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src/contents/notebook.tsx
View file @ 3fd292fd
import H1 from "../components/Headings";
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">
<strong>
<H1 text="Ich bin ein Header!"/>
</strong>
<i>
<p> Ich bin ein Paragraph. </p>
</i>
<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
......@@ -7,7 +7,7 @@ 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 text="A big thank you to all our sponsors and partners!"></H1>
<br/>
......@@ -96,34 +96,34 @@ export function Partners() {
<div className="row align-items-center">
<div className="col">
<a className="sponsor-container" href="https://www.zymoresearch.com/">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/zymo.png"/>
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/zymo.png"/>
</a>
</div>
<div className="col">
<a className="sponsor-container" href="https://www.stemcell.com/">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/stemcell-logo.png"/>
<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">
<a className="sponsor-container" href="https://www.drwolffgroup.com/en/">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/logo-wolff.png"/>
<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="https://bio.nrw.de/">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/bionrw-logo.png"/>
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/bionrw-logo.png"/>
</a>
</div>
</div>
......@@ -136,17 +136,17 @@ export function Partners() {
<div className="row align-items-center">
<div className="col">
<a className="sponsor-container" href="https://www.promega.com">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/promega-gelb.png"/>
<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="https://www.microsynth.com">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/microsynth-logo.png"/>
<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="https://www.neb.com/en/">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/neb-logo.png"/>
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/neb-logo.png"/>
</a>
</div>
</div>
......@@ -165,43 +165,43 @@ export function Partners() {
<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">
<a className="sponsor-container" href="https://v-bio.ventures/">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/vbio-logo.png"/>
<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">
<a className="sponsor-container" href="https://www.mn-net.com/">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/mn-logo.png"/>
<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">
<a className="sponsor-container" href="https://www.fiz-biotech.de/">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/fiz-logo.png"/>
<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">
<a className="sponsor-container" href="https://www.cellsignal.com/">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/cell-signaling-technology-logo.png"/>
<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">
<a className="sponsor-container" href="https://gasb.de/">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/gasb-logo.jpg"/>
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/gasb-logo.jpg"/>
</a>
</div>
</div>
......@@ -214,41 +214,41 @@ export function Partners() {
<div className="row align-items-center">
<div className="col">
<a className="sponsor-container" href="https://www.asimov.com/">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/asimov-colorful.png"/>
<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="https://algenium.de/algenium/">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/algenium.png"/>
<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="https://2024.igem.wiki/gu-frankfurt/">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/logos-team/other-teams/gu-frankfurt-logo.png"/>
<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="https://2024.igem.wiki/hamburg/">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/logos-team/other-teams/igem-hamburg-logo.png"/>
<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="https://www.stud-scicom.de/">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/studscicom-logo.png"/>
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/studscicom-logo.png"/>
</a>
</div>
</div>
......@@ -279,22 +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">
<a className="sponsor-container" href="https://www.sarstedt.com/en/">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/sarstedt-logo.png"/>
<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="https://cordenpharma.com/">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/corden-pharma-logo.png"/>
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/corden-pharma-logo.png"/>
</a>
</div>
</div>
......@@ -304,12 +304,12 @@ export function Partners() {
</div>
<div className="col">
<a className="sponsor-container" href="https://www.capricorn-scientific.com/en">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/capricorn-logo.png"/>
<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="https://www.twistbioscience.com/">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/twist-bioscience-logo.png"/>
<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">
......
src/contents/parts.tsx
View file @ 3fd292fd
import { LoremMedium } from "../components/Loremipsum";
import { Section, Subesction } from "../components/sections";
import { PartTable } from "../components/Table";
import { useTabNavigation } from "../utils/TabNavigation";
import { BasicParts, CompositeParts } from "../data/parts";
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";
export function Parts() {
useTabNavigation();
let headcols = ["Part Name", "Registry Code", "Part Description", "length [bp]", "type"]
return (
return (
<div className="col">
<Section title="Introduction" id="Introduction">
<Subesction title="Description" id="Introduction1">
<LoremMedium/>
<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="Characterization" id="Introduction2">
<LoremMedium/>
<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>
<Section title="Process" id="Process">
<Subesction title="EC" id="Process1">
<LoremMedium/>
<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="Design and Build" id="Process2">
<LoremMedium/>
<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>
<Section title="Experiments" id="Experiments">
<Subesction title="Cloning" id="Experiments1">
<LoremMedium/>
<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="Nikase-Assay" id="Experiments2">
<LoremMedium/>
<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>
</Subesction>
<Subesction title="Activity Experiments" id="Experiments3">
<LoremMedium/>
<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="Plasmids" id="Parts Collection1">
<LoremMedium/>
</Subesction>
<Subesction title="Basic Parts" id="Parts Collection2">
<Subesction title="Basic Parts" id="Parts Collection1">
<PartTable cols={headcols} data={BasicParts}/>
</Subesction>
<Subesction title="Composite Parts" id="Parts Collection3">
<LoremMedium/>
</Subesction>
</Section>
<Section title="References" id="References">
<ol>
<PartSources/>
</ol>
</Section>
</div>
);
......
src/contents/project-documentation.tsx
View file @ 3fd292fd
import { Calendar } from "../components/Calendar/Calendar";
import { H2 } from "../components/Headings";
import { PDF } from "../components/Pdfs";
import { useTabNavigation } from "../utils/TabNavigation";
......@@ -6,7 +7,8 @@ export function ProDesc() {
useTabNavigation();
return (
<div className="col">
<Calendar/>
<H2 text="Our Meeting Protocols"/>
<PDF link="https://static.igem.wiki/teams/5247/pdfs/meetings.pdf" name="meetings.pdf"/>
</div>
);
}
src/contents/results.tsx
View file @ 3fd292fd
import { H4 } from "../components/Headings";
import { LoremMedium } from "../components/Loremipsum";
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 (
<>
<Section title="Abstract" id="Abstract">
<p>We have successfully demonstrated a <b>proof of concept</b> for our gene therapy approach targeting cystic fibrosis.
In initial experiments, HEK cells carrying a 3-base deletion analogous to the F508del mutation were transfected
with our prime editing complex. The results met our expectations, confirming the viability of our approach for
precise gene correction. Based on these findings, we optimized the prime editing complex, leading to the creation
of PrimeGuide, a more compact and efficient editing tool. </p>
<p>Central to our <b>delivery system</b> is <b>AirBuddy</b>, a lung-specific lipid nanoparticle designed to stabilize and protect
the prime editing complex during transport to lung ionocytes. AirBuddy ensures that the protein complex is
delivered specifically to lung cells, enhancing the efficiency of the gene-editing process. By modifying the
lipid nanoparticle with protective features, we achieved increased stability, ensuring effective delivery to the
target cells. </p>
<p>We further optimized the fusion protein, <b>PrimeGuide</b>, to streamline its components, resulting in a smaller and
more efficient prime editing complex. This improvement significantly enhances the precision of the gene editing
process, reducing off-target effects and increasing the overall success of mutation correction. </p>
<p>In subsequent experiments, HEK cells carrying the CFTR F508del mutation were successfully transfected with the
optimized prime editing complex. Our results indicated successful correction of the mutation, confirming the
potential of our approach for treating cystic fibrosis. </p>
<p>
Additionally, we explored downstream applications. Primary cell cultures were treated with lipid nanoparticles to
introduce a reporter RNA. We also established 2D cultures transfected with YFP, a sodium-sensitive reporter protein,
to assess ion channel functionality. Finally, in CFTR-deficient organoids, our system facilitated repair of the CFTR
channel, evidenced by an increase in organoid volume upon treatment. This suggests successful functional restoration
of CFTR activity.
</p>
<Subesction title="Introduction" id="Abstract1">
<p>Cystic fibrosis (CF) is a severe genetic disorder caused by mutations in the CFTR gene, most commonly the F508del
mutation. This mutation leads to defective ion channels in lung cells, causing mucus buildup, chronic lung infections,
and progressive respiratory failure. Current therapies primarily target symptoms, but a definitive cure remains elusive.
