
openFromOtherPage(tabId)({ currentTarget: document.getElementById(tabId)! });
}
}, [location.search]);
import { H2 } from "../components/Headings";
import { useTabNavigation } from "../utils/TabNavigation";
export function Impressum() {
useTabNavigation();
return (
<>
<div className="row">
<div className="col">
<h2>Impressum</h2>
<H2 id="impressum" text="Impressum"/>
<hr />
</div>
</div>
......@@ -49,7 +22,7 @@ export function Impressum() {
33615 Bielefeld<br />
<br />
<b>Contact</b><br />
E-mail: team2024@igem-bielefeld.de<br />
E-mail: team2024[at]igem-bielefeld[dot]de<br />
<br />
<b>Supervisory Authority</b><br />
Bielefeld University - Center for Biotechnology (CeBiTec)
......
src/contents/index.tsx
View file @ 3fd292fd
......@@ -5,77 +5,60 @@ export * from "./Home.tsx";
export * from "./team.tsx";
export * from "./attributions.tsx";
// Project
export * from "./contribution.tsx";
export * from "./Contribution/contribution.tsx";
export * from "./description.tsx";
export * from "../sidebars/descS.tsx"
export * from "./engineering.tsx";
export * from "./experiments.tsx";
export * from "./notebook.tsx";
export * from "./results.tsx";
// Safety
export * from "./safety.tsx";
// Human Practices
export * from "./human-practices.tsx";
export * from "./Human Practices/human-practices.tsx";
export * from "../sidebars/hpS.tsx"
export * from "./Bfh.tsx";
export * from "./wiki.tsx";
export * from "./drylab.tsx";
export * from "./impressum.tsx";
export * from "./measurement.tsx";
export * from "./partners.tsx";
export * from "./supplementary-material.tsx";
export * from "./interviews.tsx";
export * from "../headers/bhf-h.tsx"
export * from "../headers/attribution-h.tsx"
export * from "../headers/cont-h.tsx"
export * from "../headers/desc-h.tsx"
export * from "../headers/home-h.tsx"
export * from "../headers/exp-h.tsx"
export * from "../headers/hp-h.tsx"
export * from "../headers/imp-h.tsx"
export * from "../headers/note-h.tsx"
export * from "../headers/res-h.tsx"
export * from "../headers/safe-h.tsx"
export * from "../headers/team-h.tsx"
export * from "../headers/wiki-h.tsx"
export * from "../headers/ints-h.tsx"
export * from "../headers/spons-h.tsx"
export * from "../headers/dry-h.tsx"
export * from "../headers/eng-h.tsx"
export * from "../headers/sup-h.tsx"
export * from "../headers/mes-h.tsx"
export * from "./parts.tsx";
export * from "../headers/part-h.tsx"
export * from "./proof.tsx";
export * from "../headers/proof-h.tsx"
export * from "./project-documentation.tsx";
export * from "../headers/prodesc-h.tsx"
export * from "./design.tsx";
export * from "../headers/des-h.tsx"
export * from "./judging.tsx";
export * from "../headers/judge-h.tsx"
export * from "./ethics.tsx";
export * from "../headers/eth-h.tsx"
export * from "./example.tsx";
export * from "./education.tsx";
export * from "../headers/edu-h.tsx"
export * from "../sidebars/engS.tsx"
export * from "../sidebars/intS.tsx"
export * from "../sidebars/safeS.tsx"
export * from "../sidebars/none.tsx"
export * from "../sidebars/ethS.tsx"
export * from "./survey.tsx";
export * from "../headers/sur-h.tsx"
export * from "./collaborations.tsx";
export * from "../headers/coll-h.tsx"
export * from "./igem-bielefeld.tsx";
export * from "../headers/ibie-h.tsx"
src/contents/interviews.tsx
View file @ 3fd292fd
......@@ -2,7 +2,8 @@ import { ButtonOne } from "../components/Buttons";
import { QaBox, SpecialQaBox } from "../components/Boxes";
import { useEffect } from "react";
import { useLocation } from "react-router-dom";
import { openFromOtherPage } from "../components/Buttons";
import { H3 } from "../components/Headings";
import { openFromOtherPage } from "../utils/openFromOtherpAge";
export function Ints() {
const location = useLocation();
useEffect(() => {
......@@ -33,107 +34,8 @@ export function Ints() {
return (
<>
<br/>
<section className="col" id="maxH">
<h3 id="max">Max Beckmann</h3>
<hr/>
<div className="row">
<div className="col-2">
<div className="t-tag Patient">
Patient
</div>
</div>
<div className="col">Original language: German</div>
</div>
<br/>
<div className="row">
<div className="col">
<div className="col">
<ButtonOne text="Erstes Interview" open="maxinv1"></ButtonOne>
</div>
<br/>
<div className="col">
<ButtonOne text="Zweites Interview" open="maxinv2"></ButtonOne>
</div>
</div>
<div className="col-3">
<img className="interview-img" src="https://static.igem.wiki/teams/5247/placeholders/placehilderperson.jpeg"/>
</div>
</div>
<div className="col cycletab" id="maxinv1" style={{display: "block"}}>
<h2>Notes from the first interview</h2>
<br/>
<QaBox
q="Question"
a="Answer"
/>
</div>
<div className="col cycletab" id="maxinv2" style={{display: "none"}}>
<h2>Notes from the second interview</h2>
<br/>
<QaBox
q="Question"
a="Answer"
/>
</div>
</section>
<br/>
<section className="col" id="olariuH">
<h3 id="olariu">Cristian-Gabriel Olariu</h3>
<hr/>
<div className="row">
<div className="col-2">
<div className="t-tag Medical">
Pediatrician
</div>
</div>
<div className="col">Original language: German</div>
</div>
<br/>
<div className="row">
<div className="col-3">
<img className="interview-img" src="https://static.igem.wiki/teams/5247/placeholders/placehilderperson.jpeg"/>
</div>
<div className="col">
</div>
</div>
<div className="col">
<h2>Notes from the interview</h2>
<br/>
<QaBox
q="Question"
a="Answer"
/>
</div>
</section>
<br/>
<section className="col" id="joshuaH">
<h3 id="joshua">Joshua Bauder</h3>
<hr/>
<div className="row">
<div className="col-2">
<div className="t-tag Patient">
Parent
</div>
</div>
<div className="col">Original language: English</div>
</div>
<br/>
<div className="row">
<div className="col"></div>
<div className="col-3">
<img className="interview-img" src="https://static.igem.wiki/teams/5247/placeholders/placehilderperson.jpeg"/>
</div>
</div>
<div className="col">
<h2>Notes from the interview</h2>
<br/>
<QaBox
q="Question"
a="Answer"
/>
</div>
</section>
<br/>
<section className="col" id="juliaH">
<h3 id="julia">Julia</h3>
......@@ -149,13 +51,13 @@ export function Ints() {
<br/>
<div className="row">
<div className="col-3">
<img className="interview-img" src="https://static.igem.wiki/teams/5247/placeholders/placehilderperson.jpeg"/>
<img className="interview-img" src="https://static.igem.wiki/teams/5247/photos/hp/julia.jpg"/>
</div>
<div className="col">
</div>
</div>
<div className="col">
<h2>Notes from the interview</h2>
<H3 id="julianotes" text="Notes from the interview"/>
<br/>
<QaBox
q="Question"
......@@ -183,7 +85,7 @@ export function Ints() {
</div>
</div>
<div className="col">
<h2>Notes from the interview</h2>
<H3 id="nicolenotes" text="Notes from the interview"/>
<br/>
<QaBox
q="Question"
......@@ -206,20 +108,20 @@ export function Ints() {
<br/>
<div className="row">
<div className="col-3">
<img className="interview-img" src="https://static.igem.wiki/teams/5247/placeholders/placehilderperson.jpeg"/>
<img className="interview-img" src="https://static.igem.wiki/teams/5247/photos/hp/mattijs.jpg"/>
</div>
<div className="col">
<div className="col">
<div className="col ">
<ButtonOne text="Erstes Interview" open="mattijsinv1"></ButtonOne>
</div>
<br/>
<div className="col">
<div className="col ">
<ButtonOne text="Zweites Interview" open="mattijsinv2"></ButtonOne>
</div>
</div>
</div>
<div className="col cycletab" id="mattijsinv1" style={{display: "block"}}>
<h2>Notes from the first interview</h2>
<H3 id="mattijsnotes1" text="Notes from the first interview"/>
<br/>
<QaBox
q="Question"
......@@ -227,7 +129,7 @@ export function Ints() {
/>
</div>
<div className="col cycletab" id="mattijsinv2" style={{display: "none"}}>
<h2>Notes from the second interview</h2>
<H3 id="mattijsnotes2" text="Notes from the second interview"/>
<br/>
<QaBox
q="Question"
......@@ -252,7 +154,7 @@ export function Ints() {
<div className="col">
</div>
<div className="col-3">
<img className="interview-img" src="https://static.igem.wiki/teams/5247/placeholders/placehilderperson.jpeg"/>
<img className="interview-img" src="https://static.igem.wiki/teams/5247/photos/hp/hp-katrin-portrait.jpg"/>
</div>
</div>
<div className="col">
......@@ -272,7 +174,7 @@ export function Ints() {
/>
<QaBox
q="How many patients do you treat?"
a="We currently have 8 children with cystic fibrosis in our medical practice, which is quite a lot. However, if you compare this number with other diseases, it is rather a small number. We have slightly more children with cystic fibrosis in our practice because we specialize in it, among other diseases."
a="We currently have 8 children with Cystic Fibrosis in our medical practice, which is quite a lot. However, if you compare this number with other diseases, it is rather a small number. We have slightly more children with Cystic Fibrosis in our practice because we specialize in it, among other diseases."
/>
<SpecialQaBox
q="What kind of exercises do you do?">
......@@ -324,8 +226,8 @@ export function Ints() {
a="Pancreatic complaints are rarely treated with physiotherapy, unless it is an inflammation. In such cases, the patient is admitted to a hospital. Massage or taping the intestines with kinesiology tape helps with constipation and works very well. "
/>
<QaBox
q="Are there any special hygiene guidelines for you when working with cystic fibrosis patients? "
a="Hygiene guidelines are very important when working with cystic fibrosis patients. A distinction is made between children with and without infections (Pseudomonas). Regular nasal swabs are taken and only children with or without infections are treated in the practice on any given day. Ventilation, patients wearing masks while infected and disinfection of the facilities are essential. Children infected with multi-resistant germs are not allowed to enter the practice; in such cases, physiotherapists visit the patients' homes. "
q="Are there any special hygiene guidelines for you when working with Cystic Fibrosis patients? "
a="Hygiene guidelines are very important when working with Cystic Fibrosis patients. A distinction is made between children with and without infections (Pseudomonas). Regular nasal swabs are taken and only children with or without infections are treated in the practice on any given day. Ventilation, patients wearing masks while infected and disinfection of the facilities are essential. Children infected with multi-resistant germs are not allowed to enter the practice; in such cases, physiotherapists visit the patients' homes. "
/>
<QaBox
q="Are the specific exercises customized? And if so, how do you know which therapy is the right one for which patient (based on laboratory values, tests, different mutation patterns...)? "
......@@ -337,7 +239,7 @@ export function Ints() {
/>
<QaBox
q="How many physiotherapists offer muco-therapy? "
a="The exact number of physiotherapists offering cystic fibrosis therapy is unknown. However, there are several child therapists in the region providing this therapy. "
a="The exact number of physiotherapists offering Cystic Fibrosis therapy is unknown. However, there are several child therapists in the region providing this therapy. "
/>
<QaBox
q="How are the relatives educated? "
......@@ -365,7 +267,7 @@ export function Ints() {
<br/>
<div className="row">
<div className="col-3">
<img className="interview-img" src="https://static.igem.wiki/teams/5247/placeholders/placehilderperson.jpeg"/>
<img className="interview-img" src="https://static.igem.wiki/teams/5247/photos/hp/berens.jpg"/>
</div>
<div className="col">
</div>
......@@ -433,7 +335,7 @@ export function Ints() {
q="Who could help us with the Patch-Clamp measurements? "
a="The Patch-Clamp devices are heavily utilized in our working group, so you probably cannot perform measurements on your own. However, postdocs could support you for some measurements. Dr. Oliver Dräger is available as a contact person of my working group. "
/>
</div>
<p> Test aus dem Wiki </p> </div>
</section>
<br/>
</>
......
src/contents/judging.tsx
View file @ 3fd292fd
import { useEffect } from "react";
import { useLocation } from "react-router-dom";
import { openFromOtherPage } from "../components/Buttons";
export function Judging() {
const location = useLocation();
useEffect(() => {
const params = new URLSearchParams(location.search);
const collapseId = params.get('collapseId');
const tabId = params.get('tab');
// Scroll to the section specified by collapseId
if (collapseId) {
const collapseElement = document.getElementById(collapseId);
if (collapseElement) {
const elementTop = collapseElement.getBoundingClientRect().top + window.pageYOffset;
const offset = window.innerHeight / 2 - collapseElement.offsetHeight / 2;
const scrollPosition = elementTop - offset;
window.scrollTo({
top: scrollPosition,
behavior: 'smooth',
});
}
}
// Open the tab specified by tabId
if (tabId) {
openFromOtherPage(tabId)({ currentTarget: document.getElementById(tabId)! });
}
}, [location.search]);
import { H3, H4 } from "../components/Headings";
import { BlockQuoteB } from "../components/Quotes";
import { Section } from "../components/sections";
import { useNavigation } from "../utils";
import { useTabNavigation } from "../utils/TabNavigation";
export function Judging() {
useTabNavigation();
const {goToPagesAndOpenTab} = useNavigation();
return (
<>
<div className="row">
<Section title="Overview" id="Overview">
<BlockQuoteB text="Judging for iGEM is one of the highlights on my professional calendar…" cite="Dr. Nancy Burgess, Director of Judging at iGEM HQ"/>
<p>The iGEM competition celebrates innovation in synthetic biology, offering teams the chance to compete for a range of awards based on their achievements in
various categories. Judging is based on how well teams integrate scientific rigor with societal impact, safety, and creativity. We aim to compete for
several prestigious awards, including <b>Best Integrated Human Practices</b>, <b>Safety and Security Award</b>, and <b>Best New Basic Part</b>. Additionally, we seek recognition for our <b>therapeutic project</b> and will strive for excellence in <b>Best Wiki</b>, <b>Best Presentation</b>, and the <b>iGEMers prize</b>. </p>
<div className="row align-items-center">
<div className="col">
<img src="https://static.igem.wiki/teams/5247/photos/other/therapeutic-award.svg" />
</div>
<div className="col">
<img src="https://static.igem.wiki/teams/5247/photos/other/ihp-award.svg" />
</div>
<div className="col">
<img src="https://static.igem.wiki/teams/5247/photos/other/safety-award.svg" />
</div>
<div className="col">
<img src="https://static.igem.wiki/teams/5247/photos/other/basic-part-award.svg" />
</div>
<div className="col">
<img src="https://static.igem.wiki/teams/5247/photos/other/wiki-award.svg" />
</div>
</div>
</Section>
<div className="row align-items-center">
<div className="col">
<iframe title="Bielefeld-CeBiTec: Next-Generation Prime Editing as Cystic Fibrosis Gene Therapy (2024) - Team Presentation [English]" width="560" height="315" src="https://video.igem.org/videos/embed/479e7f99-6931-47bc-9193-d4367beba4f2" frameBorder="0" allowFullScreen sandbox="allow-same-origin allow-scripts allow-popups allow-forms"></iframe>
</div>
</div>
<div className="row">
<Section title="Best Therapeutic Project" id="Best Therapeutic Project">
<p>Our project is a dual therapeutic approach targeting Cystic Fibrosis (CF), specifically the most common mutation, F508del. We aim to develop a curative solution by correcting the genetic defect using Prime Editing Technology while enhancing the folding of the CFTR protein. Additionally, we developed a Lipid Nanoparticle for the cell-specific targeting of lung epithelial cells. This project directly addresses the unmet therapeutic needs of CF patients, providing a long-term and potentially curative solution. </p>
<h4>Has the team clearly defined the therapeutic problem they are addressing? </h4>
<p>We are addressing Cystic Fibrosis (CF), a severe genetic disorder caused by mutations in the CFTR gene, particularly the F508del mutation. This mutation leads to thick mucus accumulation in vital organs, causing chronic infections and damage, particularly in the lungs. Our focus on the F508del mutation, which affects around 90 % of CF patients in Europe and beyond, ensures our project targets a well-defined and widespread therapeutic need. </p>
<h4>How well is the therapeutic mechanism of action understood and demonstrated? </h4>
<p>Our project employs a dual approach: </p>
<ol>
<li>orrecting the F508del mutation using our Prime Editing Technology PrimeGuide, a well understood gene editing tool designed to repair defective genes</li>
<li>delivering of our mRNA therapeutic via specialized Lipid Nanoparticles AirBuddy, ensuring direct application in lung epithelial cells</li>
</ol>
<h4>Has the team validated the effectiveness of their therapeutic approach? </h4>
<p>We tested the therapeutic efficacy <i>in vitro</i> using patient-derived primary epithelial cells in Air Liquid Culture (ALI) and Apical-Out Organoids carrying the F508del mutation. These cells were treated with PreCyse to assess the genetic correction, while downstream applications will validate the improved folding and function of the CFTR protein. Our experimental design allows us to gather proof-of-concept data, demonstrating the viability of both gene correction and efficient delivery. </p>
<h4>Does the project consider off-target effects or unintended consequences?</h4>
<p>By incorporating FANZOR, a new eukaryotic RNA-binding DNA-endonucleases, we enhance the precision of Prime Editing. Site-directed mutagenesis enable nickasification of our CasX and FANZOR candidates. This enables the usage as Prime Editor, while enhancing efficiency and decreasing PE complex size. We have designed our system to minimize unintended consequences, ensuring that our therapeutic intervention is safe for clinical application. </p>
<h4>What potential does the project have for real-world therapeutic applications? </h4>
<p>Our dual approach has significant potential for real-world therapeutic applications. PreCyse provides a curative genetic solution, improve immediate physiological functions. The project addresses a crucial need in CF therapy by targeting both the root genetic cause and the resulting protein dysfunction, offering a more comprehensive solution than current treatments. </p>
<h4>How well did the team integrate safety into their therapeutic design? </h4>
<p>Safety is a priority in our project. The use of FANZOR enhances the specificity of our gene-editing approach, reducing the likelihood of off-target gene modifications. Additionally, we are committed to rigorous <i>in vitro</i> testing before advancing to <i>in vivo</i> models, ensuring that our therapeutic interventions do not pose risks to patients. For this, we developed an CF-specific fluorescence-based Reporter System mimicking the CF-specific genomic alteration. </p>
<h4>To what extent has the team considered patient impact? </h4>
<p>We have closely aligned our project with patient needs by targeting the F508del mutation, which affects a significant portion of CF patients worldwide. Our therapeutic solution aims to improve quality of life by addressing the root cause of CF and reducing the burden of lifelong treatments. We are also in contact with CF patients and have gathered insights from healthcare providers to ensure our therapy is patient-centric. PreCyse offers a long-time solution, while being cost-efficient and easy-to-use. </p>
<p>Our innovative therapeutic project leverages the power of gene editing and protein modulation to offer a potentially curative solution for Cystic Fibrosis, particularly for the prevalent F508del mutation. With a well-validated scientific foundation, comprehensive safety measures, and a strong focus on patient needs, we believe our project exemplifies the qualities deserving of the <b>Best Therapeutic Project Award</b>. Our dual approach provides a template for future genetic disease treatments, paving the way for more effective and personalized medical solutions. </p>
</Section>
<Section title="Best Integrated Human Practice" id="Best Integrated Human Practice">
<p>Our team’s project at Bielefeld-CeBiTec integrates Human Practices into every aspect of our work, ensuring that our synthetic biology innovation aligns with societal needs, ethical considerations, and environmental sustainability. By proactively engaging with a broad range of stakeholders, including scientists, regulatory bodies, and community groups, we shaped our project to have meaningful and responsible impacts. Our commitment to understanding the broader implications of our work positions us as a strong candidate for the <b>Best Integrated Human Practices Award</b>. </p>
<h4>Integration of Human Practices </h4>
<p>We actively engaged with a diverse array of stakeholders throughout our project, from CF patients like <a onClick={() => goToPagesAndOpenTab('maxfirst', '/human-practices')}>Max Beckmann</a> to clinicians such as <a onClick={() => goToPagesAndOpenTab('olariu', '/human-practices')}>Dr. Olariu</a> , physiotherapists like <a onClick={() => goToPagesAndOpenTab('westhoffinv', '/human-practices')}>Katrin Westhoff</a>, and industry experts including <a onClick={() => goToPagesAndOpenTab('rnhale', '/human-practices')}>Dr. Benjamin Winkeljann</a> and <a onClick={() => goToPagesAndOpenTab('kolonkofirst', '/human-practices')}>Dr. Katharina Kolonko</a>. Each of these collaborations directly informed and shaped our therapeutic solution. Max Beckmann’s firsthand experience provided us with invaluable insights into the daily challenges of living with CF, helping us align our gene therapy with patient needs. Additionally, clinicians and physiotherapists guided us toward a treatment strategy that is practical, accessible, and effective for different patient demographics. </p>
<h4>Inspiration to Others </h4>
<p>Our project serves as a model for how synthetic biology can be leveraged to meet real-world needs while maintaining a patient-centered focus. By integrating feedback from patients and medical professionals, we demonstrate that cutting-edge science can coexist with empathy and responsibility. Our focus on inclusivity, scalability, and addressing global disparities in CF treatment sets a precedent for future iGEM teams looking to make a meaningful impact on health challenges. </p>
<h4>Documentation for Future Teams </h4>
<p>We have meticulously documented every aspect of our Human Practices integration, providing future teams with a clear roadmap for how to incorporate stakeholder feedback into a therapeutic project. This includes detailed case studies on our interactions with CF patients, medical professionals, and industry experts, along with the adjustments made to our project based on their input. Our thorough documentation ensures that others can learn from our approach and build upon our findings. </p>
<h4>Thoughtful Implementation </h4>
<p>Our project has been deeply informed by ethical, environmental, and societal considerations. For example, our consultation with Prof. Dr. Olariu emphasized the importance of mental health in CF care, leading us to incorporate psychosocial elements into our therapy design. Additionally, we aligned our solution with global healthcare disparities, ensuring our gene therapy can benefit underrepresented populations. Through engagement with regulatory bodies, we ensured that our project complies with all relevant biosafety and legal standards. </p>
<h4>Incorporation of Diverse Stakeholder Views </h4>
<p>We engaged with a broad range of stakeholders, including patients, healthcare providers, researchers, and regulatory experts, ensuring that each perspective played a role in shaping the final therapeutic solution. For instance, feedback from physiotherapist Katrin Westhoff confirmed the necessity of an inhalation-based therapy that is easy for younger patients to use. Collaborations with industry experts like Dr. Benjamin Winkeljann enabled us to optimize the technical aspects of our treatment for scalability and environmental sustainability. </p>
<h4>Creating a Responsible and Beneficial Project </h4>
<p>Our gene therapy for CF addresses not only the scientific challenges but also the societal need for accessible, patient-centered healthcare solutions. By incorporating human practices at every stage, we ensured that our project is ethically responsible and beneficial to society. The feedback from diverse stakeholders helped us refine our approach, ensuring that the solution is sustainable, inclusive, and addresses the global disparities in CF care. The long-term positive impact on the CF community—both in terms of health outcomes and accessibility—demonstrates our project’s commitment to responsible innovation. </p>
<p>Our project exemplifies the seamless integration of Human Practices into every aspect of synthetic biology research. Through collaborations with patients, healthcare professionals, and experts across different fields, we have developed a gene therapy for Cystic Fibrosis that is scientifically innovative, ethically sound, and deeply aligned with the needs of the global CF community. Our commitment to inclusivity, ethical reflection, and environmental sustainability makes us a strong candidate for the <b>Best Integrated Human Practices Award</b>, showcasing our dedication to responsible and impactful synthetic biology. </p>
</Section>
<Section title="Safety & Security" id="Safety & Security">
<p>Our project focuses on advancing biosafety and biosecurity in synthetic biology through the development and implementation of robust safety mechanisms. As part of our PreCyse project, aimed at developing a prime-editing complex to correct the F508del mutation in Cystic Fibrosis (CF), we place great emphasis on safety at every stage of research. With a commitment to responsible innovation, we have ensured that all phases of our work adhere to the highest safety standards, aiming to minimize both environmental and human health risks. Our approach incorporates novel containment systems, rigorous validation processes, and carefully planned checkpoints during experiments to push the boundaries of biosafety in synthetic biology. </p>
<h4>Contribution to Biosafety and Biosecurity </h4>
<p>We developed a comprehensive biosafety plan to minimize risks associated with our synthetic biology project. This includes biocontainment measures and protocols to prevent the accidental release of genetically modified organisms (GMOs) into the environment. By integrating both physical and genetic safeguards, we have ensured that our project contributes to safer synthetic biology applications. For example, our final construct will be tested in primary cultures of nasal epithelial cells from CF patients and healthy individuals, with carefully planned checkpoints for continuous monitoring and timely adjustments. </p>
<p>As part of our commitment to advancing safe and ethical practices in synthetic biology and patient care, we have made several key contributions to support our project and the wider community. Firstly, we developed a <b>questionnaire to evaluate the medical history of Cystic Fibrosis (CF) patients</b>, which has been specifically adapted for the collection of primary human nasal epithelial cells (hNECs). This tool ensures that essential health information is gathered in a systematic and ethical manner, aiding the accurate collection of samples while safeguarding patient well-being. </p>
<p>Secondly, we created <b>Best Practices for safe primary culture handling</b>. This guide outlines the necessary safety protocols for working with primary cell cultures, ensuring that all procedures are conducted with minimal risk of contamination and exposure to harmful pathogens. These practices promote safety for both laboratory personnel and the integrity of the biological materials.</p>
<p>Additionally, we developed a <b>Hygiene Concept for Immunocompromised Individuals</b> in consultation with a CF patient. This concept addresses the specific needs of individuals with weakened immune systems, such as those with CF, HIV, or certain cancers. It focuses on reducing health risks in high-foot-traffic public spaces, especially public restrooms, by implementing tailored hygiene measures. These guidelines aim to protect immunocompromised individuals by creating safer environments within university settings and beyond, contributing to broader public health initiatives. </p>
<h4>Characterization and Validation </h4>
<p>We rigorously tested the safety mechanisms built into our project like the disruption of the PAM sequence and pegRNA design. These mechanisms were validated through controlled experiments, ensuring reliable performance under various conditions. Furthermore, to ensure the safety and precision of our results, we introduced a series of carefully planned experimental milestones. These allow for the continuous validation of the Prime Editing complex, addressing potential issues immediately, which minimizes risk and improves the overall quality of our work. </p>
<h4>Building on Existing Knowledge </h4>
<p>Our work builds upon established biosafety frameworks, specifically improving known genetic containment systems. We incorporated lessons from previous iGEM teams and academic research to refine these tools, making them more effective in controlling gene flow and reducing unintended consequences. By drawing from the existing knowledge base, we ensured our biosafety mechanisms, such as riboswitch and PAM disruption, are both scalable and reliable, significantly advancing biosafety technologies. </p>
<h4>Risk Management </h4>
<p>From the outset, we conducted a thorough risk assessment to identify potential hazards, including those associated with handling GMOs and gene transfer. In response, we implemented stringent lab protocols and biocontainment systems to ensure the safety of our team and the surrounding environment. This risk management approach extends throughout the lifecycle of the project, from the design to the final validation stages. We also adhered to good laboratory practices, such as sterilization and controlled access, which ensures compliance with all relevant biosafety regulations. </p>
<h4>Real-World Applications </h4>
<p>We designed our project with real-world biosafety concerns in mind, particularly the potential environmental impact of GMOs. Our approach ensures that our technology can be safely applied outside the lab, with biocontainment strategies that prevent unintended release. For instance, by designing lipid nanoparticles (LNPs) that selectively fuse with lung epithelial cells, we reduce the risk of unwanted off-target interactions. Additionally, we considered dual-use concerns, ensuring our technology cannot be easily misappropriated for harmful purposes, making our project a model for responsible synthetic biology. </p>
<h4>Check-Ins and iGEM Compliance </h4>
<p>In alignment with iGEM’s emphasis on biosafety, we submitted Check-Ins for the components and organisms not covered by the iGEM White list. These formal evaluations ensured that all aspects of our project were thoroughly assessed and approved by the iGEM Safety Committee. We maintained active communication with the committee to ensure compliance with all iGEM standards, reflecting our dedication to biosafety and biosecurity. </p>
<h4>Laboratory and Safety Practices </h4>
<p>All experiments were conducted at Bielefeld University in Prof. Dr. Kristian Müller's laboratory, following BSL-1 (and BSL-2 if needed) standard operating procedures. The team participated in mandatory safety briefings and adhered to rigorous safety measures, including regulations concerning hazardous substances, genetic engineering, and the handling of biological materials. Our lab activities were meticulously planned to minimize risk and ensure data integrity. </p>
<p>By building on existing knowledge, rigorously testing our biosafety measures, and proactively managing risks, we have developed a project that significantly contributes to the field of biosafety and biosecurity. Our comprehensive approach ensures that our technology can be safely applied in real-world scenarios, and we believe this sets a new standard for responsible synthetic biology. Our efforts, particularly in the development of the Prime Editing complex for CF and the associated biosafety protocols, merit recognition for the <b>Safety and Security Award</b>. </p>
</Section>
<Section title="Best New Basic Part" id="Best New Basic Part">
<p>Our project focuses on optimizing prime editing for the Cystic Fibrosis-causing CFTR F508del mutation, which represents one of the most significant applications for prime editing. Prime editing is a precise and safe gene editing technique, but its efficiency varies greatly depending on the genomic locus. Through our development of a context-specific Prime Editor Activity Reporter (PEAR) system, we have successfully optimized this advanced technology, paving the way for more effective genomic targeting, not just for Cystic Fibrosis but for future synthetic biology applications as well. </p>
<h4> Engineering and Testing for Contextual Precision </h4>
<p>The challenges posed by low editing efficiency and the difficulty in distinguishing successful edits from background noise led us to design a highly sensitive reporter system. After an initial unsuccessful attempt using a fluorescent reporter, we shifted to the PEAR system proved to be much more adaptable and relevant to our needs. </p>
<h4>Iterative Development </h4>
<p>In our first attempt, we explored a fluorescence-based reporter that targeted GFP. While this helped us visualize prime editing in action, we quickly realized that the distance of the mutation site from the PAM sequence in the CFTR gene meant that this system wasn’t suitable for our specific genomic target. We then moved to the PEAR system, which was more flexible and sensitive, as its editing factors aligned with those required for genomic contexts. </p>
<p>Through multiple iterations, including tests in HEK293 cells, epithelial cells, and human-derived primary cells, we refined our system. Our experiments proved the successful use of the PEAR reporter in detecting prime editing activity for CFTR F508del with minimal noise, making it ideal for detecting edits in specific genomic loci with high sensitivity. </p>
<h4>Modularity and Broader Applications </h4>
<p>Recognizing the limitations of the original PEAR plasmid, especially in terms of modification flexibility and compatibility with BioBrick standards, we created a more modular and accessible system. Our new version includes an oligonucleotide-based golden gate cloning site, enabling quick modification and broader applications to other genomic targets and prime editor variants. </p>
<p>Our updated PEAR system is now compatible with RFC[1000] standards, offering future iGEM teams a versatile tool that can be applied to a wide range of editing scenarios. This is not only a significant advancement for CFTR-related research but also for any gene editing project requiring high precision and sensitivity. </p>
<h4>Contribution to the Synthetic Biology Community </h4>
<p>We believe our part contributes significantly to the synthetic biology community by providing an optimized system that can be easily adapted to various gene editing contexts. By improving the modularity and ease of use, we are confident that this part will be a valuable tool for future teams looking to optimize their own prime editing approaches. </p>
<p>Our work has demonstrated that prime editing can be made more efficient and reliable, especially in difficult-to-edit regions like CFTR. Through rigorous testing and validation, we ensured that our part meets high standards for both experimental reliability and community utility. As a result, we position ourselves as strong candidates for the <b>Best New Basic Part Award</b>.</p>
</Section>
<Section title="Conclusion" id="Conclusion">
<p>In conclusion, our project exemplifies the best of synthetic biology, combining cutting-edge science with ethical responsibility and a deep
commitment to societal impact. By integrating human practices into every stage of our work, ensuring the highest standards of biosafety, and contributing
valuable tools to the community, we have set a new standard for innovation in the field. Our therapeutic approach for Cystic Fibrosis has the potential
to revolutionize treatment for this life-threatening condition, while our contributions to biosafety and part development will benefit the broader
synthetic biology ecosystem. We are confident that our project is deserving of recognition in multiple award categories, including <b>Best Therapeutic Project</b>, <b>Best Integrated Human Practices</b>, <b>Safety and Security Award</b>, and <b>Best New Basic Part</b>. Through our work, we hope to inspire future iGEM teams to pursue solutions that are both scientifically excellent and socially responsible. </p>
</Section>
<Section title="Judging Session" id="Judging Session">
<div className="row align-items-center">
<div className="col">
<iframe title="Bielefeld-CeBiTec: Judging Session (2024)" width="560" height="315" src="https://video.igem.org/videos/embed/d90ef3d2-1bb5-426e-8ab7-8e644d1a22a5" frameBorder="0" allowFullScreen sandbox="allow-same-origin allow-scripts allow-popups allow-forms"></iframe>
</div>
</div>
<H3 text="Judging Feedback" id="Judging Session1"></H3>
<H4 text="Judge 1"></H4>
<div className="row feedbackbfh">
<div className="col b-lg">
<ul>
<li>
I could see engineering principles and mindsets even in the presentation video. It is a very well-engineered and designed project. Every progress started with some hypotheses. The team tested many hypotheses; many didn’t work but they were able to learn and improve.
</li>
<li>
Documentation is very detailed in general. Specifically, the project description comes with reviews of cystic fibrosis and some basic terms so that anyone with basic molecular biology knowledge would be able to understand the project. The graphics, where there are, also facilitate understanding of technology.
</li>
<li>
The human practices are amazing, significantly influencing key project decisions. They went beyond obvious issues (e.g., how health insurance might cover gene therapies, ethics associated with handling patient-derived cells, biobank), showing compelling evidence that it is responsible and good. The same can be said on the team’s safety work.
</li>
</ul>
</div>
<div className="col b-lo">
<ul>
<li>
I don’t see how the proposed new basic part BBa_K5247135 can be useful for the wider community. It is designed with a very specific purpose in mind, of which the characterization is also not done in calibrated units. Basically, one can test different pegRNAs for the F508del mutation in the CFTR gene with fluorescence of the internal positive control arbitrarily set as 100%.
</li>
<li>
I appreciate the extensive documentation of human practices. The way different feedback or research was incorporated is clear but the rationale for the stakeholders chosen is not always clear. It becomes sort of a laundry list of everything that affected the team with too many sections and subtitles.
</li>
<li>
I’m impressed by the fact that the team has access to some sophisticated technology like cryogenic electron microscopy but it’s not clear why these characterizations are necessary, i.e., what the research question is. In fact, the team would then observe various problems associated with these methods. For example, “for SEM analysis, the samples were dried and observed under vacuum, which probably have affected the structure and shape of the LNPs.” If this is the case, then why would the team do it from the beginning?
</li>
<li>
The contributions of the safety work (i.e. primary cultures, biosafety measures, innovative safety mechanisms and regulatory compliance), particularly to what extent they build on existing resources and standards, particularly those from the iGEM community, are not very clear.