To address this, we aim to develop a targeted gene therapy utilizing prime editing technology. This approach focuses
on correcting the CFTR F508del mutation with precision and efficiency. Our innovative system integrates a
next-generation prime editing complex (PrimeGuide) with a lung-specific delivery platform (AirBuddy) to achieve
targeted and stable delivery to lung ionocytes. </p>
<p>Our initial proof of concept successfully demonstrated the functionality of the prime editing complex in HEK cells
carrying a 3-base deletion analogous to the F508del mutation. Building on these promising results, we optimized the
prime editing complex, creating PrimeGuide, a smaller and more effective editing tool. Additionally, we developed
AirBuddy, a lung-specific lipid nanoparticle system designed to protect and transport the prime editing complex
directly to lung cells. </p>
<p>This project not only focuses on mutation correction but also validates the gene therapy’s functional restoration
in relevant cell models, including primary cultures and organoids. By exploring downstream applications, we aim to
offer a promising therapeutic option for cystic fibrosis, potentially paving the way for similar approaches in
personalized medicine. </p>
</Subesction>
<Subesction title="Goals and Milestones" id="Abstract2">
<p><b>Develop a gene therapy</b> for cystic fibrosis to correct the CFTR F508del mutation using prime editing technology. </p>
<p><b>Optimize the prime editing complex</b> (PrimeGuide) to increase efficiency, precision, and reduce off-target effects. </p>
<p><b>Create a lung-specific delivery system</b> (AirBuddy) for stable and targeted delivery of the prime editing complex to lung ionocytes.</p>
<p><b>Validate mutation correction</b> in CFTR mutant cells and primary human cultures. </p>
<p><b>Demonstrate functional recovery</b>of the CFTR channel in treated cells and organoids, confirming the therapeutic potential. </p>
</Subesction>
<><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">
<H4 text="acc. to David Liu (Anzalone et al. 2019)"/>
<LoremMedium/>
</Subesction>
<Subesction title="Proof of Concept" id="Experimental Design1">
<h4>acc. to David Liu (Anzalone et al. 2019)</h4>
<H4 text="Goals"/>
<p>Develop a targeted gene therapy for cystic fibrosis using prime editing technology. </p>
<H4 text="Workflow"/>
<p></p>
<H4 text="Conclusion "/>
<LoremMedium/>
</Subesction>
<Subesction title="PrimeGuide " id="Experimental Design2">
<H4 text="Goals"/>
<p>Optimize the prime editing complex (PrimeGuide) for efficient correction of the CFTR F508del mutation. </p>
<H4 text="Workflow"/>
<p></p>
<H4 text="Conclusion "/>
<LoremMedium/>
</Subesction>
<Subesction title="LNP Synthesis " id="Experimental Design3">
<H4 text="Goals"/>
<p></p>
<H4 text="Workflow"/>
<p></p>
<H4 text="Conclusion "/>
<LoremMedium/>
</Subesction>
<Subesction title="Cellculture " id="Experimental Design4">
<H4 text="Goals"/>
<p></p>
<H4 text="Workflow"/>
<p></p>
<H4 text="Conclusion "/>
<LoremMedium/>
</Subesction>
<Subesction title="Downstream Applications " id="Experimental Design5">
<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"/>
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<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>
<H4 text="Cayman LNP"/>
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<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"/>
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<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"/>
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<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>
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<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"/>
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<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>
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<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"/>
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<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>
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<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 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"/>
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<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>
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<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>
</div>
</div>
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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></p>
<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"/>
<p></p>
<H4 text="Conclusion "/>
<LoremMedium/>
</Subesction>
<div className="row align-items-center">
<div className="col">
<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"/>
<div className="row align-items-center">
<div className="col">
<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"
/>
</div>
<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"/>
<div className='row align-items-center'>
<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/>
</>
);
}
src/contents/safety.tsx
View file @ 3fd292fd
import { H4, H5 } from "../components/Headings";
import { LoremMedium } from "../components/Loremipsum";
import PreCyse from "../components/precyse";
import { SupScrollLink } from "../components/ScrollLink";
import { Section, Subesction } from "../components/sections";
import Collapsible from "../components/Collapsible";
import { useNavigation } from "../utils";
import { TwoLinePDF } from "../components/Pdfs";
import { useTabNavigation } from "../utils/TabNavigation";
import { OneFigure } from "../components/Figures";
// message for test commit.
export const Safety: React.FC = () =>{
const {goToPageAndScroll, goToPageWithTabAndScroll, goToPagesAndOpenTab} = useNavigation();
useTabNavigation();
return (
<>
<Section title="Role in iGEM" id="Role">
<p>
As part of our project to develop a prime-editing complex to correct the F508del mutation in cystic fibrosis, we place great emphasis on safety at all stages of research. Our final construct will be tested in primary cultures of epithelial cells obtained from nasal swabs, isolated from both patients and healthy individuals. from nasal swabs [link primär Kulturen]. To guarantee safety and ensure the highest level of precision and reliability of our results, we have introduced a series of carefully planned checkpoints during the experiments. These milestones allow for continuous monitoring, timely adjustments and validation at each critical stage. This ensures that potential issues are identified and addressed immediately, minimizing risk and improving the overall quality of the experimental results. [link zu den Experimenten]
As part of our project <PreCyse/> to develop a prime-editing complex to correct the F508del mutation in Cystic Fibrosis, we place great emphasis on safety at all stages of research. Our final construct will be tested in <a onClick={() => goToPageAndScroll ('Cell Culture3H', '/materials-methods')}> primary cultures of nasal epithelial cells </a> obtained from nasal swabs, isolated from both patients and healthy individuals. To guarantee safety and ensure the highest level of precision and reliability of our results, we have introduced a series of carefully planned checkpoints during the experiments. These milestones allow for continuous monitoring, timely adjustments and validation at each critical stage. This ensures that potential issues are identified and addressed immediately, minimizing risk and improving the overall quality of the experimental results.
</p>
</Section>
<Section title="Check-Ins" id="Check-Ins">
<div>
<p>
iGEM places great emphasis on biosafety, ensuring that all projects adhere to strict safety standards. One of these measures is the iGEM White List, which includes organisms and parts that are pre-approved for use based on their safety profile. Any components or organisms not covered by this White List must be submitted as 'Check-ins' to the iGEM Safety Committee for approval. Check-ins are formal safety evaluations that allow the committee to assess the potential risks and ensure proper containment and handling procedures are in place. Although we used some parts and organisms that were not included on the White List, these were assessed as critical for our project and submitted as Check-ins to the iGEM Safety Committee. Furthermore, we were in active exchange with the committee throughout the process. The Check-ins provide a clear picture of the biosafety aspects of our project, reflecting our commitment to safety and compliance with iGEM standards.
</p>
<p>
We adhere to good laboratory practices by ensuring proper handling of materials, effective emergency procedures, and correct waste disposal methods. This commitment guarantees a safe and compliant research environment. Our project, which involved a wide range of techniques was conducted in strict compliance with safety regulations. All experiments were carried out in Prof. Dr. Kristian Müller’s laboratory at Bielefeld University, following BSL-1 standard operating procedures. Properly equipped facilities are crucial to prevent contamination, exposure, or accidental release of modified organisms, ensuring the highest level of safety in our laboratories.
Before commencing laboratory work, all participants were required to attend a mandatory safety briefing. In compliance with German regulations, each team member's participation had to be confirmed with a personal signature. The briefing, conducted by Prof. Dr. Kristian Müller must be renewed annually in accordance with §12 ArbSchG. It covered the following areas:
</p>
<p>
General laboratory safety
</p>
<p>
Regulations regarding hazardous and toxic substances
</p>
<p>
Regulations concerning biological materials
</p>
<p>
Regulations on genetic engineering. In addition to the general safety briefing, specific instructions for the safe operation of each device were provided. The Safety and Security Officer within the laboratory highlighted the potential hazards and necessary precautionary measures. We have focused on planning our laboratory activities to minimize risk for safer practices. This ensures not only the safe and proper use of equipment but also the generation of reliable data. To meet all safety requirements, additional safety protocols have been put in place for all targeted areas of the laboratory equipment.
</p>
</div>
</Section>
<Section title="Check-Ins" id="Check-Ins">
<div>
<p>
As part of our project to develop a prime-editing complex to correct the F508del mutation in cystic fibrosis, we place great emphasis on safety at all stages of research. Our final construct will be tested in primary cultures of epithelial cells obtained from nasal swabs, isolated from both patients and healthy individuals. from nasal swabs [link primär Kulturen]. To guarantee safety and ensure the highest level of precision and reliability of our results, we have introduced a series of carefully planned checkpoints during the experiments. These milestones allow for continuous monitoring, timely adjustments and validation at each critical stage. This ensures that potential issues are identified and addressed immediately, minimizing risk and improving the overall quality of the experimental results. [link zu den Experimenten] . iGEM places great emphasis on biosafety, ensuring that all projects adhere to strict safety standards. One of these measures is the iGEM White List, which includes organisms and parts that are pre-approved for use based on their safety profile. Any components or organisms not covered by this White List must be submitted as 'Check-ins' to the iGEM Safety Committee for approval. Check-ins are formal safety evaluations that allow the committee to assess the potential risks and ensure proper containment and handling procedures are in place. Although we used some parts and organisms that were not included on the White List, these were assessed as critical for our project and submitted as Check-ins to the iGEM Safety Committee. Furthermore, we were in active exchange with the committee throughout the process. The Check-ins provide a clear picture of the biosafety aspects of our project, reflecting our commitment to safety and compliance with iGEM standards.