</li>
</ul>
</div>
</div>
<H4 text="Judge 2"></H4>
<div className="row feedbackbfh">
<div className="col b-lg">
<p>I enjoyed browsing through the Wiki, very clear and easy to understand and consume. Commendable demonstration of human practices and good integration of feedback. The Project Presentation is professionally done, enjoyable to watch. I highly encourage the tactic of team members walking around and engaging with judges/other teams. I loved the confidence. Wiki has mobile version, which is well appreciated. </p>
</div>
<div className="col b-lo">
<p>I enjoyed the Calendar feature in Project Documentation, but the dates did not always work. There are still some Lorem Ipsum paragraphs remaining (Results, for instance). Visuals in the Registry part are too big and are not well visible.</p>
</div>
</div>
<H4 text="Judge 3"></H4>
<div className="row feedbackbfh">
<div className="col b-lg">
<p>Very well done team Bielefed-CeBiTec. I thought that your project design was very well thought out and was able to clearly see the impact on quality of life that your project would help restore in CF patients. Over the course of this past year, you have done an impressive amount of work, and I think tailoring your project to not only focus on accuracy of your guide RNA but also ensuring the best possible delivery mechanisms to ensure cell uptake was a smart move if you were to track this for market. I also appreciated just how thorough your wiki presents your work, and the amount of effort put into your human practices, engaging with not only members of academia and healthcare, but actual people living with CF. Each page sufficiently attributed and cited original ideas, and the flow from top to bottom of the page made logical sense and made my life as a judge easier. Well done overall, and best of luck as you continue working with this!</p>
</div>
<div className="col b-lo">
<p>There were a couple of notes that stood out to me regarding your wiki and promotional video that I would like to highlight for next year's team. With your individual part designs and part pages, I did not see sufficient data within the part page itself to get excited about the work and how the part can help future iGEMers. Based on your wiki, I know you did a lot of testing and have the capacity to demonstrate part success; however, remember to present your research items in a way that is mindful to someone who is outside of your experimental realm. Ask yourself the following questions: 1) What specifically was successful about my part design? 2) If quantifying data and showing that data in another means (e.g. microscopy images) do these images match? 3) What caveats and troubleshooting would be necessary to bring this part further? Another note, and it is minor, but in your human practices and promotion of the BFH European meetup, you bring attention to connections and participation by two high-profile iGEM members (Nemanja - VP, Tracy - Former Director of Judging). While it is okay to demonstrate their attendance (using videos of them, describing what they do), it feels borderline like favoritism in that they were showing up to your event, in particular with you highlighting their attendance, not necessarily what they presented on and how it impacted you and the other teams in attendance. Be mindful of this going forward.</p>
</div>
</div>
<H4 text="Judge 4"></H4>
<div className="row feedbackbfh">
<div className="col b-lg">
<p>Your project is very good; I like the approach, as we are used to seeing more invasive approaches in medicine. Your project was also very well defended; you answered the questions confidently, which caught my attention. You never hesitated. It was a lot of work, and you managed to shape it well. I hope you can continue with the project and turn it into a startup</p>
</div>
<div className="col b-lo">
<p>It would be useful to delve into the potential challenges related to the biocompatibility and degradability of chitosan nanoparticles in the human body, as ensuring long-term safety and efficacy is essential for therapies of this kind. It would also be interesting to explore methods and experiments to further improve the stability of the nanoparticles, evaluate different pH conditions, and determine which peripheral cells or tissues could be harmed or could benefit. Let's remember that the human body is not an isolated system; many of these interactions may lead to allergies, unwanted responses, or affect the organism's suitability. Cultivating peripheral tissues with the same system would also be a very intriguing approach.</p>
</div>
</div>
<H4 text="Judge 5"></H4>
<div className="row feedbackbfh">
<div className="col b-lg">
<p>You demonstrated a strong commitment to biosafety by outlining detailed safety protocols and addressing both environmental and human health risks. Your proactive approach to identifying potential hazards and implementing safety measures highlights your responsibility in managing synthetic biology risks. Great work!</p>
</div>
<div className="col b-lo">
<p>Your Materials and Methods section provides a solid foundation of your technical work, but could benefit from more detailed explanations of some experimental design choices. Additionally, including a section that discusses challenges encountered and how you have addressed it would offer valuable insights for future teams attempting to replicate or build on yourwork. This would also help illustrate the adaptability and problem-solving aspects of your project</p>
</div>
</div>
<H4 text="Judge 6"></H4>
<div className="row feedbackbfh">
<div className="col b-lg">
<p>The integration of Prime Editing and the AirBuddy delivery system showcases a cutting-edge approach to gene therapy. This not only demonstrates technical innovation but also highlights your commitment to improving treatment efficacy and precision.</p>
</div>
<div className="col b-lo">
<p>While the therapy aims for long-term correction, discussing how you plan to evaluate durability and stability of the genetic correction over time could provide a clearer understanding of the treatment’s potential benefits.</p>
</div>
</div>
</Section>
</>
);
}
src/contents/measurement.tsx deleted 100644 → 0
View file @ 9143d0b0
import { useEffect } from "react";
import { useLocation } from "react-router-dom";
import { openFromOtherPage } from "../components/Buttons";
export function Measurement() {
const location = useLocation();
useEffect(() => {
const params = new URLSearchParams(location.search);
const collapseId = params.get('collapseId');
const tabId = params.get('tab');
// Scroll to the section specified by collapseId
if (collapseId) {
const collapseElement = document.getElementById(collapseId);
if (collapseElement) {
const elementTop = collapseElement.getBoundingClientRect().top + window.pageYOffset;
const offset = window.innerHeight / 2 - collapseElement.offsetHeight / 2;
const scrollPosition = elementTop - offset;
window.scrollTo({
top: scrollPosition,
behavior: 'smooth',
});
}
}
// Open the tab specified by tabId
if (tabId) {
openFromOtherPage(tabId)({ currentTarget: document.getElementById(tabId)! });
}
}, [location.search]);
return (
<>
<div className="row">
<div className="col">
</div>
</div>
<div className="row">
</div>
</>
);
}
\ No newline at end of file
src/contents/methods.tsx 0 → 100644
View file @ 3fd292fd
import { Section, Subesction } from "../components/sections";
import { useTabNavigation } from "../utils/TabNavigation";
import {H4} from "../components/Headings";
import MethodSources from "../sources/methods-sources";
import { useNavigation } from "../utils";
import { OneFigure } from "../components/Figures";
export function Methods() {
const {goToPagesAndOpenTab} = useNavigation ();
useTabNavigation();
return (
<>
<Section title="Introduction" id="Introduction">
<p>This section highlights the key materials and methods pivotal to advancing our project with the primary goal to develop an efficient prime editing technology to correct the F508del mutation in the CFTR gene by the delivery to lung epithelial cells using optimized lipid nanoparticles (LNPs) via pulmonary administration. We utilized patch clamp electrophysiology to precisely measure ion channel activity, providing crucial insights into cellular function and the impact of genetic modifications on CFTR performance. Additionally, our cell culture models of lung epithelial cells allowed us to test both the delivery and efficacy of our gene-editing system under conditions that closely mimic the <i>in vivo</i> environment. To ensure that our LNPs were both effective and safe, we performed extensive LNP cytotoxicity and characterization experiments, evaluating their biocompatibility, stability, and efficiency in delivering the editing technology. Each of these methodologies was carefully selected to optimize the delivery process and maximize the therapeutic potential of our approach.</p>
</Section>
<Section title="Patch Clamp" id="Patch Clamp">
<Subesction title="Patch Clamp: A Key Tool in Electrophysiology" id="Patch Clamp1">
<p>The patch clamp technique is a highly sensitive method for measuring ionic currents through individual ion channels in cells, making it a cornerstone of electrophysiological research. Initially developed by Erwin Neher and Bert Sakmann in the 1970s [1], this technique has evolved into various configurations, including the Whole-Cell and Single-Channel recordings [2], which provide critical insights into the functional properties of ion channels. </p>
</Subesction>
<Subesction title="Principles of the Patch Clamp Technique" id="Patch Clamp2">
<p>Patch clamp recording involves the use of a glass micropipette which is manufactured from a glass capillary through the use of a Micropipette Puller. The micropipette is then filled with an electrolyte solution, which is subsequently brought into contact with the cell membrane. By applying gentle suction, a high-resistance seal called giga seal is formed between the pipette tip and the membrane patch. This enables the measurement of ionic currents with minimal noise interference [3]. <strong>Whole-Cell Configuration</strong> records currents from the entire cell by rupturing the membrane patch, accessing the intracellular environment, and is useful for analysing overall ion channel activity and cellular responses. <strong>Single-Channel Recording</strong> measures currents through individual ion channels without rupturing the membrane, enabling high-resolution study of channel conductance, gating, and selectivity [2].</p>
<div className="figure-wrapper">
<figure>
<div className="row align-items-center">
<div className="col">
<iframe title="Bielefeld-CeBiTec: Patch Clamp Measurement (2024)" width="560" height="315" src="https://video.igem.org/videos/embed/0d948e57-5997-430a-a2df-815b71a2fc67?autoplay=1" frameBorder="0" allowFullScreen={true} sandbox="allow-same-origin allow-scripts allow-popups allow-forms"></iframe>
</div>
</div>
<figcaption> <b>Figure 1. </b> Microscopic recording of micropipette sealing of a HEK293 cell. </figcaption>
</figure>
</div>
<p>The success of patch clamp experiments heavily depends on the composition of the solutions used. Typically, two main types of solutions are employed: The <strong>Pipette Solution</strong> in the micropipette mimics the intracellular environments, while the <strong>Bath Solution</strong> surrounds the cell and usually contains components that replicate the extracellular environment. Both solutions are meticulously designed to reflect the physiological conditions under which the cells operate, thereby ensuring that the measurements accurately reflect ion channel activity in a natural setting [2].</p>
<OneFigure
pic1="https://static.igem.wiki/teams/5247/photos/for-wiki-texts/meth-patch-clamp/bild-meth-patch-clamp.png"
alt1="Patch Clamp Setup."
description="Patch Clamp setup."
num={2}
/>
</Subesction>
<Subesction title="Application in CFTR gene Prime Editing validation" id="Patch Clamp3">
<p>In our ongoing research project focusing on the treatment of Cystic Fibrosis, our patch clamp measurements, performed in collaboration with Dr. Oliver Dräger from the Cellular Neurophysiology working group at Bielefeld University, serve as a powerful validation tool for the assessment of the functional correction of the CFTR gene, particularly the common F508del mutation, via prime editing. The patch clamp technique can be employed in this context to measure the resulting chloride ion channel activity which is altered by the mutation [4]. Whole-Cell recordings were performed to assess whether the corrected CFTR channels function similarly to those in healthy cells. If the chloride ion currents in the edited cells approach levels of healthy cells, this would strongly suggest successful gene editing and validate the functionality of our therapeutic approach.</p>
</Subesction>
</Section>
<Section title="Cell Culture" id="Cell Culture">
<Subesction title="HEK293 and HEK293T cell lines" id="Cell Culture1">
<p>For testing our prime editing approach, we needed an easy-to-handle cell line with a measurable high expression of CFTR and the CFTR F508del mutation. When talking to Mattijs Bulcaen from the Laboratory of Molecular Virology and Gene Therapy at KU Leuven, he recommended to use HEK293T cell lines overexpressing CFTR they had used. HEK293 cells are a very common immortalized human cell line derived from the kidneys of a female embryo. They are particularly suited to research due to their convenient handling and transfection properties. Basic HEK293 cells were provided to us by the Cellular and Molecular Biotechnology working group at Bielefeld University led by Prof. Dr. Kristian Müller, who is also one of the Principal Investigators of our team. HEK293T cells express an additional tsA1609 allele of the SV40 large T-antigen, allowing for replication of vectors containing the SV40 origin of replication[5]. Besides the native CFTR gene, which is not expressed in HEK cells, the HEK293T cell lines used in Leuven carry another copy of the gene embedded in an expression cassette. The cassette includes a CMV promoter, which is a standard promoter used for gene overexpression in human cells derived from the human Cytomegalovirus[6], as well as a puromycin resistance co-expressed with the CFTR allowing for continuous selection of CFTR expressing cells. The whole construct was stably inserted into the genome using lentiviral transduction[7][8]. </p>
<OneFigure
pic1="https://static.igem.wiki/teams/5247/photos/for-wiki-texts/meth-used-cells/mikroskopie-hek293t.png"
alt1="Phase contrast image of HEK293T at 20x magnification."
description="Phase contrast image of HEK293T at 20x magnification."
num={3}
bg="white"
/>
</Subesction>
<Subesction title="CFBE41o- cell line" id="Cell Culture2">
<p>The CFBE41o- cell line, derived from bronchial epithelial cells of a one-year-old Cystic Fibrosis patient, serves as a vital model for studying Cystic Fibrosis. These cells closely mimic the physiological environment of the airway epithelium, allowing for more accurate studies on how CFTR mutations affect cell function and response to treatments. They were immortalized through calcium-phosphate-mediated transfection using a replication-defective pSVori plasmid that carries the simian virus 40 large T-antigen (SV40-LT). The plasmid's defective origin of replication prevents viral propagation, thus preserving essential physiological characteristics of the cells while enabling them to develop differentiated morphologies. CFBE41o- cells are homozygous for the F508del CFTR mutation [9]. We are happy we got this cell line with permission from <a onClick={() => goToPagesAndOpenTab('ignatova', '/human-practices')}>Prof. Dr. Ignatova</a>, who is leader of a working group at the Institute for Biochemistry and Molecular Biology of Hamburg University and an iGEM supporter since a long time [10]. </p>
</Subesction>
<Subesction title="Human nasal epithelial cells (hNECs)" id="Cell Culture3">
<p>Human nasal epithelial cells were obtained by nasal brushing, a minimally invasive method. These cells function/act as primary cultures. Cultivated in air-liquid interface (ALI) cultures and apical-out airway organoids (AOAO), they serve as a suitable model to visualise the functional epithelium of the airways in a differentiated form. The <i>in vivo</i> aspects of an airway disease, such as CF, can be modelled using donors with those airway diseases [11]. This model is therefore particularly suitable for testing our prime editing complex. </p>
<div className="figure-wrapper">
<figure>
<div className="row align-items-center">
<div className="col">
<iframe title="Bielefeld-CeBiTec: ALI cell culture (2024) [English]" width="500" height="315" src="https://video.igem.org/videos/embed/ff557f5a-94be-45e6-90ca-0affa14423e3?autoplay=1&amp;muted=1" frameBorder="0" allowFullScreen={true} sandbox="allow-same-origin allow-scripts allow-popups allow-forms"></iframe>
</div>
<div className="col">
<iframe title="Bielefeld-CeBiTec: AOAO cell culture (2024) [English]" width="500" height="315" src="https://video.igem.org/videos/embed/058d83cf-ab09-476e-9ab2-30cd114fbc0c?autoplay=1&amp;muted=1" frameBorder="0" allowFullScreen={true} sandbox="allow-same-origin allow-scripts allow-popups allow-forms"></iframe>
</div>
</div>
<figcaption>
<div className="row align-items-center">
<div className="col">
<b>Figure 4.</b> ALI cultures of hNECs: The active cilia beat frequency of differentiated human nasal epithelial cells (hNECs) in air-liquid interface (ALI) culture is visible. This ciliary movement is crucial for mucociliary transport, which contributes to the clearance of particles and pathogens in the respiratory tract.
</div>
<div className="col">
<b>Figure 5.</b> Apical-Out Airway Organoid (AOAO) culture: Visible apical-out airway organoids in action. These 3D structures, which mimic the airway epithelium, allow detailed study of cellular processes such as mucociliary transport and secretory activities, in which cilia and vesicles play a key role.
</div>
</div>
</figcaption>
</figure>
</div>
</Subesction>
</Section>
<Section title="LNPs" id="LNPs">
<Subesction title="Cytotoxicity Tests" id="Cytotoxicity Tests">
<H4 text="Assessing the Safety of Our LNPs "></H4>
<p>Ensuring the safety and thorough characterization of our LNPs was a central part of our project, as these particles are intended for use in biological systems. We implemented a comprehensive range of assays and techniques to assess their biosafety and physical properties, ensuring their suitability for applications such as drug delivery and gene therapy. Below is an overview of the key steps we took in our assessment.</p>
<H4 text="MTT Assay"></H4>
<div className='row align-items-center'>
<div className='col'>
<OneFigure
pic1="https://static.igem.wiki/teams/5247/integrated-human-practices/mttassay.webp"
alt1="PC1"
description="MTT Assay: Formation of purple formazan crystals by living cells."
num={6}
/>
</div>
<div className='col'>
<p>To evaluate the cytotoxicity of our LNPs, we conducted an MTT assay, which measures the metabolic activity of cells. This assay is based on the ability of living cells to reduce MTT, a yellow tetrazolium salt, into purple formazan crystals through NAD(P)H-dependent enzymes. Cells were treated with various concentrations of LNPs, and after dissolving the formazan crystals with DMSO, we measured absorbance. Higher absorbance values indicate greater cell viability. Our results showed no significant reduction in cell viability across all LNP concentrations, demonstrating that the LNPs did not induce cytotoxic effects. This finding is crucial for ensuring that the LNPs are safe for biological use, supporting their potential in clinical applications such as drug delivery and gene therapy. Overall, the MTT assay provided strong evidence of the biocompatibility of our LNPs. </p>
</div>
</div>
<H4 text="Proliferation Assay to Monitor Long-Term Safety"></H4>
<p>In addition to assessing immediate cytotoxicity, we also evaluated the long-term safety of the LNPs by conducting a proliferation assay. This assay tracked cell division and growth over time to determine whether the LNPs impacted cellular function. Our results showed that LNP-treated cells had similar growth rates to untreated controls, indicating that the LNPs do not interfere with normal cell processes. This further confirms their biocompatibility and suitability for use in biological systems.</p>
</Subesction>
<Subesction title="Flow Cytometry" id="flow cytometry">
<p>To assess the transfection efficiency of our LNPs, we used flow cytometry. This method involved tagging the LNPs with fluorescent markers and measuring their ability to deliver genetic material into target cells. The flow cytometry results provided quantitative insights into how effectively the LNPs transfected cells, helping us optimize their design for gene therapy applications. </p>
</Subesction>
<Subesction title="In-Depth Characterization of LNPs" id="In-Depth Characterization of LNPs">
<H4 text="Dynamic Light Scattering (DLS) and Zeta Potential"/>
<p>The hydrodynamic radius (𝑅𝐻) of the vesicles and LNPs was determined through angle-dependent photon correlation spectroscopy (PCS) at 𝑇=20°C. Samples were measured in NMR tubes using a 3D LS Spectrometer Pro (LS Instruments, Fribourg, Switzerland), which was equipped with a HeNe Laser (632.8 nm, 1145P; JDSU, Milpitas, CA, USA), a decaline index-matching vat, an automated goniometer, and two detectors. Measurements were performed in a 3D cross-mode to eliminate multiple scattering effects, covering a scattering angle range of 30° to 120° in increments of 10°, with a measuring time of three intervals of 120 s per angle. The autocorrelation function of the scattered light intensity was generated using a multiple-τ digital correlator and analyzed via inverse Laplace transformation (CONTIN) to determine the mean relaxation rate (Γ). From these data, the hydrodynamic radius (𝑅𝐻) was calculated using the Stokes–Einstein equation:
𝑅𝐻=𝑘𝐵⋅𝑇/6𝜋𝜂𝐷𝑇 where 𝑘𝐵 is the Boltzmann constant, T is the temperature, η is the solvent viscosity, and DT
is the translational diffusion coefficient. The value of 𝐷𝑇 was obtained from the slope of the linear relationship between the relaxation rate (Γ) and
the squared magnitude of the scattering vector (𝑞2) as defined by:Γ =𝐷𝑇⋅𝑞2Γ.
The viscosity of water was calculated based on the temperature to provide accurate measurements for the given conditions.
To complement the PCS analysis, dynamic light scattering (DLS) was used to determine the size distribution and polydispersity index (PDI) of the LNPs. DLS measurements confirmed that the LNPs had a consistent size distribution with minimal aggregation, which is crucial for their stability and effectiveness. Furthermore, we assessed the zeta potential of the LNPs to evaluate their surface charge. A high zeta potential value indicated that the LNPs were stable in suspension, a necessary condition for maintaining their functionality in biological environments.
Overall, the combination of PCS, DLS, and zeta potential measurements provided a comprehensive characterization of the LNPs, confirming their hydrodynamic properties, stability, and suitability for drug delivery applications. </p>
<div className='row align-items-center'>
<div className='col'>
<OneFigure
pic1="https://static.igem.wiki/teams/5247/photos/lab/dls-methods.webp"
alt1="Dynamic Light Scattering (DLS) measurement set-up."
description="Dynamic Light Scattering (DLS) measurement setup."
num={7}
/>
</div>
</div>
<H4 text="SEM and Cryo-EM for Structural Analysis"></H4>
<div className='row align-items-center'>
<div className='col'>
<p>For the cryogenic electron microscopy (Cryo-EM) analysis, samples were vitrified on holey carbon TEM grids (Lacey Carbon Film coated, 200 Mesh; Science Services, München, Germany) using a Leica blotting and plunging device (Leica EM GP, Leica Mikrosysteme Vertrieb GmbH, Wetzlar, Germany). The grids were rapidly plunged into liquid ethane cooled by liquid nitrogen to ensure sufficiently fast cooling. After vitrification, the grids were transferred to a cryo transfer and tomography holder (Fischione Model 2550, E.A. Fischione Instruments, Pittsburgh, USA).
TEM images were acquired using a JEOL JEM-2200FS electron microscope (JEOL, Freising, Germany) equipped with a cold field emission electron gun, operated at an acceleration voltage of 200 kV. All images were captured digitally using a bottom-mounted camera (Gatan OneView, Gatan, Pleasanton, USA) and processed with a digital imaging processing system (Digital Micrograph GMS 3, Gatan, Pleasanton, USA).
In addition to Cryo-EM, we employed scanning electron microscopy (SEM) to further characterize the morphology and surface structure of the LNPs. SEM provided high-resolution images that confirmed the spherical shape and uniformity of the LNPs.</p>
</div>
<div className='col'>
<OneFigure
pic1="https://static.igem.wiki/teams/5247/delivery/plasmatem.webp"
alt1="PC1"
description="Sample preparation for SEM: Sputtering in Argon plasma."
num={8}
/>
</div>
</div>
</Subesction>
<Subesction title="Conclusion" id="Conclusion">
<H4 text="Importance of Safety in LNP Development"></H4>
<p>Testing the safety of our LNPs was a critical step in their development. LNPs are increasingly being used in cutting-edge therapies, such as mRNA vaccines and targeted drug delivery systems. For these technologies to be viable, the nanoparticles must not harm the cells they are intended to interact with. The MTT and proliferation assays provided robust data, confirming the biocompatibility of our LNPs and reinforcing their potential for safe use in further research and clinical applications. </p>
</Subesction>
</Section>
<Section title="References" id="References">
<MethodSources/>
</Section>
</>
);
}
\ No newline at end of file
src/contents/notebook.tsx
View file @ 3fd292fd
import { useEffect } from "react";
import { useLocation } from "react-router-dom";
import { openFromOtherPage } from "../components/Buttons";
export function Notebook() {
const location = useLocation();
useEffect(() => {
const params = new URLSearchParams(location.search);
const collapseId = params.get('collapseId');
const tabId = params.get('tab');
// Scroll to the section specified by collapseId
if (collapseId) {
const collapseElement = document.getElementById(collapseId);
if (collapseElement) {
const elementTop = collapseElement.getBoundingClientRect().top + window.pageYOffset;
const offset = window.innerHeight / 2 - collapseElement.offsetHeight / 2;
const scrollPosition = elementTop - offset;
window.scrollTo({
top: scrollPosition,
behavior: 'smooth',
});
}
}
// Open the tab specified by tabId
if (tabId) {
openFromOtherPage(tabId)({ currentTarget: document.getElementById(tabId)! });
}
}, [location.search]);
import { DownloadImageButton } from "../components/Buttons";
import H2 from "../components/Headings";
import { useTabNavigation } from "../utils/TabNavigation";
export function Notebook() {
useTabNavigation();
return (
<>
<div className="row mt-4">
<div className="col-lg-8">
<div>
<H2 text="Lab Journals and Protocol collection" id="notebookH"/>
<div className="eng-box box" >
<p>Here you can have a detailed look at our lab work - just klick the front pages of the Lab Journals and Protocol Collection to download them!
</p>
</div>
<p></p>
<div className='row'>
<div className="col">
<DownloadImageButton url="https://static.igem.wiki/teams/5247/pdfs/laboratory-notebook-1-proof-of-concept-for-pe.pdf" fileName="laboratory-notebook-1-proof-of-concept-for-pe.pdf">
<img src="https://static.igem.wiki/teams/5247/lab-journals/titelseite-lab-book-1-proof-of-concept-pe.webp" style={{height: "75%", width: "auto"}}/>
</DownloadImageButton>
</div>
<div className="col">
<DownloadImageButton url="https://static.igem.wiki/teams/5247/pdfs/laboratory-notebook-2-engineering-our-prime-editing-tool-primeguide.pdf" fileName="laboratory-notebook-2-engineering-our-prime-editing-tool-primeguide.pdf">
<img src="https://static.igem.wiki/teams/5247/lab-journals/titelseite-lab-book-2-engineering-pe.webp" style={{height: "75%", width: "auto"}}/>
</DownloadImageButton>
</div>
</div>
</>
<div className='row'>
<div className="col">
<DownloadImageButton url="https://static.igem.wiki/teams/5247/pdfs/lab-book-3-primary-cultures.pdf" fileName="lab-book-3-primary-cultures.pdf">
<img src="https://static.igem.wiki/teams/5247/lab-journals/titelseite-lab-book-3-primary-cell-culture.webp" style={{height: "75%", width: "auto"}}/>
</DownloadImageButton>
</div>
<div className="col">
<DownloadImageButton url="https://static.igem.wiki/teams/5247/pdfs/lab-book-4-lnp-design-airbuddy.pdf" fileName="lab-book-4-lnp-design-airbuddy.pdf">
<img src="https://static.igem.wiki/teams/5247/lab-journals/titelseite-lab-book-4-lnp.webp" style={{height: "75%", width: "auto"}}/>
</DownloadImageButton>
</div>
</div>
<div className='row'>
<div className="col">
<DownloadImageButton url="https://static.igem.wiki/teams/5247/pdfs/lab-book-5-downstream-experiments.pdf" fileName="lab-book-5-downstream-experiments.pdf">
<img src="https://static.igem.wiki/teams/5247/lab-journals/titelseite-lab-book-5-downstream.webp" style={{height: "75%", width: "auto"}}/>
</DownloadImageButton>
</div>
<div className="col">
<DownloadImageButton url="https://static.igem.wiki/teams/5247/pdfs/protocol-collection-igem-2024.pdf" fileName="protocol-collection-igem-2024.pdf">
<img src="https://static.igem.wiki/teams/5247/lab-journals/titelseite-lab-book-sop.webp" style={{height: "75%", width: "auto"}}/>
</DownloadImageButton>
</div>
</div>
</div>
);
}
src/contents/partners.tsx
View file @ 3fd292fd
import { useEffect } from "react";
import { useLocation } from "react-router-dom";
import { openFromOtherPage } from "../components/Buttons";
import { BackUp } from "../components/Buttons";
import H1, { H2 } from "../components/Headings";
import { useTabNavigation } from "../utils/TabNavigation";
export function Partners() {
const location = useLocation();
useEffect(() => {
const params = new URLSearchParams(location.search);
const collapseId = params.get('collapseId');
const tabId = params.get('tab');
// Scroll to the section specified by collapseId
if (collapseId) {
const collapseElement = document.getElementById(collapseId);
if (collapseElement) {
const elementTop = collapseElement.getBoundingClientRect().top + window.pageYOffset;
const offset = window.innerHeight / 2 - collapseElement.offsetHeight / 2;
const scrollPosition = elementTop - offset;
window.scrollTo({
top: scrollPosition,
behavior: 'smooth',
});
}
}
// Open the tab specified by tabId
if (tabId) {
openFromOtherPage(tabId)({ currentTarget: document.getElementById(tabId)! });
}
}, [location.search]);
export function Partners() {
useTabNavigation();
return (
<>
<div id="sidebarbox" className="col-1 d-none d-lg-block"> </div>
<div id="sidebarbox" className="col-1 d-lg-block"> </div>
<br/>
<h1>A big thank you to all our sponsors and partners!</h1>
<H1 text="A big thank you to all our sponsors and partners!"></H1>
<br/>
<br/>
<br/>
{/* GOLD */}
<h2>Sponsor category Gold</h2>
<H2 id="gold" text="Sponsor category Gold"/>
<br/>
<div className="col">
<div className="row align-items-center">
<div className="col">
<div className="col">
<a className="sponsor-container sp-a" href="https://www.uni-bielefeld.de/">
<img className="img-sponsor-a" src="https://static.igem.wiki/teams/5247/logos-team/uni-bielefeld-dunkel.png"/>
</a>
......@@ -65,16 +39,19 @@ export function Partners() {
</div>
<br/>
<div className="row align-items-center">
<div id="zeiss-portrait" className="col-5 sponsor-portrait">
<a href="http://www.zeiss.de/naturwissenschaften">
<img id="zeiss-portrait-logo" src="https://static.igem.wiki/teams/5247/sponsors/zeiss.png"></img>
</a>
<div className="col-5 sponsor-portrait">
<a href="http://www.zeiss.de/naturwissenschaften">
<div id="zeiss-portrait" className="col">
<img id="zeiss-portrait-logo" src="https://static.igem.wiki/teams/5247/sponsors/zeiss.png"></img>
</div>
</a>
</div>
<div id="zeiss-text" className="col sponsor-text-right">
<h4>ZEISS ist Technologie. ZEISS ist Optik und Innovation.</h4>
<br/>
<p> Wir entwickeln, fertigen und vertreiben für unsere Kunden in einer Vielzahl von Geschäftsfeldern hochinnovative Produkte und Lösungen – und loten dabei die Grenzen des Machbaren aus. Als weltweit führendes Technologieunternehmen, mit einer starken Marke und mit einem Portfolio, das auf Wachstumsfelder der Zukunft wie Digitalisierung, Gesundheit und Industrie 4.0 ausgerichtet ist, gestalten wir die Zukunft weit über die optische und optoelektronische Branche hinaus. Grundlage für den Erfolg und den weiteren kontinuierlichen Ausbau der Technologie und Marktführerschaft von ZEISS sind die nachhaltig hohen Aufwendungen für Forschung und Entwicklung. Hauptstandort des 1846 in Jena gegründeten Unternehmens ist Oberkochen, Deutschland. Alleinige Eigentümerin der Dachgesellschaft, der Carl Zeiss AG, ist die Carl-Zeiss-Stiftung, eine der größten deutschen Stiftungen zur Förderung der Wissenschaft.</p>
</div>
<p>We develop, manufacture and sell highly innovative products and solutions for our customers in a wide range of business areas - and in doing so, we push the boundaries of what is possible. As a leading global technology company with a strong brand and a portfolio that is geared towards future growth areas such as digitalization, healthcare and Industry 4.0, we are shaping the future far beyond the optical and optoelectronic sector. ZEISS' success and the continued expansion of its technology and market leadership are based on its sustained high level of investment in research and development. Founded in Jena in 1846, the company is headquartered in Oberkochen, Germany. The sole owner of the parent company, Carl Zeiss AG, is the Carl Zeiss Foundation, one of the largest German foundations for the promotion of science.</p>
</div>
</div>
<br/> <br/>
<div className="row align-items-center">
......@@ -113,59 +90,63 @@ export function Partners() {
</div>
<br/> <br/>
{/* SILVER */}
<h2>Sponsor category silver</h2>
<H2 text="Sponsor category silver" id="silver"/>
<br/> <br/>
<div id="project-portrait" className="sponsor-portrait" style={{padding: "20px"}}>
<div className="row align-items-center">
<div className="col">
<a className="sponsor-container" href="">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/zymo.png"/>
<a className="sponsor-container" href="https://www.zymoresearch.com/">
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/zymo.png"/>
</a>
</div>
<div className="col">
Stemcell
<a className="sponsor-container" href="https://www.stemcell.com/">
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/stemcell-logo.png"/>
</a>
</div>
<div className="col">
<a className="sponsor-container" href="https://www.plasmidfactory.com/">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/plasmidfactory.png"/>
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/plasmidfactory.png"/>
</a>
</div>
</div>
<div className="row align-items-center">
<div className="col">
Wolff
<div className="col">
<a className="sponsor-container" href="https://www.drwolffgroup.com/en/">
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/logo-wolff.png"/>
</a>
</div>
<div className="col">
<a className="sponsor-container" href="https://snapgene.com">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/snapgene.png"/>
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/snapgene.png"/>
</a>
</div>
<div className="col">
<a className="sponsor-container" href="">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/bionrw-logo.png"/>
<a className="sponsor-container" href="https://bio.nrw.de/">
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/bionrw-logo.png"/>
</a>
</div>
</div>
</div>
<br/> <br/>
{/* BRONZE */}
<h2>Sponsor category bronze</h2>
<H2 text="Sponsor category bronze" id="bronze"></H2>
<br/> <br/>
<div id="project-portrait" className="sponsor-portrait" style={{padding: "20px"}}>
<div className="row align-items-center">
<div className="col">
<a className="sponsor-container" href="www.promega.com">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/promega-gelb.png"/>
<a className="sponsor-container" href="https://www.promega.com">
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/promega-gelb.png"/>
</a>
</div>
<div className="col">
<a className="sponsor-container" href="">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/microsynth-logo.png"/>
<a className="sponsor-container" href="https://www.microsynth.com">
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/microsynth-logo.png"/>
</a>
</div>
<div className="col">
<a className="sponsor-container" href="">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/neb-logo.png"/>
<a className="sponsor-container" href="https://www.neb.com/en/">
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/neb-logo.png"/>
</a>
</div>
</div>
......@@ -173,45 +154,55 @@ export function Partners() {
<br/> <br/>
<div className="row align-items-center">
<div id="project-text" className="col sponsor-text-left">
<h2>BFH MeetUp sponsors...</h2>
<H2 text="BFH MeetUp sponsors" id="bfh-sponsors"/>
<br/>
<p></p>
</div>
<div id="project-portrait" className="col-6 sponsor-portrait" style={{padding: "20px"}}>
<div id="project-portrait" className="col-5 sponsor-portrait" style={{padding: "20px"}}>
<div className="row align-items-center">
</div>
<div className="row align-items-center">
<div className="col">
<a className="sponsor-container" href="https://www.gip.com/home/">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/gip.png" />
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/gip.png" />
</a>
</div>
<div className="col">
<a className="sponsor-container" href="https://www.jenabioscience.com/">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/jbs-dunkelgruen-text.png"/>
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/jbs-dunkelgruen-text.png"/>
</a>
</div>
</div>
<div className="row align-items-center">
<div className="col">
V Bio
<a className="sponsor-container" href="https://v-bio.ventures/">
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/vbio-logo.png"/>
</a>
</div>
<div className="col">
MN
<div className="col">
<a className="sponsor-container" href="https://www.mn-net.com/">
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/mn-logo.png"/>
</a>
</div>
</div>
<div className="row align-items-center">
<div className="col">
fiz
<a className="sponsor-container" href="https://www.fiz-biotech.de/">
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/fiz-logo.png"/>
</a>
</div>
<div className="col">
cell signaling technologies
<div className="col">
<a className="sponsor-container" href="https://www.cellsignal.com/">
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/cell-signaling-technology-logo.png"/>
</a>
</div>
</div>
<div className="row align-items-center">
<div className="col">
GASB
<a className="sponsor-container" href="https://gasb.de/">
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/gasb-logo.jpg"/>
</a>
</div>
</div>
......@@ -222,54 +213,54 @@ export function Partners() {
<div id="meetup-portrait" className="col-5 sponsor-portrait" style={{padding: "20px"}}>
<div className="row align-items-center">
<div className="col">
<a className="sponsor-container" href="">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/asimov-colorful.png"/>
<a className="sponsor-container" href="https://www.asimov.com/">
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/asimov-colorful.png"/>
</a>
</div>
<div className="col">
<a className="sponsor-container" href="https://www.uni-bielefeld.de/fakultaeten/technische-fakultaet/arbeitsgruppen/multiscale-bioengineering/campusbrauerei/">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/campus-brauerei.png"/>
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/campus-brauerei.png"/>
</a>
</div>
<div className="col">
<a className="sponsor-container" href="">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/algenium.png"/>
<a className="sponsor-container" href="https://algenium.de/algenium/">
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/algenium.png"/>
</a>
</div>
</div>
<div className="row align-items-center">
<div className="col">
<a className="sponsor-container" href="">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/logos-team/other-teams/gu-frankfurt-logo.png"/>
<a className="sponsor-container" href="https://2024.igem.wiki/gu-frankfurt/">
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/logos-team/other-teams/gu-frankfurt-logo.png"/>
</a>
</div>
<div className="col">
<a className="sponsor-container" href="https://bts-ev.de/">
<img className="img-sponsor" src="https://static.igem.wiki/teams/5247/sponsors/bts.png"/>
<img className="img-sponsor side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/bts.png"/>
</a>
</div>
<div className="col">
<a className="sponsor-container" href="">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/logos-team/other-teams/igem-hamburg-logo.png"/>
<a className="sponsor-container" href="https://2024.igem.wiki/hamburg/">
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/logos-team/other-teams/igem-hamburg-logo.png"/>
</a>
</div>
</div>
<div className="row align-items-center">
<div className="col">
<a className="sponsor-container" href="">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/studscicom-logo.png"/>
<a className="sponsor-container" href="https://www.stud-scicom.de/">
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/studscicom-logo.png"/>
</a>
</div>
</div>
</div>
<div id="meetup-text" className="col sponsor-text-right" style={{textAlign: "right"}}>
<h2> ... and collaborators </h2>
<H2 text="and collaborators " id="bfh-collabs"/>
<br/>
</div>
</div>
<br/> <br/>
<h2>Other collaborators</h2>
<H2 text="Other collaborators" id="otehrs"/>
<br/> <br/>
<div className="row align-items-center">
<div id="idt-text" className="col sponsor-text-left">
......@@ -279,7 +270,7 @@ export function Partners() {
</div>
<div id="" className="col-5 sponsor-portrait">
<a href="https://www.cfvww.org/">
<img id="idt-portrait-logo" src="https://static.igem.wiki/teams/5247/sponsors/cfvestslogo.png"></img>
<img id="vests-portrait-logo" src="https://static.igem.wiki/teams/5247/sponsors/cfvestslogo.png"></img>
</a>
</div>
</div>
......@@ -288,20 +279,22 @@ export function Partners() {
<div className="row align-items-center">
<div className="col">
<a className="sponsor-container" href="https://www.carlroth.de/">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/roth.jpg"/>
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/roth.jpg"/>
</a>
</div>
<div className="col">
<a className="sponsor-container" href="https://www.uni-bielefeld.de/fakultaeten/technische-fakultaet/">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/techfak.jpg"/>
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/techfak.jpg"/>
</a>
</div>
<div className="col">
Sarstedt
<a className="sponsor-container" href="https://www.sarstedt.com/en/">
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/sarstedt-logo.png"/>
</a>
</div>
<div className="col">
<a className="sponsor-container" href="">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/corden-pharma-logo.png"/>
<a className="sponsor-container" href="https://cordenpharma.com/">
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/corden-pharma-logo.png"/>
</a>
</div>
</div>
......@@ -310,13 +303,13 @@ export function Partners() {
{/* leer */}
</div>
<div className="col">
<a className="sponsor-container" href="">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/capricorn-logo.png"/>
<a className="sponsor-container" href="https://www.capricorn-scientific.com/en">
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/capricorn-logo.png"/>
</a>
</div>
<div className="col">
<a className="sponsor-container" href="">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/twist-bioscience-logo.png"/>
<a className="sponsor-container" href="https://www.twistbioscience.com/">
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/twist-bioscience-logo.png"/>
</a>
</div>
<div className="col">
......@@ -324,7 +317,7 @@ export function Partners() {
</div>
</div>
</div>
<BackUp/>
......