The main safety measures we have implemented include:
</p>
<p>
<strong>Compliance with S1 conditions:</strong> Working in S1 laboratories ensures that only organisms in the lowest risk group are used, minimizing the risk to humans and the environment.
</p>
<p>
<strong>Sterile working practices:</strong> To avoid contamination, we have implemented strict hygiene measures, including the disinfection of work surfaces and the correct disposal of biological waste.
</p>
<p>
<strong>Controlled access:</strong> Access to laboratories was strictly regulated to ensure that only trained personnel worked with the genetically modified organisms and cell lines.
</p>
<p>
<strong>Documentation:</strong> All work steps, materials used and cell lines were carefully documented to ensure traceability and safety.
</p>
<p>
<strong>Safe handling of cell lines:</strong> The cell lines used for experiments were handled in accordance with the applicable safety regulations. This included regular checks for contamination and the safe storage and disposal of cell cultures.
</p>
<H4 text="Check-in for the Prime-Editing Komplex "></H4>
<p>
<strong>Reverse transcriptase:</strong> Reverse transcriptase plays a central role in prime editing by specifically inserting the correction as DNA at the inserted nick using an RNA template provided by pegRNA. The correction of the complementary DNA strand then takes place via the natural cell repair mechanisms. This ensures an exact correction of the target sequence. We checked the reverse transcriptase to ensure it could perform precise genome editing without introducing unintended mutations. This was important to minimize the risk of off-target effects that could lead to unexpected or harmful consequences.
</p>
<p>
<strong>pegRNA (Prime Editing Guide RNA):</strong> The pegRNA is a multifunctional RNA molecule that fulfils two essential tasks. Firstly, it serves as a standard guide RNA (gRNA) that binds specifically to the target DNA and thus marks the site of editing. Secondly, it contains an RNA template that encodes the desired DNA modification. This enables the precise integration of the genetic modifications at the target site. We evaluated pegRNA for its ability to specifically target and modified the intended DNA sequence. Ensuring its specificity was crucial to avoid the potential disruption of other genes.
</p>
<p>
<strong>Nickase Cas9, CasX, Fanzor (SpuFz1):</strong> These modified nucleases are designed to cut only one strand of DNA. This leads to controlled and precise editing of the genome, as cutting only one strand minimizes the risk of unwanted double-strand breaks. CasX and Fanzor offer smaller alternatives to Cas9, which is particularly advantageous for use in cells or organisms where space and efficiency requirements in terms of the transport system are an issue. Fanzor, being a newly introduced endonuclease, was particularly scrutinized in our project to ensure its safety and effectiveness in different cellular contexts.
This prime-editing complex thus represents a precise and efficient method for gene editing. By combining these components, genetic modifications can be performed with minimal side effects
</p>
<H4 text="Check-in for Cloning"></H4>
<p>
For our cloning experiments and the development of our prime editing complexes, we have amplified various plasmids in <i>E. coli</i> K-12 strains (DH5α,10-Beta) When working with microbial strains such as <i>E. coli</i> K-12 strains, a it's important to consider potential risks associated with their use, even though they are generally regarded as safe in laboratory settings. All experiments were performed under strict S1 conditions, following all relevant safety protocols. Below you will find an overview of the <i>E. coli</i> K-12 strains for our cloning experiments, submitted by us as a checkin and the specific safety measures:
</p>
<p>
<strong><i>E. coli K-12</i> strains (DH5α, 10-Beta):</strong> Although these strains are non-pathogenic and have been modified to minimize the risk of spreading antibiotic resistance, there remains a low risk of horizontal gene transfer, where genetic material could be transferred to other microorganisms, potentially leading to the spread of resistance genes or other traits. If accidentally released into the environment, <i>E. coli</i> K-12 strains could potentially interact with native microbial communities. While they are typically outcompeted in natural environments, there's a remote possibility of ecological disruption, particularly in microenvironments where they could find a niche.While these strains are non-virulent, they still pose a minimal risk to humans, particularly immunocompromised individuals, through accidental ingestion or inhalation in a laboratory setting.
</p>
<p>
We submitted the yeast strain <i>Pichia pastoris</i> (SMD1163) for the protein expression of Fanzor.
</p>
<p>
<strong><i>Pichia pastoris</i> (SMD1163):</strong> <i>Pichia pastoris</i> (SMD1163) is a widely used yeast strain for the expression of recombinant proteins. It is characterized by a methanol-inducible expression system (AOX1 promoter) and high cell growth rates, which makes it ideal for industrial applications. The strain can be easily genetically manipulated and can perform post-translational modifications, which supports correct protein production.
When working with <i>Pichia pastoris</i> (SMD1163), various safety-relevant aspects must be observed. Although the organism is considered non-pathogenic and biologically safe (S1), skin contact and aerosol formation should be avoided to minimize the risk of infection or allergic reactions. When using genetically modified strains, it is important to follow the relevant GMO guidelines to prevent uncontrolled release. In addition, handling chemicals such as methanol requires special precautions as they are toxic and highly flammable. The disposal of cell cultures and waste must also be carried out in accordance with biosafety regulations, especially in the case of genetically modified organisms.
</p>
<H4 text="Check-in for Testing in cell lines "></H4>
<p>
In our project, we paid attention to safety at every step, especially when working with specific cell lines [link Zellinien]. All experiments were performed under strict S1 conditions, following all relevant safety protocols. Given the sensitivity of the human cell lines we used, we placed great emphasis on controlled and well-designed workflows. All transfections were performed in our own transfection laboratory to ensure a high level of safety and compliance. Below you will find an overview of the cell lines submitted by us as a checkin and the specific safety measures:
HEK293T-3HA-CFTR.
The HEK293T-3HA-CFTR cell line is based on HEK293T cells expressing an additional tsA1609 allele of the SV40 large T antigen. This allele enables the replication of vectors containing the SV40 origin of replication. In addition to the native CFTR gene, which is not expressed in HEK cells, the HEK293T-3HA-CFTR cell line from Leuven carries another copy of the CFTR gene embedded in an expression cassette. This cassette contains a CMV promoter, which is derived from the human cytomegalovirus and is frequently used for the overexpression of genes in human cells. In addition, the cassette contains a puromycin resistance gene that is co-expressed with CFTR, allowing continuous selection of CFTR-expressing cells.
</p>
<p>
<strong>HEK293T-3HA-F508del-CFTR cell line:</strong> The HEK293T-3HA-F508del-CFTR cell line is a modified HEK293T cell line that carries the F508del mutation in the CFTR gene, which is responsible for the most common mutation in cystic fibrosis. This mutation leads to a defective CFTR protein that impairs the normal function of the chloride channel. The cell line is therefore ideal for studying the effects of this mutation and for evaluating potential therapies for cystic fibrosis.
</p>
<p>
<strong>CFBE41o- cell line:</strong> The CFBE41o- cell line, derived from the bronchial epithelial cells of a cystic fibrosis patient, is homozygous for the ΔF508-CFTR mutation and was essential for our cystic fibrosis research. A reduced CFTR expression level is present. The cell line carries the CFTR defect and can therefore represent a patient with CF. The cell line is used to test our mechanism. These cells were immortalized with a replication-defective plasmid that retains their physiological properties.
When working with the HEK293T and CFBE41o- cell lines, it’s important to consider the minimal risks associated with their use. While not harmful on their own, the genetic modifications in HEK293T cells require careful handling to prevent accidental release or exposure. These cells, engineered to overexpress CFTR, including the F508del mutation, necessitate strict safety measures like regular monitoring and proper waste disposal to comply with S1 laboratory standards. Similarly, CFBE41o- cells, due to their genetic modifications and disease relevance, require careful handling to avoid cross-contamination and ensure biosafety.
</p>
<p>
<strong>Human nasal epithelial cells (hNECs):</strong> Human nasal epithelial cells (hNECs) were harvested using a nasal brush, a minimally invasive procedure, and cultured in air-liquid interface (ALI) cultures to model the airway epithelium. Human nasal epithelial cells (hNECs) were obtained using a nasal brush, a minimally invasive technique, and then cultured in air-liquid interface (ALI) cultures to model the airway epithelium. Using these primary cultures, derived from donors with airway diseases such as cystic fibrosis, we were able to simulate the in vivo conditions of such diseases.