src/contents/parts.tsx
View file @ 3fd292fd
import { useEffect } from "react";
import { useLocation } from "react-router-dom";
import { openFromOtherPage } from "../components/Buttons";
export function Parts() {
const location = useLocation();
useEffect(() => {
const params = new URLSearchParams(location.search);
const collapseId = params.get('collapseId');
const tabId = params.get('tab');
import { Section, Subesction } from "../components/sections";
import { PartTable } from "../components/Table";
import { useTabNavigation } from "../utils/TabNavigation";
import { BasicParts } from "../data/parts";
import { H4 } from "../components/Headings";
import PartSources from "../sources/part-sources";
import { SupScrollLink } from "../components/ScrollLink";
import { OneFigure, TwoVertical } from "../components/Figures";
// Scroll to the section specified by collapseId
if (collapseId) {
const collapseElement = document.getElementById(collapseId);
if (collapseElement) {
const elementTop = collapseElement.getBoundingClientRect().top + window.pageYOffset;
const offset = window.innerHeight / 2 - collapseElement.offsetHeight / 2;
const scrollPosition = elementTop - offset;
export function Parts() {
useTabNavigation();
let headcols = ["Part Name", "Registry Code", "Part Description", "length [bp]", "type"]
return (
<div className="col">
<Section title="Description" id="Description">
<Subesction title="Introduction" id="Description1">
<p>In the context of Cystic Fibrosis, the F508del mutation represents a significant challenge for correction. The efficacy of current gene editing technologies hinges on the availability of precise tools to ensure the success of treatment strategies. In view of the above, we have developed a novel reporter system that is specifically tailored to the F508del mutation in the CFTR gene. The objective is to provide a high degree of comparability to the genomic context of this mutation, while maintaining ease of use. This system allows researchers to test and screen Prime Editors and various pegRNAs (prime editing guideRNAs), particularly in the context of the F508del mutation. By closely mimicking the genomic environment, it is believed that this tool will offer enhanced utility in the selection of optimal Prime Editing strategies. </p>
</Subesction>
<Subesction title="Prime Editor and pegRNA Testing" id="Description2">
<p>The principal feature of the reporter system is its capacity to assess and quantify the efficacy of diverse Prime Editors, with a particular focus on pegRNAs. In its default state, the system expresses a non-functional GFP due to the disruption of the splice site. However, if a Prime Editor successfully restores the mutation to its correct form, the splice site is repaired and functional GFP is expressed, thereby allowing for fluorescent detection. This fluorescence serves as a reliable indicator of successful prime editing. </p>
<p>The modified GFP sequence was cloned into the pDAS12124_PEAR-GFP-preedited plasmid, which was then transfected into HEK cells to initiate the pegRNA screening process. The capacity to observe the restoration of functional GFP provides a definitive indication of the efficacy of both the Prime Editor and the specific pegRNA variant under examination. Furthermore, the considerable degree of similarity between the reporter system and the actual genomic context of the CFTR mutation renders the screening process highly pertinent to the optimisation of specific applications. </p>
</Subesction>
<Subesction title="Conclusion" id="Description3">
<p>This reporter system represents a substantial advancement in the study and correction of the CFTR F508del mutation. The design of the system allows for the straightforward screening of an array of Prime Editor and pegRNA constructs, while maintaining a high degree of comparability to the genomic context. By closely emulating the CFTR gene environment, particularly in the context of the F508del mutation, researchers are able to identify the most efficient pegRNAs and Prime Editors, offering a promising approach for developing more effective gene-editing treatments for Cystic Fibrosis.<SupScrollLink label="1"/> </p>
</Subesction>
</Section>
window.scrollTo({
top: scrollPosition,
behavior: 'smooth',
});
}
}
<Section title="Characterization" id="Characterization">
<Subesction title="Design and Functionality" id="Characterization1">
<p>The reporter system has been designed with the specific intention of facilitating a more comparable genomic context for the F508del mutation, particularly for the purpose of testing the efficacy of different pegRNA variants and prime editors. The system provides a highly reliable platform for screening a variety of pegRNAs, thereby facilitating the identification of the most effective variant for correcting the F508del mutation</p>
<p>The system is constructed around a plasmid structure, specifically pDAS12124_PEAR-GFP_GGTdel_edited, from which a modified version of GFP (Green Fluorescent Protein) has been derived. The green fluorescent protein (GFP) is composed of two exons, separated by a Vim gene intron in its natural state. In the absence of the intron, the GFP is expressed and fluoresces. However, the GFP sequence was modified to introduce a three-base-pair deletion, specifically in the junction between Exon 1 and the Vim gene intron. This deletion affects the last base of Exon 1 and the first two bases of the intron, effectively disrupting the splice site. As a result, the intron is no longer correctly spliced out, leading to the expression of a non-functional GFP that does not fluoresce. </p>
</Subesction>
<Subesction title="Adaptions for CFTR F508del mutation comparibility" id="Characterization2">
<p>In addition to the introduction of the three-base-pair deletion, the intron sequence was further altered with the objective of enhancing the comparability of the system to the CFTR genomic context. Specifically, 27 base pairs were replaced downstream of the splice site with a sequence derived from the CFTR gene in the region of the F508del mutation. This modification guarantees that the gRNA spacer employed in our system is identical to the one found in the actual genomic context of the CFTR mutation. </p>
<p>The only notable differences between the system and the genomic sequence are observed in the RTT (Reverse Transcript Template) and PBS (Primer Binding Site), which have been calibrated with silent mutations to maintain comparability in GC content with the native CFTR gene. These silent mutations do not affect the encoded protein but optimise the system's mimicry of the CFTR gene. </p>
<OneFigure
pic1="https://static.igem.wiki/teams/5247/thaw/reporter-insert.webp"
alt1="Illustration of our constructed reporter system"
description="Illustration of our constructed reporter system"
num={1}
/>
</Subesction>
</Section>
// Open the tab specified by tabId
if (tabId) {
openFromOtherPage(tabId)({ currentTarget: document.getElementById(tabId)! });
}
}, [location.search]);
<Section title="Experiments" id="Experiments">
<Subesction title="Cloning" id="Experiments1">
<p>The synthesised fragment was cloned into pDAS12124_PEAR-GFP-preedited plasmid using Gibson assembly, thus providing a vector with which the desired tests could be performed in HEK293 cells. The correctness of the cloning was determined by two methods: the correct size of the cloned plasmid was confirmed by gel electrophoresis, while the correct orientation and complete cloning were confirmed by Sanger sequencing. </p>
<H4 text="Workflow "/>
<p>The creation and validation of the CF-specific reporter system commenced with the selection and subsequent outgrowth of E. coli DH5α strains that contain the pDAS12124 plasmid. The initial stage of the process entails the isolation and purification of the pDAS12124 plasmid through the utilisation of conventional plasmid preparation methodologies, thereby ensuring its sterility and facilitating seamless downstream applications. Subsequent to the design of the CF-specific reporter system, the sequence was obtained from IDT. Upon its receipt, the fragment was amplified through polymerase chain reaction (PCR) to produce a sufficient quantity of material for the subsequent cloning phases. With the reporter system fragment ready, the pDAS12124_PEAR-GFP-preedited plasmid was digested using NheI and XhoI restriction enzymes. This cuts out the GFP cassette, creating the required entry point for the integration of the DNA fragment of the reporter system. To prevent the backbone from re-ligating, the sample is treated with phosphatase, ensuring the plasmid remains open for the upcoming Gibson assembly. </p>
<p>Subsequently, a purification process is conducted to extract the plasmid backbone and concentrate the samples. This facilitates the integration of the amplified reporter system into the prepared pDAS12124_PEAR-GFP-preedited backbone, which is then subjected to the Gibson assembly process. This assembly process results in the creation of the novel pDAS12124_PEAR-GFP_GGTdel_edited plasmid, which incorporates the CF-specific reporter system. </p>
<p>Subsequently, the pDAS12124_PEAR-GFP_GGTdel_edited plasmid is transformed into E. coli DH5α cells for propagation. To confirm the successful integration of the reporter fragment, colony PCR (cPCR) is performed on the transformed colonies. The positive colonies, identified by cPCR, are selected and grown in LBCm50 medium for further analysis. </p>
<p>The final validation step involves preparing the pDAS12124_PEAR-GFP_GGTdel_edited plasmid from the positive colonies and verifying the correct insertion of the reporter fragment using Sanger sequencing. This ensures the fragment is inserted in the correct orientation and that the CF-specific reporter system has been successfully constructed without any errors. </p>
<OneFigure
pic1="https://static.igem.wiki/teams/5247/thaw/cloning-reporter.webp"
alt1=""
description="Cloning strategy of our reporter system for functional expression"
num={2}
/>
</Subesction>
<Subesction title="pegRNA Screening" id="Experiments2">
<p>In connection with the optimisation of prime editing with regard to the F508del mutation, it was necessary to compare different pegRNAs, as their optimal structure always depends on the application context. We therefore designed and cloned 14 variants of pegRNAs for the target of the reporter system and then tested them on the reporter system using the PE2 system. </p>
<p>For pegRNA screening, we co-transfected the HEK293 cells with our modified reporter plasmid, the pegRNA expressing plasmid and pCMV-PE2. We were then able to measure the fluorescence after 72 hours using FACS and evaluate which pegRNA showed the highest efficiency. </p>
<OneFigure
pic1="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/bild9.png"
alt1=""
description="Percentage of fluorescent HEK293 cells 72 h after transfection with various pegRNAs (pegRNA1-14) normalized to pDAS12124 pre-edited as internal positive control as result of flow cytometry analysis"
num={3}
/>
<p>We also co-transfected the CFBE41o- with our modified reporter plasmid, the plasmid expressing pegRNA04 as well as pCMV-PE6c. As a result, we observed fluorescence, indicating successful editing of the reporter plasmid. The negative controls transfected with only one of the plasmids each showed no fluorescence, routing out other factors. This gave us validation, that our pegRNAs work not only in HEK, but also in epithelial cells that express CFTR F508del. </p>
<TwoVertical
description="Microscopy results after 24h or 48h. Transfection of pDAS12124-preedited with lipofectamine 3000 was successfully done in CFBE41o- cell line and visible after 48h. CFBE41o- cell line was transfected with pDAS-IDT with Lipofectamine 3000 and afterwards with LNPs including PE6c and pegRNA4 and was after 24h fluorescence visible."
num={4}
bg="white"
alt1=""
pic1="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/bild10-1.png"
pic2="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/bild10-2.png"
/>
<p>Based on the results, we were able to select 4 possible candidates and one negative example, whose attributes we then used to create pegRNAs for the CFTR target. The next step is to test these pegRNAs using CFBE41o- cells by again co-transfecting these with three plasmids: reporter plasmid, pegRNA expressing plasmid and pCMV-PE6c, and measuring fluorescence after 72 hours. </p>
return (
<>
<div className="row">
<div className="col">
</div>
</div>
<div className="row">
</div>
</>
</Subesction>
<Subesction title="Future Experiment: Nickase Assay" id="Experiments3">
<p>In the next series of experiments, we would like to investigate various mutation candidates, in particular the possible SpuFz1 nickases (BBa_K5247101- BBa_K5247104) and various PlmCasx nickase variants (BBa_K5247105- BBa_K5247107), in more detail using the PE6c system. </p>
{/* Bild Nikase */}
<H4 text="nSpuFz1 "/>
<p>The nSpuFz1 variants are expressed in yeast strain Pichia pastoris (SMD1163), which we obtained from Nils Berelsmann{/* [link zu HP-Timeline]. */} In advance, the corresponding genes were cloned into a suitable expression vector, pPIC9K, via Gibson Assembly to ensure efficient expression of the nickases. The cloning as well as the subsequent expression and purification of the nickases were carried out according to a detailed protocol<SupScrollLink label="2"/> under the expert guidance of Hakan Soytürk[link zu HP timeline]. </p>
<H4 text="nPlmCasX "/>
<p>The nPlmCasX variants are expressed in E. coli strain BL21D3, which we obtained from AG Müller{/* [link zu HP-Timeline] */}. In advance, the corresponding genes were cloned into a suitable expression vector, pZMB1029, via Gibson Assembly to ensure efficient expression of the nickases. The cloning as well as the subsequent expression and purification of the nickases were carried out according to a detailed protocol<SupScrollLink label="2"/> . </p>
{/* Bild beide */}
<p>After successful purification, the isolated nickases are comprehensively analyzed according to verify their activity and efficiency. These analyses will serve to evaluate the functionality and suitability of the nickases for specific applications in prime editing. Subsequently, detailed characterization experiments are planned to determine the properties of the nickases, if functional, including their specificity, editing activity and potential for use in precise gene editing procedures. </p>
<p>Validation of the nickases will be performed in different cell lines to confirm their efficiency and reliability in a cellular context. These validation steps are crucial to further investigate the potential of Prime Guide for therapeutic applications. </p>
</Subesction>
</Section>
<Section title="Parts Collection" id="Parts Collection">
<Subesction title="Basic Parts" id="Parts Collection1">
<PartTable cols={headcols} data={BasicParts}/>
</Subesction>
</Section>
<Section title="References" id="References">
<ol>
<PartSources/>
</ol>
</Section>
</div>
);
}
\ No newline at end of file
src/contents/project-documentation.tsx 0 → 100644
View file @ 3fd292fd
import { H2 } from "../components/Headings";
import { PDF } from "../components/Pdfs";
import { useTabNavigation } from "../utils/TabNavigation";
export function ProDesc() {
useTabNavigation();
return (
<div className="col">
<H2 text="Our Meeting Protocols"/>
<PDF link="https://static.igem.wiki/teams/5247/pdfs/meetings.pdf" name="meetings.pdf"/>
</div>
);
}
\ No newline at end of file
src/contents/proof.tsx deleted 100644 → 0
View file @ 9143d0b0
import { useEffect } from "react";
import { useLocation } from "react-router-dom";
import { openFromOtherPage } from "../components/Buttons";
export function Proof() {
const location = useLocation();
useEffect(() => {
const params = new URLSearchParams(location.search);
const collapseId = params.get('collapseId');
const tabId = params.get('tab');
// Scroll to the section specified by collapseId
if (collapseId) {
const collapseElement = document.getElementById(collapseId);
if (collapseElement) {
const elementTop = collapseElement.getBoundingClientRect().top + window.pageYOffset;
const offset = window.innerHeight / 2 - collapseElement.offsetHeight / 2;
const scrollPosition = elementTop - offset;
window.scrollTo({
top: scrollPosition,
behavior: 'smooth',
});
}
}
// Open the tab specified by tabId
if (tabId) {
openFromOtherPage(tabId)({ currentTarget: document.getElementById(tabId)! });
}
}, [location.search]);
return (
<>
<section id="prelim-2000">
<h2>Preliminary test with lipofectamine 2000</h2>
<h3>Seeding</h3>
<h3>Transfection</h3>
<h3>Microscopy</h3>
<table>
<tr>
<th>Plasmid</th>
<th>Expected results</th>
<th>Results</th>
</tr>
<tr>
<th></th>
<th></th>
<th></th>
</tr>
</table>
</section>
<section id="pos-control-2000">
<h2>Positiv control and optimization</h2>
<h3>Seeding</h3>
<h3>Transfection</h3>
<h3>Microscopy</h3>
<table>
<tr>
<th>Plasmid</th>
<th>Expected results</th>
<th>Results</th>
</tr>
<tr>
<th></th>
<th></th>
<th></th>
</tr>
</table>
</section>
<section id="pos-control-3000">
<h2>Positiv control and optimization</h2>
<h3>Seeding</h3>
<h3>Transfection</h3>
<h3>Microscopy</h3>
<table>
<tr>
<th>Plasmid</th>
<th>Expected results</th>
<th>Results</th>
</tr>
<tr>
<th></th>
<th></th>
<th></th>
</tr>
</table>
</section>
<section id="poc-3000">
<h2>Preliminary test with lipofectamine 2000</h2>
<h3>Seeding</h3>
<h3>Transfection</h3>
<h3>Microscopy</h3>
<table>
<tr>
<th>Plasmid</th>
<th>Expected results</th>
<th>Results</th>
</tr>
<tr>
<th></th>
<th></th>
<th></th>
</tr>
</table>
</section>
</>
);
}
\ No newline at end of file
src/contents/results.tsx
View file @ 3fd292fd
import { useEffect } from "react";
import { useLocation } from "react-router-dom";
import { openFromOtherPage } from "../components/Buttons";
export function Results() {
const location = useLocation();
useEffect(() => {
const params = new URLSearchParams(location.search);
const collapseId = params.get('collapseId');
const tabId = params.get('tab');
// Scroll to the section specified by collapseId
if (collapseId) {
const collapseElement = document.getElementById(collapseId);
if (collapseElement) {
const elementTop = collapseElement.getBoundingClientRect().top + window.pageYOffset;
const offset = window.innerHeight / 2 - collapseElement.offsetHeight / 2;
const scrollPosition = elementTop - offset;
window.scrollTo({
top: scrollPosition,
behavior: 'smooth',
});
}
}
// Open the tab specified by tabId
if (tabId) {
openFromOtherPage(tabId)({ currentTarget: document.getElementById(tabId)! });