Due to the sensitive nature of these primary human cells, we performed all experiments with hNECs in our S2 laboratory, where increased safety precautions were taken. This included strict safety controls, safe handling of samples and proper disposal of materials after testing. In particular, the hNECs underwent HHH (Triple H: HIV, HCV and HBV) testing to ensure that no contamination occurred during sample collection or experimentation. These tests included sterility testing, viability assessments and contamination testing to ensure the safety and integrity of both the samples and the laboratory environment. After a negative HHH test, the primary cultures can be treated as S1. In addition, the nasal epithelial cells were handled with the utmost care during collection, ensuring that all procedures were performed under sterile conditions to avoid any risk of contaminationFor this purpose, the intensive examination of ethical questions was fundamental and a constant companion of our project. The numerous results from the interviews in the areas of: Ethics, storage and training in the handling of samples have been summarized in a guideline for patient consent for Germany and are intended to provide iGEM teams with the scope, critical examination and observance of iGEM rules, international and national guidelines.
</p>
<H4 text="Checkin for Delivery "></H4>
<p>
Our finished construct is designed to be delivered into the lung via an inhaler using lipid nanoparticles (LNPs). To be more spezific a selective organ-targeting (SORT)- LNPs were developed to deliver mRNA specifically to the lung, with special measures taken to increase biocompatibility and safety. Since the LNP composition is very specific and also differs from other formulas, we submitted the LNP as a checkin:
</p>
<p>
<strong>LNP:</strong> These LNPs are then taken up by epithelial cells through endocytosis, releasing the construct into the cytosol. We carefully evaluated the potential risks, including unintended immune responses and the need for precise dosing to minimize side effects. In addition, we have conducted an in-depth analysis of the dual-use potential of our technology. Dual-use refers to the possibility that scientific advances can be used for both civilian and military purposes. Therefore, we have implemented strict safety protocols and ethical guidelines to ensure that our technology is used exclusively for peaceful and therapeutic applications.
</p>
</div>
</Section>
<Section title="Our Lab" id="Our Lab">
<p>
As part of our laboratory activities for our PreCyse project, we worked in various laboratories. For general lab work and cloning experiments, you can find some pictures of our laboratories below:
</p>
<H4 text="Our Cloning Lab"></H4>
<p>
Our Cloning-laboratory is divided into different work areas to ensure that the experiments run smoothly and efficiently. These include the gel station, the PCR station, the transformation section and the measurement area. Each area is specially equipped for the respective method, and the corresponding experiments were carried out exclusively in the designated stations. In this way, we ensure that our work is carried out under optimal conditions and with the greatest possible precision.
</p>
<H4 text="Our Cell Culture Lab "></H4>
<p>
In our cell culture laboratory, we work under sterile conditions to ensure optimal growth conditions for human cell lines. Among other things, we carry out transfections in order to introduce genetic material into cells and investigate their behavior. Strict protocols and state-of-the-art technology ensure the precision and reproducibility of our experiments.
</p>
<p>
In our S2 laboratory, the harvested nasal epithelial cells that serve as primary cultures undergo a comprehensive HHH test (link zu primär Kulturen) to ensure their safety and suitability for further experiments. This test is crucial to ensure that we can subsequently work safely with these cells in the S1 range without the risk of contamination or unwanted release of biological material.
</p>
<div className="col">
<img src="https://static.igem.wiki/teams/5247/photos/biosafety/s2/s2-lab.jpeg" width="50%" height="50%"/>
</div>
</Section>
<Section title="Biosafety" id="Biosafety">
<Subesction title="Safety aspects of our PrimeGuide" id="Biosafety1">
<p>
The biosafety of our Prime Editing complex has been a top priority throughout the entire development process. We have therefore tried to optimise all parts that influence the biosecurity of our system as much as possible. To ensure maximum biosecurity, we have created and tested many designs, as well as extensively researched alternatives and/or additional elements that contribute to biosecurity.
</p>
<H4 text="PAM disrupt" ></H4>
<p>
A key safety mechanism incorporated in our design of the Prime Editing complex is the disruption of the PAM sequence. For the nickase enzyme to function properly, it must bind directly to the DNA strand, a process that is facilitated by the presence of a specific sequence called the PAM (Protospacer Adjacent Motif). This critical interaction occurs through the recognition of the PAM sequence by the nickase itself. To achieve PAM disruption, the pegRNA (prime editing guide RNA) is specifically designed in a way so that the PAM sequence is situated within the reverse transcription template (RTT) of the pegRNA. By introducing a silent mutation within the RT template into the PAM sequence. Therefore the PAM sequence is effectively eliminated after the gene editing process is successfully completed [1]. As a result of that, the PAM sequence is no longer present on the DNA strand, preventing the nickase from binding again at the same location. This reduction in repeated or undesired binding of the nickase enhances the safety of our prime editing complex, minimizing the risk of unintended edits or off-target effects in subsequent steps. Ultimately, this feature contributes very much to the overall safety and reliability of the prime editing process.
</p>
<div className="row">
<div className="col">
<p>
A key safety mechanism incorporated in our design of the Prime Editing complex is the disruption of the PAM sequence{/* [Link PAM text] */}. For the nickase enzyme to function properly, it must bind directly to the DNA strand, a process that is facilitated by the presence of a specific sequence called the PAM (Protospacer Adjacent Motif). This critical interaction occurs through the recognition of the PAM sequence by the nickase itself. To achieve PAM disruption, the pegRNA (prime editing guide RNA) is specifically designed in a way so that the PAM sequence is situated within the reverse transcription template (RTT) of the pegRNA. By introducing a silent mutation within the RT template into the PAM sequence. Therefore the PAM sequence is effectively eliminated after the gene editing process is successfully completed<SupScrollLink label="1"/>. As a result of that, the PAM sequence is no longer present on the DNA strand, preventing the nickase from binding again at the same location. This reduction in repeated or undesired binding of the nickase enhances the safety of our prime editing complex, minimizing the risk of unintended edits or off-target effects in subsequent steps. Ultimately, this feature contributes very much to the overall safety and reliability of the prime editing process.
</p>
</div>
<div className="col">
<OneFigure
pic1="https://static.igem.wiki/teams/5247/photos/biosafety/wiki/bild-1.webp"
num={1}
description="Illustration of the introduction of silent mutations leading to the PAM disrupt"
alt1="Illustration of the introduction of silent mutations leading to the PAM disrupt"
/>
</div>
</div>
<H4 text="pegRNA design - Spacer"></H4>
<p>
Biosafety is also guaranteed by the careful selection of the spacer, which plays a critical role in guiding the complex to its intended target site [2]. To ensure both precision and safety, we meticulously chose and rigorously checked the spacer using the CRISPick software [3]. This allowed us to evaluate whether our Spacer would be likely to target other regions than our target site and therefore allowing us to analyse and predict potential off-target effects, ensuring that erroneous edits are minimised. By optimising the spacer selection, we have not only significantly enhanced the overall editing efficiency, striking a balance between precision and performance, but especially ensured the utmost accuracy in directing the Prime Editor, further contributing to the safety of the editing process. [Bild 1]
Biosafety is also guaranteed by the careful selection of the spacer, which plays a critical role in guiding the complex to its intended target site<SupScrollLink label="2"/>. To ensure both precision and safety, we meticulously chose and rigorously checked the spacer using the <a href="https://www.synthego.com/products/bioinformatics/crispr-design-tool">CRISPick software</a><SupScrollLink label="3"/>. This allowed us to evaluate whether our Spacer would be likely to target other regions than our target site and therefore allowing us to analyse and predict potential off-target effects, ensuring that erroneous edits are minimised. By optimising the spacer selection, we have not only significantly enhanced the overall editing efficiency, striking a balance between precision and performance, but especially ensured the utmost accuracy in directing the Prime Editor, further contributing to the safety of the editing process.
</p>
<H4 text="Riboswitch"></H4>
<p>
Riboswitches are segments of an RNA strand that bind to small molecules, causing them to change their secondary structure by forming hairpin structures. This process regulates gene expression at the translation level by preventing ribosomes from binding at the RBS and translating the coding region on the RNA strand. 0For our project we also considered an ion-sensitive riboswitch, specifically dependent on sodium ions (Na⁺), as a regulatory mechanism. The secondary structure of this riboswitch prevents the binding of ribosomes to the ribosome binding site (RBS) under normal conditions, thus inhibiting the translation of the subsequent mRNA. When sodium ions bind to the riboswitch, a structural change occurs, exposing the RBS, which allows for the translation of the mRNA and the production of our fusion protein which is the main component of our prime editing system and therefore of enormous importance for it to work [4]. In the context of the CFTR mutation and its effects on the cell, the elevated Na⁺ levels play a crucial role. Due to the dysfunctional CFTR channel, which fails to properly function as a chloride channel, the ENaC channel (epithelial sodium channel) becomes upregulated. This upregulation results in an increased transport of sodium ions into the cell, leading to a higher intracellular sodium concentration. This elevated Na⁺ concentration creates a specific ionic environment that could potentially be utilized to regulate our Prime-Editing complex in a targeted manner. Given these specific ionic changes in the cell, we could have a disease-specific regulation of our Prime-Editing system based on the ionic situation typical of this condition. However, despite the initial promise of this approach, after further research, we concluded that the riboswitch, even considering the ion levels within epithelial cells, is overall too nonspecific and therefore too unreliable as a regulatory mechanism. Although the ion levels in CFTR cells are much lower, there are still low concentrations of sodium ions, which can lead to the riboswitch not being completely switched off.
[Bild 2]
As a further approach to developing alternative riboswitch variants, we considered the possibility of an RNA-regulated riboswitch targeting the defective mRNA sequence of the genetically defective CFTR gene. The basic idea behind this concept was that the riboswitch specifically binds to a region on the CFTR mRNA containing the F508Δ mutation. This binding should induce a structural change in the riboswitch on our prime editing complex’s mRNA that ultimately leads to exposure of the RBS to allow translation of the downstream sequence. This mechanism would be designed to react specifically to the defective CFTR mRNA and only cause a change in the secondary structure in the presence of the specific mutation. The riboswitch could thus ensure selective and disease-specific activation of our prime editing complex, which would be of particular interest in the context of genetic diseases such as cystic fibrosis. However, we did not pursue this approach any further. A major reason for this was the lack of sufficient literature providing a sound scientific basis for this specific application of a riboswitch. In
Riboswitches are segments of an RNA strand that bind to small molecules, causing them to change their secondary structure by forming hairpin structures. This process regulates gene expression at the translation level by preventing ribosomes from binding at the RBS and translating the coding region on the RNA strand. 0For our project we also considered an ion-sensitive riboswitch, specifically dependent on sodium ions (Na⁺), as a regulatory mechanism. The secondary structure of this riboswitch prevents the binding of ribosomes to the ribosome binding site (RBS) under normal conditions, thus inhibiting the translation of the subsequent mRNA. When sodium ions bind to the riboswitch, a structural change occurs, exposing the RBS, which allows for the translation of the mRNA and the production of our fusion protein which is the main component of our prime editing system and therefore of enormous importance for it to work<SupScrollLink label="4"/>. In the context of the CFTR mutation and its effects on the cell, the elevated Na⁺ levels play a crucial role. Due to the dysfunctional CFTR channel, which fails to properly function as a chloride channel, the ENaC channel (epithelial sodium channel) becomes upregulated. This upregulation results in an increased transport of sodium ions into the cell, leading to a higher intracellular sodium concentration. This elevated Na⁺ concentration creates a specific ionic environment that could potentially be utilized to regulate our Prime-Editing complex in a targeted manner. Given these specific ionic changes in the cell, we could have a disease-specific regulation of our Prime-Editing system based on the ionic situation typical of this condition. However, despite the initial promise of this approach, after further research, we concluded that the riboswitch, even considering the ion levels within epithelial cells, is overall too nonspecific and therefore too unreliable as a regulatory mechanism. Although the ion levels in CFTR cells are much lower, there are still low concentrations of sodium ions, which can lead to the riboswitch not being completely switched off.
</p>
<OneFigure
pic1="https://static.igem.wiki/teams/5247/photos/biosafety/wiki/bild-2.png"
num={2}
description="Illustration of the mechanism of action of the riboswitch"
alt1="Illustration of the mechanism of action of the riboswitch"
/>
<p>
As a further approach to developing alternative riboswitch variants, we considered the possibility of an RNA-regulated riboswitch targeting the defective mRNA sequence of the genetically defective CFTR gene. The basic idea behind this concept was that the riboswitch specifically binds to a region on the CFTR mRNA containing the F508Δ mutation. This binding should induce a structural change in the riboswitch on our prime editing complex’s mRNA that ultimately leads to exposure of the RBS to allow translation of the downstream sequence. This mechanism would be designed to react specifically to the defective CFTR mRNA and only cause a change in the secondary structure in the presence of the specific mutation. The riboswitch could thus ensure selective and disease-specific activation of our prime editing complex, which would be of particular interest in the context of genetic diseases such as Cystic Fibrosis. However, we did not pursue this approach any further. A major reason for this was the lack of sufficient literature providing a sound scientific basis for this specific application of a riboswitch. In
addition, our research steered us in a different direction, particularly with regard to the alternative mechanism involving the XBP1 intron to regulate the prime editing system. This alternative seemed more promising and was based on an established regulatory mechanism that is triggered by cellular stress and specifically responds to misfolding processes.
</p>
<H4 text="XBP1 Intron"></H4>
<p>
After extensive research, we discovered a regulatory system in eukaryotic cells, the XBP1 mechanism. The activation of XBP1 is an important mechanism that occurs as part of the Unfolded Protein Response (UPR), a cellular stress response triggered by the accumulation of misfolded proteins in the endoplasmic reticulum (ER). The ER is a key cellular component responsible for protein folding and transport. When many misfolded proteins accumulate in the ER, a specific regulatory mechanism is activated to reduce the stress on the ER. XBP1 activation is controlled by a protein called IRE1α, which is embedded in the ER membrane. IRE1α acts as a sensor for protein misfolding stress in the ER. Once IRE1α detects misfolded proteins, it dimerizes and becomes activated through autophosphorylation. This activation switches on the endoribonuclease activity of IRE1α, which is a crucial step in the activation of XBP1. The mRNA for XBP1 is continuously transcribed in the nucleus and transported to the cytoplasm, where it contains an intron that is not normally spliced out. This intron contains a stop codon, preventing the translation of a functional XBP1 protein. However, when ER stress activates IRE1α, the endoribonuclease domain of IRE1α splices this intron out of the XBP1 mRNA. This is an unconventional splicing event, as it occurs in the cytoplasm rather than in the nucleus. Once the intron is removed, the spliced XBP1 mRNA can be translated into a functional XBP1 protein. This activated XBP1 acts as a transcription factor, turning on genes that increase the protein-folding capacity of the ER and promote the degradation of misfolded proteins. In this way, XBP1 helps the cell cope with ER stress and restore balance in the protein-folding process. Thus, this mechanism originally functions within the cell in the context of ER stress to maintain ER function when protein folding is disrupted. [5] [6] Our idea was therefore to integrate this intron into the mRNA encoding our prime-editing complex and thus use this mechanism to ensure that a functional prime editor is only synthesized when there is a high accumulation of misfolded proteins in the cell (similar to F508del). This would therefore represent an optimal safety aspect, as our fusion protein, which is essential for prime editing, cannot be fully synthesised as long as the genetic defect is not present in the cell. Accordingly, this provides the security that no healthy cells, as well as correctly edited cells, cannot be edited, which is an enormous contribution to biosafety. However, there was too much uncertainty about the extent to which other factors, such as misfolded proteins that are not associated with the CFTR protein, play a role in this mechanism. And since we could not and did not want to take the risk of such factors initiating the system, we decided against using it. To clarify this unknown correlation, we have considered a future experiment in which we want to switch this intron in front of a fluorescent marker and express it in cells with defective CFTR in order to confirm/investigate the dependence of intron splicing and the presence of CFTR F508del.
After extensive research, we discovered a regulatory system in eukaryotic cells, the XBP1 mechanism. The activation of XBP1 is an important mechanism that occurs as part of the Unfolded Protein Response (UPR), a cellular stress response triggered by the accumulation of misfolded proteins in the endoplasmic reticulum (ER). The ER is a key cellular component responsible for protein folding and transport. When many misfolded proteins accumulate in the ER, a specific regulatory mechanism is activated to reduce the stress on the ER. XBP1 activation is controlled by a protein called IRE1α, which is embedded in the ER membrane. IRE1α acts as a sensor for protein misfolding stress in the ER. Once IRE1α detects misfolded proteins, it dimerizes and becomes activated through autophosphorylation. This activation switches on the endoribonuclease activity of IRE1α, which is a crucial step in the activation of XBP1. The mRNA for XBP1 is continuously transcribed in the nucleus and transported to the cytoplasm, where it contains an intron that is not normally spliced out. This intron contains a stop codon, preventing the translation of a functional XBP1 protein. However, when ER stress activates IRE1α, the endoribonuclease domain of IRE1α splices this intron out of the XBP1 mRNA. This is an unconventional splicing event, as it occurs in the cytoplasm rather than in the nucleus. Once the intron is removed, the spliced XBP1 mRNA can be translated into a functional XBP1 protein. This activated XBP1 acts as a transcription factor, turning on genes that increase the protein-folding capacity of the ER and promote the degradation of misfolded proteins. In this way, XBP1 helps the cell cope with ER stress and restore balance in the protein-folding process. Thus, this mechanism originally functions within the cell in the context of ER stress to maintain ER function when protein folding is disrupted<SupScrollLink label="5"/><sup>,</sup><SupScrollLink label="6"/>. Our idea was therefore to integrate this intron into the mRNA encoding our prime-editing complex and thus use this mechanism to ensure that a functional prime editor is only synthesized when there is a high accumulation of misfolded proteins in the cell (similar to F508del). This would therefore represent an optimal safety aspect, as our fusion protein, which is essential for prime editing, cannot be fully synthesised as long as the genetic defect is not present in the cell. Accordingly, this provides the security that no healthy cells, as well as correctly edited cells, cannot be edited, which is an enormous contribution to biosafety. However, there was too much uncertainty about the extent to which other factors, such as misfolded proteins that are not associated with the CFTR protein, play a role in this mechanism. And since we could not and did not want to take the risk of such factors initiating the system, we decided against using it. To clarify this unknown correlation, we have considered a future experiment in which we want to switch this intron in front of a fluorescent marker and express it in cells with defective CFTR in order to confirm/investigate the dependence of intron splicing and the presence of CFTR F508del.