}
}, [location.search]);
import { H4 } from "../components/Headings";
import { Section, Subesction } from "../components/sections";
import { useTabNavigation } from "../utils/TabNavigation";
import { H5 } from "../components/Headings";
import { useNavigation } from "../utils";
import { DownloadLink } from "../components/Buttons";
import { OneFigure, ThreeVertical, TwoHorizontal, TwoVertical } from "../components/Figures";
import { ResTable } from "../components/Table";
import { headercols, resultdata } from "../data/results-table";
import { SupScrollLink } from "../components/ScrollLink";
import ResultSources from "../sources/res-sources";
export function Results() {
useTabNavigation();
const {goToPagesAndOpenTab} = useNavigation ();
const {goToPageAndScroll} = useNavigation();
return (
<>
<div className="row mt-4">
<div className="col-lg-5">
<><Section title="Abstract" id="Abstract">
<p>For the prime editing of <strong>Cystic Fibrosis (CF)</strong>, we on the one hand optimized a prime editing complex and on the other hand developed an efficient delivery system. For testing, we set up cell culture with model cell lines as well as primary cells taken from team members and a patient.</p>
<p>For editing, we first compared different existing prime editors <strong>(pCMV-PE2, pMLV-PE_CO-Mini, pCMV-PE6c)</strong> and constructed a reporter plasmid simulating the CFTR context. In addition and to further enhance the editing process, we designed various pegRNAs tailored to our construct incorporating features such as <strong>silent edits</strong>, for a lower mismatch repair, and a 3′ stabilizing stem loop <strong>(tevopreQ1)</strong>. The aim was to identify the most effective pegRNA for our specific target, which is why pegRNA especially for <strong>CFTR F508del mutation</strong> were designed. </p>
<p>As proof of concept, we transfected these constructs in <strong>HEK293 and CFB41o- cells</strong> and observed significant prime editing of our reporter via fluorescence microscopy. We identified the PE6c editor and our pegRNA variant 4 as optimal. This resulted in our <strong>New Basic Part</strong>, <strong>PEAR_CFTR</strong>. Furthermore, we extended our approach to primary human nasal epithelial cells generated from our own nasal epithelial cells through nasal swabs. By cultivating them in Air Liquid Culture (ALI) and Apical-Out Airway Organoids (AOAO), we successfully tested our technologies <i>in vitro</i>, mimicking the <i>in vivo</i> situation.</p>
<p>Furthermore, we successfully designed and cloned novel nickases of <strong>Fanzor</strong>, which is special because of its smaller size and eukaryotic origin. This serves as valuable tool for future genome editing applications. </p>
<p>For delivery, lipid nanoparticles (LNPs) are a highly effective and versatile delivery system, valued for their larger cargo capacity, biocompatibility, and ability to protect RNA from degradation. To deliver our Prime Editing construct as pegRNA and mRNA, we optimized a <strong>Selective ORgan Targeting (SORT) LNP</strong> for targeted delivery to the lungs by using the cationic helper lipid DOTAP and encapsulating a stable <strong>chitosan-RNA complex</strong>, achieving significant breakthroughs in transfection of <i>in vitro</i> lung epithelial cells. </p>
<p>We began by testing three different LNP formulations, starting with the <strong>Cayman LipidLaunch LNP-102 Exploration Kit</strong>. We confirmed by fluorescence microscopy, where Minicircle DNA effectively transfected HEK293 cells. Further experiments with the <strong>Corden LNP Stater Kit #2</strong> failed to achieve successful transfection, likely due to increased cytotoxicity from a more cytotoxic PEG component. Our successful formulation was a <strong>lung-specific SORT LNP</strong>, which demonstrated excellent stability, as confirmed by Zeta potential measurements. Dynamic light scattering (DLS) analysis revealed an optimal particle size of closely smaller than 200 nm, aligning with literature and supporting the ability of the LNPs to penetrate deep lung regions via inhalation. Flow cytometry analysis showed that the SORT LNP had 14 times higher transfection efficiency compared to traditional transfection methods. Moreover, a MTT assay revealed that the SORT LNP, along with Cayman LNPs, exhibited the lowest cytotoxicity, thanks to the use of low-molecular-weight PEG components. </p>
<p>To further enhance the stability of the LNPs for inhalation, we incorporated <strong>chitosan-RNA complexes</strong>, which provide thermal stability and protect RNA from degradation by RNases. Integration of these complexes into the SORT LNP resulted in a lung-specific delivery platform with superior stability. Using this system, we achieved highly efficient transfection of a bronchial cell line from a Cystic Fibrosis patient (CFBE41o- with F508del mutation), demonstrating the potential of this approach for targeted gene delivery to lung epithelial cells. These results highlight the remarkable efficiency, stability and specificity of our optimized SORT LNP formulation, positioning it as a promising platform for lung-specific genetic therapies. </p>
</Section>
<Section title="Experimental Design" id="Experimental Design">
<Subesction title="Proof of Concept" id="Experimental Design1">
<H5 text="Workflow"/>
<p>The prepared pDAS12124-preedited plasmid serves as a positive control to validate the success of the experiment. A technical control with the pZMB938 plasmid confirms successful transfection of the cells. In the main part of the experiment, pDAS12489-2in1 and pCMV-PE2 are co-transfected. Successful transfection is visualised by GFP signals.</p>
<H5 text="Conclusion"/>
<p>The microscopy data validates our proof of concept. Compared to our internal positive control, pDAS12124-preedited (see Figure 1), less cells co-transfected with pDAS12489 and pCMV-PE2 (see Figure 1) showed fluorescence. Contrary to our expectations, the technical transfection control with pZMB938 showed lower transfection efficiency. All negative controls showed no fluorescence.</p>
<ThreeVertical
description="Microscopy of HEK293 72h post transfection with lipofectamine 2000. Transfection with technical positive control pZMB938, internal positive control pDAS12124-preedited, co-transfection of pDAS12489 with pCMV-PE2, NTC, PE2 as control and pDAS12489 as control. All controls are negative and both positve controls as well as pDAS12489+pCMV-PE2 show fluorescence signals. BF = Brightfield"
num={1}
pic1="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/bild1-1.png"
alt1="pZMB"
bg="white"
pic2="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/bild1-2.png"
pic3="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/bild1-3.png"
/>
<H4 text="Transfection optimization"/>
<H5 text="Workflow"/>
<p>To optimise transfection efficiency, different dilutions and concentrations of DNA were used to find the best transfection conditions.</p>
<H5 text="Conclusion"/>
<p>All 4 different transfection conditions were done with pZMB938 and showed good results, but best result were done when lipofectamine 2000 was diluted 1:10 and 1000 ng DNA was transfected.</p>
<TwoVertical
description="Microscopy of HEK293 72h post transfection with lipofectamin 2000. Transfection with 1:10 or 1:25 diluted lipofectamine and 800 ng or 1000 ng of out technical positive control pZMB938. BF = Brightfield"
num={2}
bg="white"
alt1="1000ng DNA with 1:10 and 1:25 Lipofectamine 2000"
pic1="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/bild2-1.png"
pic2="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/bild2-2.png"
/>
<H5 text="Workflow"/>
<p>Transfection with Lipofectamine 3000 was performed because of the probably better performance and transfection rate. The prepared pDAS12124-preedited plasmid serves as a positive control to validate the success of the experiment. A technical control with the pZMB938 plasmid confirmed successful transfection of the cells as before. In the main part of the experiment, pDAS12489-2in1 and pCMV-PE2 were co-transfected. Successful transfection and prime editing was detected by GFP signals.</p>
<H5 text="Conclusion"/>
<p>Internal control and technical control showed higher transfection efficiency then in previous experiments, therefore transfection with lipofectamine 3000 seems to be more efficient than transfection with lipofectamine 2000. The fluorescence of pDAS12189+pCMV-PE2 was still quite low. All negative controls are showed no fluorescence.</p>
<ThreeVertical
description="Microscopy of HEK293 72h post transfection with lipofectamine 2000. Transfection with technical positive control pZMB938, internal positive control pDAS12124-preedited, co-transfection of pDAS12489 with pCMV-PE2, NTC, PE2 as control and pDAS12489 as control. All controls are negative and both positve controls as well as pDAS12489+pCMV-PE2 show fluorescence signals. BF = Brightfield"
num={3}
bg="white"
alt1="pZMB and pDAS12124"
pic1="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/bild3-1.png"
pic2="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/bild3-2.png"
pic3="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/bild3-3.png"
/>
<H5 text="Workflow"/>
<p>Again a preliminary test with the technical positive control was conducted potentially optimize our transfection protocol and to train the handling.</p>
<H5 text="Conclusion"/>
<p>The results of the test were not as good as expected. Nearly no transfection efficiency was visible. This could be due to too old HEK293 cells</p>
<TwoVertical
description="Microscopy of HEK293 72h post transfection with lipofectaine 3000. Transfection of 500 ng or 1000 ng of our technical positive control pZMB938 with 1 µl or 1.5 µl of lipofectamine 3000. BF = Brightfield"
num={4}
bg="white"
alt1="500ng DNA 1 µl or 1.5 µl of lipofectamine 3000"
pic1="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/bild4-1.png"
pic2="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/bild4-2.png"
/>
<H5 text="Workflow"/>
<p>HEK cells were thawed and another prelimary test was conducted. In this test two different transfection agents were used (Lipofectamine 3000 & CaCl<sub>2</sub>) to check which one is better suited for our experiments. The literature uses lipofectamine 3000 but CaCl<sub>2</sub> transfection is much cheaper.</p>
<H5 text="Conclusion"/>
<p>Both transfections are working out well but the efficiency of the lipofectamine transfection was much higher.</p>
<OneFigure
pic1="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/bild5.png"
num={5}
bg="white"
alt1="Microscopy of HEK293 72h post transfection with lipofectamine 3000 with 1000 ng or 1500 ng technical positive control pZMB938. Both transfections show fluorescence signals"
description="Microscopy of HEK293 72h post transfection with lipofectamine 3000 with 1000 ng or 1500 ng technical positive control pZMB938. Both transfections show fluorescence signals. BF = Brightfield"
/>
<TwoVertical
description="Microscopy of HEK293 72h post transfection with CaCl<sub>2</sub> with 500 ng, 1000 ng or 1500 ng pZMB938. All transfections show fluorescence signals. BF = Brightfield"
num={6}
bg="white"
alt1=""
pic1="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/bild6-1.png"
pic2="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/bild6-2.png"
/>
<H5 text="Workflow"/>
<p>One last time the transfection of pDAS12189+pCMV-PE2 was conducted. Although our proof-of-concept already showed successful editing the first time, we repeated the experiment to get better transfection efficiency.</p>
<H5 text="Conclusion"/>
<p>The transfection efficiency was much better. Our proof-of-concept was working correctly. The reporter system pDAS12189 only led to production of a fluorescent signal when co transfected with a prime editing complex as pCMV-PE2.</p>
<TwoVertical
alt1=""
description="Microscopy of HEK293 72h post transfection with lipofectamine 2000. Transfection with technical positive control pZMB938, internal positive control pDAS12124-preedited, co-transfection of pDAS12489 with pCMV-PE2, NTC, PE2 as control and pDAS12489 as control. All controls are negative and both positve controls as well as pDAS12489+pCMV-PE2 show fluorescence signals. BF = Brightfield"
num={7}
bg="white"
pic1="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/bild7-1.png"
pic2="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/bild7-2.png"
/>
</Subesction>
<Subesction title="Prime Guide" id="Experimental Design2">
<H4 text="Initial testing of pegRNAs and Evaluation of silent edits"/>
<H5 text="Workflow"/>
<p>With this experiment we wanted to compare the efficiency of pegRNAs with and without silent edits.</p>
<H5 text="Conclusion"/>
<p>The Flow Cytometry analysis shows that pegRNA without silent edits (pegRNA1) had a 2.05 times higher transfection efficiency than pegRNA with silent edits (pegRNA2).</p>
<TwoHorizontal
description="Flow cytometry analysis of pegRNAs with and without silent edits. Histograms of cell count applied against fluorescence intensity of healthy HEK293 cells (left) with untransfected cells as negative control, pDAS12124 pre-edited as internal positive control and pegRNAs with (pegRNA1) and without (pegRNA2) silent edits 72 h after transfection. The portion of fluorescent cells is normalized to the internal positive control (right)."
num={8}
bg="white"
pic1="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/se-nose.png"
pic2="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/bild8.png" alt1={""} />
<H4 text="Screening of pegRNA variances"/>
<H5 text="Workflow"/>
<p>Cotransfection of pPEAR_CFTR and PE2 and also 1 of the 14 pegRNAs to compare the transfection efficiency of all of our designed pegRNAs.</p>
<H5 text="Conclusion"/>
<div className="row">
<div className="col">
<ResTable cols={headercols} data={resultdata}/>
</div>
<div className="col">
<p>The pegRNAs lead to differing amounts of cells showing fluorescence, which, assuming comparable transfection efficiencies, indicates varying prime editing efficiency. The pegRNA7 showed the highest transfection efficiency (see Figure 9).</p>
<OneFigure
pic1="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/bild9.png"
num={9}
alt1="Flow Cytometry results of all screened pegRNAs"
description="Percentage of fluorescent HEK293 cells 72 h after transfection with various pegRNAs (pegRNA1-14) normalized to pDAS12124 pre-edited as internal positive control as result of flow cytometry analysis."
/>
</div>
</div>
<H4 text="Application lung epithelial cell lines"/>
<H5 text="Workflow"/>
<p>We tried to transfect CFBE41o- cells with pDAS12124-preedited, our internal positive control, to check if a transfection of this cell line is possible. Furthermore we tried to co transfect the CFBE41o- with pPEAR_CFTR, PE6c and pegRNA4.</p>
<H5 text="Conclusion"/>
<p>Transfection of CFBE41o- with pDAS12124-preedited was successful (see Figure 10). After 24 hours a successful co transfection of pPEAR_CFTR with PE6c and pegRNA4 was visible, although the transfection efficiency was really bad (see Figure 10).</p>
<TwoVertical
alt1=""
description="Microscopy results after 24h or 48h. Transfection of pDAS12124-preedited with lipofectamine 3000 was successfully done in CFBE41o- cell line and visible after 48h. CFBE41o- cell line was transfected with pDAS-IDT with Lipofectamine 3000 and afterwards with LNPs including PE6c and pegRNA4 and was after 24h fluorescence visible. BF = Brightfield"
num={10}
bg="white"
pic1="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/bild10-1.png"
pic2="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/bild10-2.png"
/>
<p>Moreover transfection was conducted in human nasal epithelial cells (hNECs) in Air-liquid interface cultures as well as apical-out airway organoids (see Figure 11). No fluorescence was visible. </p>
<TwoVertical
alt1=""
description="Microscopy of HEK 72h post transfection with lipofectamine 3000. Co-transfection of pPEAR_CFTR with PE6c and pegRNA4. Both show no fluorescence signals. BF = Brightfield"
num={11}
bg="white"
pic1="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/ali-tr.png"
pic2="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/aoao-tr.png"
/>
<H4 text="Comparison of prime editing complexes PE2 and PE_CO-Mini"/>
<H5 text="Workflow"/>
<p>pCMV-PE2 was co transfected with pDAS12489 and pCMV-PE_CO-Mini was co transfected with pDAS12489 in HEK293 cell line.</p>
<H5 text="Conclusion"/>
<p>The Flow Cytometry results show that transfection with pCMV-PE2 as the prime editing complex had editing efficiency of 52.90% when normalized on pDAS12124-preedited. When pCMV-PE_CO-Mini was used as a prime editing complex it had a transfection efficiency of 2.54% (see Figure 11, 12).</p>
<div className="row">
<div className="col">
<TwoVertical
alt1=""
description="Microscopy of HEK 72h post transfection with lipofectamine 3000. Co-transfection of pDAS12489 with pCMV-PE2 or pDAS12489 with LV-PE_CO-Mini. Both show fluorescence signals. BF = Brightfield"
num={12}
bg="white"
pic1="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/bild11-1.png"
pic2="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/bild11-2.png"
/>
</div>
<div className="col">
<OneFigure
alt1=""
pic1="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/pe2-pe-co.png"
num={13}
bg="white"
description="Flow Cytometry analysis to compare prime editing complexes PE2 and PE_CO-Mini. Histograms of cell count applied against fluorescence intensity of healthy HEK293 cells (left) with untransfected cells as negative control, pDAS12124 pre-edited as internal positive control 72 h after transfection."
/>
</div>
</div>
<H5 text="Workflow"/>
<p>We compared the 3 different Prime Editing complexes (pCMV-PE2, pCMV-PE2_CO-Mini & pCMV-PE6c) to check which one has the best transfection efficiency.</p>
<H5 text="Conclusion"/>
<p>The Flow Cytometry measurement shows the fluorescence rate cells co-transfected with pDAS12489 and pCMV-PE6c as a prime editing complex. The editing efficiency off PE6c was by far the highest (81.88%) (see Figure 13, 14). The efficiency was 1.55 higher than the efficiency when pCMV-PE2 was used as prime editing complex (see Figure 13).</p>
<OneFigure
pic1="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/bild13.png"
num={14}
bg="white"
description="Microscopy of HEK293 72h post transfection with lipofectamine 3000 and co transfection with pCMV-PE6c and pDAS12489. BF = Brightfield"
alt1="Microscopy of HEK293 72h post transfection with lipofectamine 3000 and co transfection with pCMV-PE6c and pDAS12489."