</p>
</Subesction>
<Subesction title="Safety aspects of our Airbuddy" id="Biosafety2">
<H4 text="SORT LNP and Cytotoxicity"></H4>
<p>
We have carefully considered the biosafety aspects of our delivery system, starting with the decision between Adeno-associated viruses (AAV) or LNPs as delivery systems. Our comparison revealed that the biocompatibility and safety of LNPs are paramount for our approach. That is why we chose selective organ-targeting (SORT) lipid nanoparticles (LNPs) [7] in the context of targeted pulmonary mRNA delivery. One of our primary concerns with the LNP was the potential cytotoxicity of polyethylene glycol (PEG), a common stabilizing agent in LNP formulations. Aware of the immune responses PEG can trigger, potentially leading to cytotoxicity [8], we aimed at optimizing its concentration in our SORT LNPs to minimize such reactions while maintaining therapeutic efficacy. By the use of low molecular weight PEG, we addressed this problem. To test weather our approach succeeded, we conducted MTT and proliferation assays to ensure that our LNP posed no cytotoxicity risks.
We have carefully considered the biosafety aspects of our delivery system, starting with the decision between Adeno-associated viruses (AAV) or LNPs as delivery systems. Our comparison revealed that the biocompatibility and safety of LNPs are paramount for our approach. That is why we chose selective organ-targeting (SORT) lipid nanoparticles (LNPs){/* [Link LNP text] */}<SupScrollLink label="7"/> in the context of targeted pulmonary mRNA delivery. One of our primary concerns with the LNP was the potential cytotoxicity of polyethylene glycol (PEG), a common stabilizing agent in LNP formulations. Aware of the immune responses PEG can trigger, potentially leading to cytotoxicity<SupScrollLink label="8"/>, we aimed at optimizing its concentration in our SORT LNPs to minimize such reactions while maintaining therapeutic efficacy. By the use of low molecular weight PEG, we addressed this problem. To test weather our approach succeeded, we conducted MTT and proliferation assays to ensure that our LNP posed no cytotoxicity risks.
</p>
<H4 text="Precision of our SORT LNP"></H4>
<p>
To further improve safety, we focused on reducing off-target effects. By incorporating specific SORT molecules, such as permanently cationic lipids like DOTAP, we ensured that the nanoparticles are systematically directed to the lungs. This precise targeting is particularly beneficial for respiratory diseases, as it enhances therapeutic effectiveness while limiting the impact on non-target organs. Our outlook of antibody conjugation as surface modification of our LNP for cell type-specific delivery, more exactly club cells [9] and ionocytes [10] as CFTR-expressing lung epithelial cells, would round off this aspect.
To further improve safety, we focused on reducing off-target effects. By incorporating specific SORT molecules, such as permanently cationic lipids like <a onClick={() => goToPageWithTabAndScroll({tabId:'tab-delivery', path: '/engineering', scrollToId: "delivery-header"})}>DOTAP</a> , we ensured that the nanoparticles are systematically directed to the lungs. This precise targeting is particularly beneficial for respiratory diseases, as it enhances therapeutic effectiveness while limiting the impact on non-target organs. Our outlook of antibody conjugation as surface modification of our LNP for cell type-specific delivery, more exactly club cells<SupScrollLink label="9"/> and ionocytes<SupScrollLink label="10"/> as CFTR-expressing lung epithelial cells, would round off this aspect.
</p>
<p>
In summary, our design strategy emphasizes both safety and efficacy. The careful optimization of components like PEG 2000 and the use of targeted delivery molecules allow SORT LNPs to deliver therapeutic agents directly to the lungs, reducing systemic exposure and minimizing side effects. This targeted approach ensures more effective treatments, especially for conditions requiring localized intervention.
......@@ -175,7 +85,7 @@ export const Safety: React.FC = () =>{
<Section title="Biosecurity" id="Biosecurity">
<Subesction title="About Our Project" id="Biosecurity1">
<p>
Our project focuses on the genetic disease cystic fibrosis, specifically targeting the Delta-508 mutation. The aim is to correct this mutation using Prime Editing, a precise genome-editing technique. We have explored different strategies to optimize the Prime Editing complex for this specific application.
Our project focuses on the genetic disease Cystic Fibrosis, specifically targeting the Delta-508 mutation. The aim is to correct this mutation using Prime Editing, a precise genome-editing technique. We have explored different strategies to optimize the Prime Editing complex for this specific application.
</p>
<p>
The Prime Editing complex consists of a nickase, a reverse transcriptase, a pegRNA. The pegRNA guides the editing process by directing the complex to the target DNA sequence, allowing for precise genetic modifications. For targeted delivery, we selected LNPs to introduce the mRNA encoding the Prime Editing components specifically into lung epithelial cells, where the CFTR protein is highly expressed. Additionally, we investigated alternatives to the conventional Cas9 nickase, such as the smaller CasX and Fanzor, aiming to reduce the overall size of the Prime Editing complex. In our optimization efforts, we also explored smaller reverse transcriptases to enhance the efficiency of the system in human cells.
......@@ -184,7 +94,7 @@ export const Safety: React.FC = () =>{
Furthermore, we have developed a modular plasmid that contains the backbone of our Prime Editing complex. The individual components can be cloned individually into the backbone. This plasmid allows us to either deliver the construct directly into target cells or transcribe the plasmid into RNA, enabling the delivery of the Prime Editing complex in the form of mRNA. The modularity of the plasmid is a key feature; specific restriction sites are included to facilitate the easy exchange of the complex's components. This design makes it straightforward to adapt the Prime Editing complex for various use cases and therapeutic requirements.
</p>
<p>
We have investigated safety mechanisms to control the prime-editing complex, including a riboswitch that responds to sodium ion concentrations, but have discarded it due to suspected insufficient sensitivity. We are currently investigating the use of the ER stress response to activate the prime editing complex only in cells with high ER stress, as is typical for cystic fibrosis. Further details can be found in the Biosafety section.
We have investigated safety mechanisms to control the prime-editing complex, including a riboswitch that responds to sodium ion concentrations, but have discarded it due to suspected insufficient sensitivity. We are currently investigating the use of the ER stress response to activate the prime editing complex only in cells with high ER stress, as is typical for Cystic Fibrosis. Further details can be found in the Biosafety section.
</p>
</Subesction>
<Subesction title="Assessing Project Risks" id="Biosecurity2">
......@@ -192,19 +102,19 @@ export const Safety: React.FC = () =>{
Given the sensitive nature of genome editing, our project presents specific biosecurity concerns that need to be assessed and mitigated.
</p>
<p>
<strong>Dual-Use Potential:</strong> One of the main biosecurity risks is the potential for dual-use of the Prime Editing technology. The system we are developing, while intended for therapeutic use, could be misused to target other genes or genomes for malicious purposes.[11] This includes the possibility of weaponizing the technology to induce harmful genetic changes in crops, animals, or even humans. The modular design of our plasmid system, although intended to facilitate optimization, could be exploited to exchange components for harmful applications, thereby increasing the risk of misuse.
<strong>Dual-Use Potential:</strong> One of the main biosecurity risks is the potential for dual-use of the Prime Editing technology. The system we are developing, while intended for therapeutic use, could be misused to target other genes or genomes for malicious purposes<SupScrollLink label="11"/>. This includes the possibility of weaponizing the technology to induce harmful genetic changes in crops, animals, or even humans. The modular design of our plasmid system, although intended to facilitate optimization, could be exploited to exchange components for harmful applications, thereby increasing the risk of misuse.
</p>
<p>
<strong>Unintendend Dissemination:</strong> Since our approach uses mRNA delivered via LNPs, there is a risk of unintended dissemination into the environment. If the LNPs are not adequately contained or disposed of, there is a possibility that they could be absorbed by non-target organisms, potentially leading to off-target genetic modifications.[12] In addition, the mRNA itself could theoretically be transferred between cells, especially if taken up by unintended hosts, raising concerns about unintentional spread in the environment.
<strong>Unintendend Dissemination:</strong> Since our approach uses mRNA delivered via LNPs, there is a risk of unintended dissemination into the environment. If the LNPs are not adequately contained or disposed of, there is a possibility that they could be absorbed by non-target organisms, potentially leading to off-target genetic modifications<SupScrollLink label="12"/>. In addition, the mRNA itself could theoretically be transferred between cells, especially if taken up by unintended hosts, raising concerns about unintentional spread in the environment.
</p>
<p>
<strong>Unauthorized Access:</strong> The genetic constructs and the detailed methodology of our Prime Editing system must be securely stored and protected.[13] If unauthorized individuals were to gain access to the plasmids, LNP formulations, or editing protocols, there is a risk of the technology being replicated or adapted for unintended, potentially harmful uses. This highlights the importance of proper biosecurity protocols in both physical and digital storage of our project materials.
<strong>Unauthorized Access:</strong> The genetic constructs and the detailed methodology of our Prime Editing system must be securely stored and protected<SupScrollLink label="13"/>. If unauthorized individuals were to gain access to the plasmids, LNP formulations, or editing protocols, there is a risk of the technology being replicated or adapted for unintended, potentially harmful uses. This highlights the importance of proper biosecurity protocols in both physical and digital storage of our project materials.
</p>
<p>
<strong>Synthetic Biology and information Sharing:</strong> The ease of synthesizing genetic material means that our project information could potentially be used to order similar constructs from commercial synthesis providers.[14] While these providers follow biosecurity guidelines, the increasing accessibility of synthetic biology raises the concern of our Prime Editing system being reproduced or modified without our knowledge. This includes potential attempts to bypass safety mechanisms or create variants that evade current regulatory frameworks.
<strong>Synthetic Biology and information Sharing:</strong> The ease of synthesizing genetic material means that our project information could potentially be used to order similar constructs from commercial synthesis providers<SupScrollLink label="14"/>. While these providers follow biosecurity guidelines, the increasing accessibility of synthetic biology raises the concern of our Prime Editing system being reproduced or modified without our knowledge. This includes potential attempts to bypass safety mechanisms or create variants that evade current regulatory frameworks.
</p>
<p>
<strong>Public Perception and Miscommunication:</strong> There is a biosecurity risk in how our project's technology is communicated to the public.[15] Miscommunication or misunderstanding of the project’s intent and capabilities could lead to misinformation, fear, or even attempts to replicate the technology outside of controlled and regulated environments. This could undermine public trust in legitimate therapeutic uses of genome-editing technologies and potentially facilitate misuse.
<strong>Public Perception and Miscommunication:</strong> There is a biosecurity risk in how our project's technology is communicated to the public<SupScrollLink label="15"/>. Miscommunication or misunderstanding of the project’s intent and capabilities could lead to misinformation, fear, or even attempts to replicate the technology outside of controlled and regulated environments. This could undermine public trust in legitimate therapeutic uses of genome-editing technologies and potentially facilitate misuse.
</p>
</Subesction>
<Subesction title="Managing Risks" id="Biosecurity3">
......@@ -216,7 +126,7 @@ export const Safety: React.FC = () =>{
Firstly, we strictly control access to all our project data, including genetic sequences, plasmid designs, and protocols. Access is limited to authorized team members and collaborators who follow strict biosecurity guidelines. Further elaboration on these access controls will be provided in the "Unauthorized Access" section.
</p>
<p>
Secondly, we intend to incorporate a safety mechanism into our Prime Editing complex that significantly limits its potential misuse. By utilizing the ER stress response pathway, we would design our therapeutic mRNA to undergo unconventional splicing only in cells experiencing high levels of protein stress. Only this unconventional splicing would convert the mRNA into a form that can be translated into the final PE complex. This mechanism ensures that the Prime Editing complex becomes active primarily in cells under such stress conditions. While this does not exclusively limit the complex to cystic fibrosis-affected cells, it considerably narrows the range of cells where activation can occur, thus preventing arbitrary application of the editing system and reducing the risk of targeting unintended cells.
Secondly, we intend to incorporate a safety mechanism into our Prime Editing complex that significantly limits its potential misuse. By utilizing the ER stress response pathway, we would design our therapeutic mRNA to undergo unconventional splicing only in cells experiencing high levels of protein stress. Only this unconventional splicing would convert the mRNA into a form that can be translated into the final PE complex. This mechanism ensures that the Prime Editing complex becomes active primarily in cells under such stress conditions. While this does not exclusively limit the complex to Cystic Fibrosis-affected cells, it considerably narrows the range of cells where activation can occur, thus preventing arbitrary application of the editing system and reducing the risk of targeting unintended cells.
</p>
<p>
Together, these measures provide a layer of protection against dual-use risks, making it more difficult for the technology to be employed outside of its intended therapeutic context.
......@@ -267,23 +177,23 @@ export const Safety: React.FC = () =>{
One of the challenges in scientific research, especially in fields like synthetic biology, is that advancements can often outpace public understanding and discourse. This can lead to confusion, fear, or mistrust if the research is not communicated effectively. To address this issue, we believe that scientific progress should occur in constant dialogue with the public.
</p>
<p>
We have adopted a strong Human Practices approach to ensure transparency and foster public engagement. Our efforts include initiatives like "MUKOmoove" and "Teuto ruft," where we have worked directly with students, educational institutions, and public organizations. Through these initiatives, we aim to explain our project, discuss its implications, and answer any questions, thus maintaining an open line of communication. By collaborating with a variety of public entities, including patient associations, educational programs, and community groups, we ensure that our research remains accessible and understandable to a broader audience.
We have adopted a strong Human Practices approach to ensure transparency and foster public engagement. Our efforts include initiatives like <a onClick={() => goToPageWithTabAndScroll ({scrollToId: 'cf-month', path: '/human-practices', tabId: 'mukomove' })}> MUKOmove </a> and <a onClick={() => goToPageWithTabAndScroll ({scrollToId: 'Der Teuto ruft!', path: '/human-practices', tabId: 'teutoruft' })}> "Der Teuto ruft!" </a> , where we have worked directly with students, educational institutions, and public organizations. Through these initiatives, we aim to explain our project, discuss its implications, and answer any questions, thus maintaining an open line of communication. By collaborating with a variety of public entities, including patient associations, educational programs, and community groups, we ensure that our research remains accessible and understandable to a broader audience.
</p>
<p>
This proactive approach helps us address potential concerns, demystify our research, and contribute to a more informed public perception of synthetic biology.
</p>
</Subesction>
</Section>
</Section>
<Section title="Bioethics" id="Bioethics">
<div>
<p>
Bioethics is an interdisciplinary field of research that addresses ethical issues pertaining to the life sciences and medical research. It plays a pivotal role in contemporary research, particularly in projects that employ human samples or data. This is due to the fact that in these cases, the protection of the rights and dignity of the people involved is of the utmost importance <SupScrollLink label="1"/> [16]. In order to ascertain the necessity for an ethics application, an interview was conducted with Eva-Maria Berens, the scientific director of the office of the Ethics Committee at Bielefeld University, as part of the current research project. Following a comprehensive review, it was concluded that an ethics application was not necessary for the specific research project. Nevertheless, a comprehensive patient consent form was developed in conjunction with Eva-Maria Berens to guarantee that the donors of their samples are adequately informed and provide their consent of their own volition. The document guarantees that all pertinent information regarding sample collection, utilisation and storage is provided in an intelligible format. Furthermore, an interview was conducted with Dr. Timm Weber, a representative of the biobank, to discuss the topic of bioethics in greater depth. During the course of the interviews, the ethical aspects of sample storage and utilisation within the biobank were discussed in detail. Particular attention was paid to the responsible handling and protection of the rights of the test subjects. The discussion of bioethics in both interviews emphasises the relevance of ethical principles for research and ensures that it is conducted in accordance with the highest ethical standards.