/>
<TwoHorizontal
alt1=""
description="Flow cytometry analysis for evaluation of performance of prime editor variants. Histograms of cell count applied against fluorescence intensity of healthy HEK293 cells (left) with untransfected cells as negative control, pDAS12124 pre-edited as internal positive control and PE6c, PE2 and PE2_Co-Mini 72 h after transfection. The portion of fluorescent cells is normalized to the internal positive control (right)."
num={15}
bg="white"
pic1="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/pe2-pe-co-pe6c.png"
pic2="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/bild12.png"
/>
</Subesction >
<Subesction title="LNP Synthesis" id="Experimental Design3">
<H4 text="RNA Synthesis"/>
<div className="row align-items-center">
<div className="col">
<OneFigure
num={16}
pic1="https://static.igem.wiki/teams/5247/delivery/results/rna-gel-final.png"
description="Gel of Denaturing RNA Gel Electrophoresis for mRNA synthesized from pcDNA 3.1 eYFP indicating successful RNA synthesis. Lane 1: Low Range Ribo Ruler, Lane 2: FLuc Control Template, Lane 3: Negative Control, Lane 4-9 mRNA from pcDNA 3.1 eYFP."
alt1="Gel of Denaturing RNA Gel Electrophoresis for mRNA synthesized from pcDNA 3.1 eYFP indicating successful RNA synthesis. Lane 1: Low Range Ribo Ruler, Lane 2: FLuc Control Template, Lane 3: Negative Control, Lane 4-9 mRNA from pcDNA 3.1 eYFP."
/>
</div>
<div className="col">
<p>We began by synthesizing mRNA <i>in vitro</i> using a plasmid with a eYFP reporter from Addgene (pcDNA 3.1 eYFP) before proceeding with the synthesis of our approximately 6000 bp prime editing RNA. This was done to test the transfection efficiency and compatibility of our LNPs. The synthesis was successful, yielding an average of 1400 ng/µl of purified mRNA from 1 µg of plasmid DNA determined by Nanodrop measurement (data not shown). The size and integrity of the synthesized RNA were confirmed using a Denaturing RNA Gel, where we expected to see a product of 900 nucleotides. As anticipated, a strong and prominent band corresponding to this size was observed (Figure 16). This mRNA was subsequently used in further LNP formulations with RNA.</p>
</div>
</div>
<div className="row mt-4">
<div className="col-lg-8">
<H4 text="Cayman LNP"/>
<div className="row align-items-center">
<div className="col">
<p>Next, we formulated the LNPs using the Cayman LipidLaunch™ LNP-102 Exploration Kit after the manufacturers protocol. The initial assembly attempt was unsuccessful, as no cloudy, bluish solution formed after mixing the lipids. Additionally, transfection of HEK293 cells with LNPs containing nucleic acids did not produce any reporter fluorescence. After consulting with expert <a onClick={() => goToPagesAndOpenTab('radukic', '/human-practices')}>Dr. Marco Radukic</a> and adjusting our LNP formulation and transfection protocols, specifically by pre-acidifying the OptiMEM medium, we were able to successfully assemble and transfect the LNPs. We also got from him Minicircle DNA from <a href="https://www.plasmidfactory.com/custom-dna/minicircle-dna/" title="PlasmidFactory" >PlasmidFactory</a> as a small plasmid carrying an eYFP gene and easy to transform, by that serving as a positive control in our experiments. Upon pipetting the components together, the solution immediately turned cloudy and bluish, indicating successful LNP formation (Figure 17).</p>
</div>
<div className="col">
<OneFigure
description="Cayman LNP Formation indicated by blue color and turbidity. Mini DNA = Minicircle DNA from PlasmidFactory."
num={17}
pic1="https://static.igem.wiki/teams/5247/delivery/results/caymanlnpblue.webp"
alt1="Cayman LNP Formation indicated by blue color and turbidity. Mini DNA = Minicircle DNA from PlasmidFactory."
/>
</div>
</div>
<H5 text="Transfection"/>
<p>To evaluate the efficiency of transfection, we performed fluorescence microscopy (Leica DMI6000 B at 20x magnification) on HEK293 cells transfected with LNP-formulated DNA and mRNA of pcDNA 3.1 eYFP, Minicircle DNA as the positive control and LNP without cargo as the negative control. Moreover, transfection via lipofectamine was tested for delivery method comparison.</p>
<p>Until 72h post-transfection, we observed in the conditions with lipofectamine alone or combined with RNA, no fluorescence, indicating unsuccessful transfection with RNA. Similarly, no fluorescence was seen in cells treated with LNPs alone or in combination with DNA or RNA. When LNPs were combined with Minicircle DNA or the cells were transfected with lipofectamine and Minicircle DNA, clear fluorescence was observed, indicating successful transfection and expression of our eYFP reporter under this condition (Figure 18). However, a strong background fluorescence from the OptiMEM medium was observed, complicating the analysis.</p>
<p>Overall, among all the tested conditions, the LNP formulation with Minicircle DNA was the only combination that resulted in noticeable fluorescence, suggesting it to be the most effective transfection method for HEK293 cells in this experiment.</p>
<OneFigure
pic1="https://static.igem.wiki/teams/5247/delivery/results/precyse/cayman.png"
num={18}
bg="white"
description="Overlay of phase contrast and fluorescence microscopic images of transfected HEK293 cells at 20x magnification 72h post-transfection with different Cayman LNP formulations recorded with Leica DMI6000 B. Lipo= lipofectamine, Mini DNA = Minicircle DNA."
alt1="Overlay of phase contrast and fluorescence microscopic images of transfected HEK293 cells at 20x magnification 72h post-transfection with different Cayman LNP formulations recorded with Leica DMI6000 B. Lipo= lipofectamine, Mini DNA = Minicircle DNA."
/>
<H5 text="SEM"/>
<div className="row align-items-center">
<div className="col">
<p>Scanning Electron Microscopy (SEM) (Phenom ProX, Thermo Fisher) was employed by us to examine the morphology and surface characteristics of Cayman LNPs. The SEM images revealed that the LNPs displayed a generally spherical morphology with a relatively smooth surface (Figure 19). The average particle size was approximately 200 nm. However, a heterogeneous distribution of particle sizes was observed, with some larger, round structures present. These larger structures could potentially indicate aggregated LNPs.</p>
</div>
<div className="col">
<OneFigure
pic1="https://static.igem.wiki/teams/5247/delivery/results/screenshot-2024-10-01-200629.png"
alt1="CayREM"
description="SEM image of Cayman LNPs (10,000x magnification) with Topography mode."
num={19}/>
</div>
</div>
<p>While many particles retained their structural integrity, the presence of these aggregates suggests that, under certain conditions, the LNPs may tend to cluster. It is important to note that for SEM analysis, the samples were dried and observed under vacuum, which probably have affected the structure and shape of the LNPs. This preparation process can introduce artifacts that would not typically be present in solution and should be considered when interpreting the results. Additionally, the contrast under vacuum conditions was too low to reliably distinguish the LNPs with sufficient detail. It provided a useful initial glimpse into the world of nanoparticles. Further complementary techniques will be needed for a more accurate and detailed characterization.</p>
<H4 text="Corden LNP"/>
<div className="row align-items-center">
<div className="col">
<OneFigure
pic1="https://static.igem.wiki/teams/5247/delivery/results/whatsapp-image-2024-09-24-at-12-57-59.jpeg"
num={20}
description="Turbidity after components of the Corden LNP have been pipetted together indicates particle formation."
alt1="Turbidity after components of the Corden LNP have been pipetted together indicates particle formation."
/>
</div>
<div className="col">
<p>During the preparation of the LNPs, the solution became turbid and bluish, indicating successful nanoparticle formation (Figure 20). This was further confirmed by cryo-EM (cryogenic electron microscopy) analysis, which revealed the presence of well-formed LNPs. Despite the successful formation of LNPs, no detectable fluorescence was observed in the cells treated with LNPs containing pcDNA 3.1 eYFP DNA or mRNA at any of the measured time points, indicating that transfection did not occur under these conditions.</p>
</div>
</div>
<H5 text="Transfection"/>
<p>Fluorescence microscopy with the Leica DMI6000 B microscope at 20x magnification was performed by us on HEK293 cells transfected with LNPs containing pcDNA 3.1 eYFP DNA and mRNA. Minicircle DNA served as the positive control, while LNPs without cargo acted as the negative control. Cells were imaged at 24h, 48h, and 72h post-transfection.</p>
<p>Retrospectively, after 72h none of the LNP-treated samples showed significant fluorescence, indicating a failure in transfection (Figure 21). The lack of fluorescence in all experimental groups, except lipofectamine and Minicircle DNA, suggests either insufficient uptake of the LNPs by the cells or a failure in expression of the YFP reporter, indicating that the Corden LNP may not suited as our delivery system. Also the deformed morphology and decreased growth are indicators for negative effects of the Cayman LNP on HEK293, probably reasoned in the employment of more cytotoxic mPEG-DSPE compared to DMG-PEG in the Cayman and SORT LNP.</p>
<OneFigure
pic1="https://static.igem.wiki/teams/5247/delivery/results/precyse/corden.png"
num={21}
bg="white"
description="Overlay of phase contrast and fluorescence microscopic images of transfected HEK293 cells at 20x magnification 72h post-transfection with different Corden LNP formulations recorded with Leica DMI6000 B. For lipofectamine (lipo) + Minicircle DNA (Mini DNA) only the fluorescence image is shown."
alt1="Overlay of phase contrast and fluorescence microscopic images of transfected HEK293 cells."
/>
<H5 text="Cryo-EM"/>
<p>Cryo-EM as a form of transmission electron microscopy (TEM) was performed by us using a JEOL JEM-2200FS electron microscope (JEOL, Freising, Germany) operating at 200kV, equipped with a cold field emission electron gun. The sample preparation and imaging were carried out at cryogenic temperatures, which allowed for the visualization of LNPs in their native hydrated state.</p>
<div className="row align-items-center">
<div className="col">
<OneFigure
pic1="https://static.igem.wiki/teams/5247/delivery/results/corden-lnp.jpg"
num={22}
description="Cryo-EM image of Corden LNPs."
alt1="Cryo-EM image of Corden LNPs."
/>
</div>
<div className="col">
<p>The images reveal the presence of spherical LNP structures with an approximate size of 100 nm (Figure 22). The LNPs appear well-formed, with uniform morphology, indicating successful nanoparticle formation. In addition to individual particles, some larger, round structures were also observed, which could represent aggregated LNPs. These aggregations are a common phenomenon in LNP systems and could be attributed to interactions between particles under certain conditions.</p>
</div>
</div>
<p>While Cryo-EM provides valuable insights into the morphology and size distribution of the LNPs, there are some limitations to this technique. The imaging process involves cryogenic freezing and exposure to high-energy electron beams, which can potentially induce minor structural artifacts. Furthermore, the thinness of the sample may limit contrast, making it difficult to fully distinguish between different LNP populations or their internal structures. Despite these limitations, Cryo-EM still offers a high-resolution view of the LNPs in their near-native state, providing essential information about their size and shape.</p>
<H4 text="SORT LNP"/>
<H5 text="Transfection"/>
<p>Already at 48h of 72 monitored hours post-transfection of HEK293 with the SORT LNP, clear and strong fluorescence was already observed when using LNPs combined with Minicircle DNA, as well as in the lipofectamine and Minicircle DNA condition, confirming successful transfection and robust expression of the eYFP reporter (Figure 23). This early detection of fluorescence highlights the efficiency of SORT LNPs as delivery system. In contrast, no fluorescence was detected in any conditions involving lipofectamine alone, lipofectamine with RNA, or LNPs combined with DNA or RNA, further emphasizing the superior performance of the Minicircle DNA formulations and highlighting the need for improvement of cargo stabilization and our cargo choice.</p>
<OneFigure
pic1="https://static.igem.wiki/teams/5247/delivery/results/precyse/sort.png"
num={23}
bg="white"
description="Overlay of phase contrast and fluorescence microscopic images of transfected HEK293 cells at 20x magnification 48h post-transfection with different SORT LNP formulations recorded with Leica DMI6000 B. Lipo = lipofectamine, Mini DNA = Minicircle DNA."
alt1="Overlay of phase contrast and fluorescence microscopic images of transfected HEK293 cells."
/>
<p>Notably, the SORT LNP formulation with Minicircle DNA showed significant transfection efficiency within 48 hours, making it the most effective delivery method tested among all three LNPs. This early and robust expression demonstrates the clear advantage of this approach for HEK293 cells.</p>
<H5 text="Flow Cytometry"/>
<div className="row align-items-center">
<p>We performed flow cytometry analysis 72h post-transfection to evaluate the transfection efficiency of the SORT LNP in HEK293. The relative percentage of fluorescent cells was determined by measuring the percentage of FITC-A+ cells, followed by normalization to the negative control and fold change calculation.</p>
<div className="col">
<OneFigure
pic1="https://static.igem.wiki/teams/5247/delivery/results/sortlnp-facs.png"
num={24}
bg="white"
description="Percentage of fluorescent cells (FITC-A+) performed 72h post-transfection of SORT LNP in HEK293. Mean +/- SEM for n=3. For statistics one-way ANOVA was performed."
alt1="Percentage of fluorescent cells post-transfection of SORT LNP in HEK293."
/>
</div>
<div className="col">
<p>The SORT LNP-transfected sample carrying Minicircle DNA exhibited a significant increase in fluorescence compared to the lipofectamine transfection of Minicircle DNA, with approximately 14 times more fluorescent cells compared to the lipofectamine-transfected sample (Figure 24). This substantial difference indicates that the transfection efficiency with LNPs is markedly higher than with lipofectamine, demonstrating the superior performance of our LNP formulation in delivering nucleic acids to HEK cells.</p>
</div>
</div>
<H5 text="Zeta Potential"/>
<div className="row align-items-center">
<div className="col">
<p>We measured both the particle size distribution and the Zeta potential using the Nanotrack Wave II. We could assume that the particles exhibit a polarized Zeta potential, which is sufficient to provide electrostatic stabilization, thereby preventing aggregation and maintaining particle stability. For effective targeting of lung cells which have negatively charged surfaces, a negative polarity is desirable meaning the LNP is positively charged, so there can be electrostatic attraction to lung epithelial cells. We were able to show that our SORT LNP has these properties regardless of the load. Furthermore we could <a href="https://chem.libretexts.org/Bookshelves/Analytical_Chemistry/Physical_Methods_in_Chemistry_and_Nano_Science_(Barron)/02%3A_Physical_and_Thermal_Analysis/2.05%3A_Zeta_Potential_Analysis" title="StabZeta" >determine the stability via the Zeta potential</a>. In detail the mean of the Zeta potential lays at 16.2 mV for the SORT LNP with Minicircle DNA as cargo, indicating incipient stability, at 59.45 mV for the SORT LNP with pcDNA 3.1 eYFP as cargo, indicating good stability and at 88.22 mV for the SORT LNP without cargo indicating excellent stability (Figure 25). The good stability of the SORT LNP with pcDNA 3.1 eYFP is crucial for our purposes, as it ensures effective delivery and performance. In contrast, the stability of the LNPs with Minicircle DNA can be considered secondary, as it primarily serves as a positive transfection control and is not central to our main objectives.</p>
</div>
<div className="col">
<OneFigure
pic1="https://static.igem.wiki/teams/5247/delivery/results/sort-zeta.webp"
num={25}
bg="white"
description="Zeta potential of SORT LNP with different cargos measured with Nanotrack Wave II indicating varying degrees of stability but most important good stability for the SORT LNP loaded with pcDNA 3.1 eYFP (LNP DNA). Mean +/- SEM for n=5. For statistics one-way ANOVA was performed."
alt1="Zeta potential of SORT LNP with different cargos."
/>
</div>
</div>
<div className="row align-items-center">
<div className="col">
<OneFigure
pic1="https://static.igem.wiki/teams/5247/delivery/results/screenshot-2024-10-01-204938.png"
num={26}
bg="white"
description="Size distribution for the SORT LNP with different cargos weighted by scattering intensity measured with Nanotrack Wave II."
alt1="Size distribution for the SORT LNP with different cargos."
/>
</div>
<div className="col">
<p>The size distribution for all three samples shows a predominantly monomodal, yet broad, distribution with diameters ranging between 50 nm and 700 nm, with the peak of the distribution lying between 150 nm and 200 nm (Figure 26). SORT LNPs without DNA exhibited larger radii, with a peak around 300 nm. The SORT LNP containing Minicircle DNA suggests the presence of larger aggregates with diameters exceeding 1 µm. The likely reason for this variable particle size distribution, despite loading with different types of DNA, could be attributed to the manufacturing method. Since the LNPs were not produced using an extruder but rather via dialysis, this is highly plausible.</p>
</div>
</div>
<H5 text="Cryo-EM"/>
<div className="row align-items-center">
<div className="col">
<p>Cryo-EM analysis was also performed of SORT LNPs by us with the same JEOL JEM-2200FS microscope at 200kV, allowing visualization of LNPs in their native hydrated state. The images show spherical LNP structures around 100 nm, with some larger aggregates also present. These aggregates likely result from interactions between particles due to the non-extrusion-based preparation method, which may explain the variability in particle size. Additionally, particles potentially representing different LNP populations or overlapped structures with low contrast were observed (Figure 27). The low sample concentration likely contributed to the limited number of visible particles.</p>
</div>
<div className="col">
<OneFigure
pic1="https://static.igem.wiki/teams/5247/delivery/results/sortcryoem.png"
num={27}
description="Cryo-EM image of SORT LNPs. The different colored outlines indicate different size populations of LNPs."
alt1="Cryo-EM image of SORT LNPs with different size populations."
/>
</div>
</div>
<p>Overall, while the Cryo-EM data confirm the presence and general morphology of LNPs that also fall within the diameter range specified by Wang et al. for SORT LNPs at smaller than 200 nm <SupScrollLink label="1"></SupScrollLink>. The variability in size and the presence of aggregates highlight potential areas for optimization, such as refining sample concentration and preparation methods to achieve more consistent particle formation.</p>
<H5 text="DLS"/>
<p>We used Dynamic Light Scattering (DLS) to assess the size distribution of our SORT LNPs by measuring the fluctuations in scattered light due to particle motion. The hydrodynamic diameter was calculated using the Stokes-Einstein equation, considering the diffusion coefficient, temperature, and viscosity of the medium.</p>
<div className="row align-items-center">
<div className="col">
<OneFigure
pic1="https://static.igem.wiki/teams/5247/delivery/results/sort-dls.webp"
num={28}
bg="white"
description="Results for hydrodynamic radius determination by DLS Measurements for our SORT LNP, indicating a radius of approximately 100 nm."
alt1="Hydrodynamic radius of SORT LNP measured by DLS."
/>
</div>
</div>
<div className="col">
<p>The results showed a hydrodynamic diameter of SORT LNPs yielding an average radius of approximately 100 nm (Figure 28). These findings are consistent with our previous applied size determination methods, such as Zeta potential and Cryo-EM, which also indicated similar particle dimensions in appropriate range for our research and medical applications.</p>
</div>
</div>
<H5 text="MTT Assay"/>
<div className="row align-items-center">
<div className="col">
<OneFigure
pic1="https://static.igem.wiki/teams/5247/fanzor/sort-mtt.webp"
num={29}
bg="white"
description="MTT Assay of LNPs from all iterations performed on HEK293 including Triton as negative control and untreated cells as positive control. Mean +/- SEM for n=6. For statistics one-way ANOVA was performed."
alt1="MTT Assay results of LNPs on HEK293 cells."
/>
</div>
<div className="col">
{/* <p>In order to evaluate the <a onClick={() => goToPageAndScroll ('Biosafety2', '/safety')}>biosafety</a> of our lung-specific LNPs, particularly concerning the choice of <a onClick={() => goToPagesAndOpenTab({collapseId: 'Col1', path: '/engineering', tabId: 'delivery' })}>PEG</a> - known to cause cytotoxicity issues - we performed MTT assays using HEK293 cells with various LNP formulations. The results demonstrated that the Cayman LNP achieved 74.90% viability and SORT LNP showed 75.01% viability, exhibiting lower cytotoxicity due to the inclusion of DMG-PEG, a less cytotoxic PEG variant compared to mPEG-2000-DSPE, which resulted in 66.69% viability in the Corden LNP (Figure t). These findings prove we made the best decision by choosing the SORT LNP as the least cytotoxic LNPs.</p> */}
<p>In order to evaluate the <a onClick={() => goToPageAndScroll ('Biosafety2', '/safety')}>biosafety</a> of our lung-specific LNPs, particularly concerning the choice of PEG - known to cause cytotoxicity issues - we performed MTT assays using HEK293 cells with various LNP formulations. The results demonstrated that the Cayman LNP achieved 74.90% viability and SORT LNP showed 75.01% viability, exhibiting lower cytotoxicity due to the inclusion of DMG-PEG, a less cytotoxic PEG variant compared to mPEG-2000-DSPE, which resulted in 66.69% viability in the Corden LNP (Figure 29). These findings prove we made the best decision by choosing the SORT LNP as the least cytotoxic LNPs.</p>
</div>
</div>
<H4 text="AirBuddy"/>
<p>Now we present our results including the chitosan-complexation of the therapeutic RNA cargo, which withdraws AirBuddy from basic lung-specific SORT LNPs.</p>
<p>We successfully transfected a well-established lung epithelial cell line CFBE41o- with our chitosan-RNA complexes using our synthesized pcDNA 3.1 eYFP mRNA, which were subsequently incubated with SORT LNPs for packaging. The goal was to encapsulate the chitosan-RNA complexes within the LNPs to enhance mRNA delivery to cells. After formulation, the LNP chitosan-RNA complexes were transfected into CFBE41o- cells to assess the efficiency of mRNA delivery and expression in a CF patient bronchial cell line being closer to the clinically relevant model.</p>
<p>We tested two different chitosan and mRNA concentrations (always used both in the same concentrations), 50 ng/µl and 500 ng/µl, in combination with our LNP to assess their impact on transfection efficiency. Interestingly, both concentrations resulted in similarly high levels of transfection, as evidenced by the fluorescence microscopy images, further validation is necessary to confirm these results.</p>
<p>Fluorescence microscopy was performed 24 hours post-transfection to evaluate the expression of the YFP reporter gene encoded by the pcDNA 3.1 eYFP mRNA. This reporter gene serves as an indicator of successful transfection and translation of the mRNA into protein. The results of the fluorescence microscopy were highly positive and aligned with our expectations, indicating efficient delivery, uptake, and expression of the mRNA in the CFBE41o- cells.</p>
<H5 text="Transfection"/>
<p>We tested two different chitosan and mRNA concentrations (always used both in the same concentrations), 50 ng/µl and 500 ng/µl, in combination with our LNP to assess their impact on transfection efficiency. Interestingly, both concentrations resulted in similarly high levels of transfection, as evidenced by the fluorescence microscopy images, further validation is necessary to confirm these results.</p>
<div className="row align-items-center">
<div className="col">
<OneFigure
pic1="https://static.igem.wiki/teams/5247/delivery/results/precyse/lnp-chito-rna500-t24.webp"
num={30}
description="Overlay of phase contrast and fluorescence microscopic images of with SORT + chitosan 500 transfected CFBE41o- cells at 20x magnification 24h post-transfection recorded with Leica DMI6000 B."
alt1="Images of SORT + chitosan 500 transfected CFBE41o- cells."
/>
</div>
<div className="col">
<p>The sample containing 500 ng/µl of chitosan and pcDNA 3.1 eYFP mRNA, encapsulated within SORT LNPs, exhibited fluorescence 24 hours post-transfection (Figure 30). This result indicates that the RNA was successfully delivered into the CFBE41o- cells, where it was transcribed and translated into the YFP protein. The presence of YFP fluorescence confirms not only successful transfection but also robust expression of the reporter gene.</p>
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<p>In the sample containing 50 ng/µl of chitosan and pcDNA 3.1 eYFP mRNA was used, which is tenfold lower than the chitosan and mRNA concentration used in the chitosan500 sample (see above). Despite the lower mRNA concentration, fluorescence was still observed (Figure 31) indicating that the mRNA was efficiently delivered and expressed in the CFBE41o- cells. This result suggests that even at lower mRNA doses, the system can achieve successful transfection and gene expression. We are planning to perform a more detailed comparison of fluorescence intensity between the chitosanRNA500 and chitosanRNA50 samples and even lower concentrations to assess the relationship between mRNA dose and expression level.</p>
</div>
<div className="col">
<OneFigure
pic1="https://static.igem.wiki/teams/5247/delivery/results/precyse/lnpchito-rna50-t24.webp"
num={31}
description="Overlay of phase contrast and fluorescence microscopic images of with SORT + chitosan 50 transfected CFBE41o- cells at 20x magnification 24h post-transfection recorded with Leica DMI6000 B."
alt1="Images of SORT + chitosan 50 transfected CFBE41o- cells."
/>
</div>
</div>
<p>The samples containing only chitosan (Figure 32) and only SORT LNP (Figure 33) without any mRNA, did not exhibit any detectable fluorescence. This result is consistent with expectations, as chitosan alone and SORT LNP alone are not fluorescent. These samples served as important negative controls to confirm that the chitosan itself and the SORT LNP itself don't interfere with the fluorescence signal.</p>
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<OneFigure
pic1="https://static.igem.wiki/teams/5247/delivery/results/precyse/chito-t24-1.jpeg"
num={32}
description="Overlay of phase contrast and fluorescence microscopic images of with SORT transfected CFBE41o- cells at 20x magnification 24h post-transfection recorded with Leica DMI6000 B."
alt1="Images of SORT transfected CFBE41o- cells."
/>
</div>
<div className="col">
<OneFigure
pic1="https://static.igem.wiki/teams/5247/delivery/results/precyse/lnp-leer-t24.webp"
num={33}
description="Overlay of phase contrast and fluorescence microscopic images of with chitosan transfected CFBE41o- cells at 20x magnification 24h post-transfection recorded with Leica DMI6000 B."
alt1="Images of chitosan transfected CFBE41o- cells."
/>
</div>
</div>
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<div className="col">
<OneFigure
pic1="https://static.igem.wiki/teams/5247/delivery/results/precyse/ntc-t24.webp"
num={34}
description="Overlay of phase contrast and fluorescence microscopic images of untransfected CFBE41o- cells at 20x magnification 24h post-transfection recorded with Leica DMI6000 B."
alt1="Images of untransfected CFBE41o- cells."
/>
</div>
<div className="col">
<p>The untreated control, which did not receive any chitosan, RNA, or LNPs, did not show any fluorescence (Figure 34). This negative control confirms that the cells themselves do not express YFP under normal conditions, and that any observed fluorescence in the experimental groups is directly attributable to the transfection and expression of the delivered RNA.</p>
</div>
</div>
<p>The results demonstrate that chitosan-RNA complexes, when packaged with SORT LNPs, can efficiently deliver mRNA into CFBE41o- cells and facilitate the expression of a YFP reporter gene. Furthermore, the absence of fluorescence in the control samples validates the specificity of the system, ensuring that the fluorescence signal is solely due to the delivered mRNA and not from other components of the system. Additional testing, such as flow cytometry analysis, is planned to provide a more quantitative assessment of transfection efficiency. This would allow us to accurately determine whether there are subtle differences between the two concentrations that were not detectable by microscopy alone.</p>
<p>Overall, this experiment highlights the potential of chitosan-RNA complexes in combination with SORT LNPs as a promising platform for mRNA delivery and gene expression in airway epithelial cells. Further investigation could focus on optimizing the mRNA dose to maximize expression levels and transfection efficiency.</p>
</Subesction >
<Subesction title="Primary Cell Culture" id="Experimental Design4">
<H4 text="Goals"/>
<p>The central aim of the project was to obtain epithelial cells from <strong>nasopharyngeal swabs</strong> and successfully culture them. The aim was to develop a cell model that is as close as possible to the physiological conditions of the human airway epithelium in the laboratory. Two different cultivation methods were used in the project: <strong>Air-Liquid Interface (ALI) culture</strong> as a 2D model and <strong>Apical-Out Airway Organoid (AOAO) culture</strong> as a 3D model. </p>
<p>The ALI culture allows the simulation of the air-side differentiation of the epithelium, so that cell interactions can be simulated in an environment that mimics the natural barrier function of the airways. In contrast, the AOAO cultures represent a more complex, three-dimensional model that more closely mimics the cellular organisation and function of the airway epithelium. </p>
<p>The cell models generated should provide a reliable basis for reproducing the in vivo conditions of the human nasal and airway epithelium as closely as possible in vitro. </p>
<H4 text="Workflow"/>
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<p>The workflow of this experiment started with the collection of primary human nasal epithelial cells (hNECs) from nasopharyngeal swabs of human volunteers. This method allowed the non-invasive isolation of cells that represent a suitable model for the respiratory epithelium. After collection, the cells were isolated and cultured in a special medium to ensure their proliferation. This step was essential to generate a sufficient number of cells for the subsequent experiments. </p>
<p>The next step was to culture the isolated cells in two different systems: the Air-Liquid Interface (ALI) culture as a 2D model and the Apical-Out Airway Organoid (AOAO) culture as a 3D model. In the ALI culture, the cells were grown on a porous membrane with the basal side of the cells in contact with a culture medium and the apical side exposed to air. This method allowed the cells to differentiate into a multilayered epithelium that reproduced different cell types, such as ciliated cells, goblet cells and basal cells, typical of the respiratory epithelium. </p>
<p>In parallel, a 3D model was established in AOAO culture in which the cells form spherical organoids with the apical side facing outwards. This 3D structure more closely mimicked the physiological conditions of the human airway epithelium and offered extended possibilities for studying cell-cell interactions in space. </p>
</div>
<div className="col">
<OneFigure
pic1="https://static.igem.wiki/teams/5247/photos/results/img-2217.webp"
num={35}
description="Schematic overview of the isolation and culture of primary human nasal epithelial cells (hNECs). First, nasopharyngeal swabs are taken to obtain the cells. The cells are then cultured in two different culture systems: Apical-Out-Airway-Organoids (AOAO) as a 3D culture (left) and Air-Liquid-Interface (ALI) as a 2D culture (right). In the 3D culture, the cells form a spherical organoid with an outward-facing apical surface, whereas in the 2D culture, the cells form a differentially structured epithelial barrier that mimics different cell types such as ciliated cells, goblet cells and basal cells."
alt1="Overview"
/>
</div>
</div>
<p>Characterisation of the cells in both culture systems was performed by a combination of microscopic examination and histological staining. In particular, haematoxylin-eosin (HE) and periodic acid-Schiff (PAS) staining was performed in the ALI culture to analyse cell differentiation and the formation of mucus-forming goblet cells. HE staining was used for general visualisation of cell morphology and cell stratification, while PAS staining was used specifically to identify the mucins in the goblet cells. These histological techniques allowed a detailed assessment of epithelial differentiation, in particular the maturation of ciliated and mucus-producing cells. </p>
<p>Once the cell cultures had been successfully established, a key objective of the project was pursued: To test gene our therapy approach in these cell models. The cells would be used to assess the efficacy and safety of new gene therapies before they could potentially be used in clinical applications. </p>
<H4 text="Conclusion"/>
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<OneFigure
pic1="https://static.igem.wiki/teams/5247/photos/results/a.webp"
num={36}
description="Histochemical staining of air liquid interface culture. A) HE staining with goblet cell visible. B) HE staining with goblet cell visible. C) Overview of differnetiated epithelium after PAS staining."
alt1="HE and PAS staining"
/>
</div>
<div className="col">
<p>The <strong>successful cultivation of primary human nasal epithelial cells (hNECs)</strong> was clearly demonstrated in both two-dimensional and three-dimensional cultures. In the ALI culture, a well-differentiated ciliated epithelium was observed microscopically, exhibiting the characteristic ciliary dynamics. Histological staining, in particular HE and PAS staining, provided confirmation of the differentiation of the cells. The multilayered epithelial structure and the presence of mucus-producing goblet cells were both clearly demonstrated (Figure 36 A, B). </p>
</div>
</div>
<p>The Apical-Out Airway Organoid culture also demonstrated successful differentiation of the cells, which formed an epithelial cell network. The cells exhibited a spherical morphology and were observed to float in the culture, thereby mimicking the natural physiological conditions of the respiratory epithelium. The various 2D and 3D models thus provide a promising foundation for further research and the application of gene therapy approaches. </p>
</Subesction >
<Subesction title="Downstream Application" id="Experimental Design5">
<p>To validate our gene editing approach by prime editing of CFTR F508del delivered to lung cells via SORT LNPs, we planned to use <a onClick={() => goToPageAndScroll ('Patch Clamp', '/materials-methods')}>Patch Clamp</a> as a downstream method. Our goal was to detect the restored conductance of the repaired CFTR by this electrophysiological method. This was made possible through the assistance of the <a onClick={() => goToPagesAndOpenTab('patchclamp', '/human-practices')}>Cellular Neurophysiology research group</a> at our university.</p>
<H4 text="Initial Measurements"/>
<div className='row align-items-center'>
<div className='col'>
<OneFigure
pic1="https://static.igem.wiki/teams/5247/photos/results/patchclamp/pc.webp"
num={37}
bg="white"
description="Current density of HEK293, HEK293T CFTR WT and HEK293T CFTR F508del showing significant differences of both HEK293T cell lines compared to HEK293 but no significant differences between them. For statistics one-way ANOVA was performed."
alt1="Current density differences between HEK293 and HEK293T CFTR cell lines"
/>
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<div className='col'>
<p>In our first set of experiments, we measured current density in <a onClick={() => goToPageAndScroll ('Cell Culture', '/materials-methods')}>HEK293T CFTR wild type (WT) and HEK293T F508del</a> cell lines, comparing them with regular HEK293. The results demonstrated significant differences in chloride ion conductance, with the CFTR-expressing cell lines showing enhanced conductivity compared to HEK293 (Figure 37). However, a drawback was that we did not observe any significant differences between the HEK293T CFTR WT and F508del cell line. This was unexpected, as the F508del mutation typically leads to a knockdown of the CFTR protein<SupScrollLink label="2"></SupScrollLink>, impairing chloride ion transport through the CFTR channel.</p>
</div>
</div>
<H4 text="Further Validation and Challenges"/>
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<div className='col'>
<p>In light of these results, we improved our experimental setup and performed additional validation experiments. Unfortunately, the repeated measurements yielded similar outcomes, confirming the absence of a significant difference between the two CFTR-expressing cell lines (Figure 38). This finding led us to consult with the research group at <a onClick={() => goToPagesAndOpenTab('mattijsvisit', '/human-practices')}>KU Leuven</a>, who established these cells lines. Although they had not conducted similar Patch Camp measurements, they suggested an alternative approach using Ussing Chamber measurements. This technique, unlike Patch Camp, does not rely on single-cell measurements but rather examines the ion currents across the entire cell monolayer, which may provide a more comprehensive view of CFTR functionality.</p>
</div>
<div className='col'>
<OneFigure
alt1=""
pic1="https://static.igem.wiki/teams/5247/photos/results/patchclamp/pc2.webp"
num={38}
bg="white"
description="Repeated validation of current density measurements in HEK293T CFTR WT and HEK293T CFTR-F508del, showing consistent results with the initial experiment. Mean +/- SEM for n=5. For statistics one-way ANOYA was performed."
/>
</div>
</div>
<Subesction title="Combining Primeguide and AirBuddy" id="Experimental Design6">
<H5 text="Transfection"/>
<div className='col'>
<p>Fluorescence microscopy was performed using the Leica DMI6000 B microscope at 20x magnification to analyze CFBE4o- cells. These cells were initially pre-transfected with pPEAR_CFTR to establish a baseline for Prime Editing activity. Subsequently, transfection of pCMV-PE6c and pegRNA4, delivered via the AirBuddy system, was carried out. LNPs without cargo acted as the negative control. Imaging was conducted at 24h, 48h, and 72h post-transfection to assess fluorescence intensity and evaluate editing efficiency over time.</p>
</div>
<div className='col'>
<OneFigure
alt1=""
pic1="https://static.igem.wiki/teams/5247/photos/results/precyse-results.webp"
num={39}
bg="white"
description="Microscopy of CFBE4o- 48h after transfection. Cells were previoulsy transfectet with pPEAR_CFTR and later transfected with pCMV-PE6c + pegRNA4 via AirBuddy. BF = Brightfield "
/>
<p>The results show a sucsessful transfection of pCMV-PE6c and pegRNA4 encapsulated in our LNPs was achieved following prior transfection with pPEAR_CFTR. The experimental setup demonstrated an increase in fluorescence intensity, indicative of enhanced Prime Editing efficiency under these conditions.
Moreover, the PreCyse results confirm the effectiveness of our strategy, showcasing the synergy between our proprietary PrimeGuide system and the AirBuddy platform. This combination has proven to be pivotal in optimizing the editing outcomes and advancing the precision of the Prime Editing process.
These findings underscore the robustness of our delivery approach and the innovative integration of the PrimeGuide and AirBuddy systems in driving Prime Editing to new levels of efficiency and accuracy.</p>
</div>
</Subesction>
</Subesction >
</Section>
<Section title="Supplementary Material" id="Supplementary Material">
<p><DownloadLink url="https://static.igem.wiki/teams/5247/pdfs/raw-data-patch-clamp.pdf" fileName="raw-data-patch-clamp.pdf" /> Supplementary Material for Patch Clamp</p>
<DownloadLink url="https://static.igem.wiki/teams/5247/pdfs/raw-data-delivery.pdf" fileName="raw-data-delivery.pdf" /> Supplementary Material for Delivery
</Section>
<ResultSources/>
</>
);
}