Bioethics is an interdisciplinary field of research that addresses ethical issues pertaining to the life sciences and medical research. It plays a pivotal role in contemporary research, particularly in projects that employ human samples or data. This is due to the fact that in these cases, the protection of the rights and dignity of the people involved is of the utmost importance<SupScrollLink label="16"/>. In order to ascertain the necessity for an ethics application, an interview was conducted with <a onClick={() => goToPagesAndOpenTab('berens', '/human-practices')}>Eva-Maria Berens</a>, the scientific director of the office of the Ethics Committee at Bielefeld University, as part of the current research project. Following a comprehensive review, it was concluded that an ethics application was not necessary for the specific research project. Nevertheless, a comprehensive patient consent form was developed in conjunction with Eva-Maria Berens to guarantee that the donors of their samples are adequately informed and provide their consent of their own volition. The document guarantees that all pertinent information regarding sample collection, utilisation and storage is provided in an intelligible format. Furthermore, an interview was conducted with <a onClick={() => goToPagesAndOpenTab('timm', '/human-practices')}>Dr. Timm Weber</a>, a representative of the biobank, to discuss the topic of bioethics in greater depth. During the course of the interviews, the ethical aspects of sample storage and utilisation within the biobank were discussed in detail. Particular attention was paid to the responsible handling and protection of the rights of the test subjects. The discussion of bioethics in both interviews emphasises the relevance of ethical principles for research and ensures that it is conducted in accordance with the highest ethical standards.
</p>
</div>
<Subesction title="Gene Therapy" id="Bioethics1">
<div>
<p>
The potential of gene therapy to treat genetic diseases is promising, but it is also associated with significant ethical issues. One of the principal challenges is ensuring the safety of the procedure and the potential for unforeseen long-term consequences. Such consequences may only become apparent years after the genetic intervention has taken place. The modification of the germline, which affects not only the individual but also future generations, is a particularly sensitive issue. This gives rise to the question of the extent to which the decisions made today will influence future generations without their consent, thereby jeopardising intergenerational justice <SupScrollLink label="2"/> [17]. Another ethical issue is the potential for misuse for eugenic purposes. While the current focus is on combating disease, future applications could be aimed at 'optimising' human traits, which could result in a worsening of social inequalities. Access to gene therapy is also a significant issue. High costs could limit access to wealthy population groups, which would reinforce existing inequalities <SupScrollLink label="3"/> [18]. The issue of informed consent is also a key aspect. Many patients do not have the necessary knowledge to fully understand the complex risks, which raises ethical questions about their decision-making capacity. Overall, the debate around gene therapy highlights that ethical considerations such as safety, justice and patient rights need to be considered alongside scientific progress <SupScrollLink label="4"/> [19].
The potential of gene therapy to treat genetic diseases is promising, but it is also associated with significant ethical issues. One of the principal challenges is ensuring the safety of the procedure and the potential for unforeseen long-term consequences. Such consequences may only become apparent years after the genetic intervention has taken place. The modification of the germline, which affects not only the individual but also future generations, is a particularly sensitive issue. This gives rise to the question of the extent to which the decisions made today will influence future generations without their consent, thereby jeopardising intergenerational justice<SupScrollLink label="17"/>. Another ethical issue is the potential for misuse for eugenic purposes. While the current focus is on combating disease, future applications could be aimed at 'optimising' human traits, which could result in a worsening of social inequalities. Access to gene therapy is also a significant issue. High costs could limit access to wealthy population groups, which would reinforce existing inequalities<SupScrollLink label="18"/>. The issue of informed consent is also a key aspect. Many patients do not have the necessary knowledge to fully understand the complex risks, which raises ethical questions about their decision-making capacity. Overall, the debate around gene therapy highlights that ethical considerations such as safety, justice and patient rights need to be considered alongside scientific progress<SupScrollLink label="19"/>.
</p>
</div>
</Subesction>
......@@ -291,14 +201,14 @@ export const Safety: React.FC = () =>{
<div>
<H4 text="Introduction of primary cultures"></H4>
<p>
A primary culture is defined as a cell culture that is isolated directly from the tissue of an organism. In our case, the organism is human. The cells are then cultivated in a controlled environment, namely an S2 laboratory <SupScrollLink label="5"/> [20]. Primary cultures are a fundamental biomedical research tool, widely regarded as indispensable due to their capacity for realistic modelling of complex cell interactions. Primary cells are derived directly from the tissue of an organism and, as a consequence, they essentially retain their original properties. Consequently, they mirror the authentic conditions of the target tissue, which is vital for accurately assessing the impact of a therapeutic agent. In contrast, HEK cells represent transformed cell lines that exhibit physiological properties distinct from those of target cells in the human body. The effect of a therapeutic agent is typically limited to a specific cell type. The investigation of cell-specific effects and reactions of an active substance is feasible with the use of primary cells, as these possess the functional characteristics inherent to the cell type under consideration. Although HEK cells are relatively straightforward to cultivate, they are less representative of a number of tissue types and may activate other signalling pathways. The authenticity of the receptors and signalling pathways is guaranteed, as primary cells show the natural expression of receptors, ion channels and other cellular mechanisms. HEK cells are often genetically modified to express specific receptors, which can be useful for simple test systems. However, this does not reflect the complex environment of a real tissue. Given the sensitivity of primary cultures to environmental influences, thus resulting in higher risk of a contamination, it is imperative that researchers employ special safety measures to ensure the safety of themselves and the integrity of the cells. Primary cultures are employed extensively in the development of vaccines, cancer research and the investigation of basic cell processes.
A primary culture is defined as a cell culture that is isolated directly from the tissue of an organism. In our case, the organism is human. The cells are then cultivated in a controlled environment, namely an S2 laboratory<SupScrollLink label="20"/>. Primary cultures are a fundamental biomedical research tool, widely regarded as indispensable due to their capacity for realistic modelling of complex cell interactions. Primary cells are derived directly from the tissue of an organism and, as a consequence, they essentially retain their original properties. Consequently, they mirror the authentic conditions of the target tissue, which is vital for accurately assessing the impact of a therapeutic agent. In contrast, HEK cells represent transformed cell lines that exhibit physiological properties distinct from those of target cells in the human body. The effect of a therapeutic agent is typically limited to a specific cell type. The investigation of cell-specific effects and reactions of an active substance is feasible with the use of primary cells, as these possess the functional characteristics inherent to the cell type under consideration. Although HEK cells are relatively straightforward to cultivate, they are less representative of a number of tissue types and may activate other signalling pathways. The authenticity of the receptors and signalling pathways is guaranteed, as primary cells show the natural expression of receptors, ion channels and other cellular mechanisms. HEK cells are often genetically modified to express specific receptors, which can be useful for simple test systems. However, this does not reflect the complex environment of a real tissue. Given the sensitivity of primary cultures to environmental influences, thus resulting in higher risk of a contamination, it is imperative that researchers employ special safety measures to ensure the safety of themselves and the integrity of the cells. Primary cultures are employed extensively in the development of vaccines, cancer research and the investigation of basic cell processes.
</p>
<H4 text="Ethics in work with primary cultures"></H4>
<p>
The term 'ethics' is used to describe the examination of moral principles that determine the behaviour of individuals or groups <SupScrollLink label="6"/> [21]. In a scientific context, the term 'ethics' encompasses the examination of the moral justifiability of actions and decisions, particularly with regard to the welfare of living beings and the responsible use of resources <SupScrollLink label="7"/> [22]. The isolation of primary cells from living organisms raises ethical questions, particularly in the case of human or animal tissue. In the context of research with animal primary cells, careful consideration must be given to the need for animal suffering and the potential benefits of the research <SupScrollLink label="8"/> [23]. An ethical dilemma frequently arises from the fact that primary cells offer the most meaningful data from a biological standpoint, yet their production is associated with challenges. In this context, the necessity of primary cell cultures is called into question, and the promotion of alternative methods, such as artificially produced tissues or organoids, is advocated where feasible. It is of crucial importance to emphasize the necessity of ethical responsibility in the collection of primary cultures. It is of the utmost importance that the procedure is carried out with consideration for the rights, and particularly the well-being of the donor. The removal of cells or tissue must be medically justifiable and, moreover, ethically justifiable in every case. To this end, the potential for research use and the possible risks and burdens for the donor must be weighed against each other to ensure careful consideration. However, it is also particularly important to ensure that the donor is involved in the entire process and is able to make an informed decision. The purpose of the research, the use of the cells and possible consequences must also be made transparent at all times.
The term 'ethics' is used to describe the examination of moral principles that determine the behaviour of individuals or groups<SupScrollLink label="21"/>. In a scientific context, the term 'ethics' encompasses the examination of the moral justifiability of actions and decisions, particularly with regard to the welfare of living beings and the responsible use of resources<SupScrollLink label="22"/>. The isolation of primary cells from living organisms raises ethical questions, particularly in the case of human or animal tissue. In the context of research with animal primary cells, careful consideration must be given to the need for animal suffering and the potential benefits of the research<SupScrollLink label="23"/>. An ethical dilemma frequently arises from the fact that primary cells offer the most meaningful data from a biological standpoint, yet their production is associated with challenges. In this context, the necessity of primary cell cultures is called into question, and the promotion of alternative methods, such as artificially produced tissues or organoids, is advocated where feasible. It is of crucial importance to emphasize the necessity of ethical responsibility in the collection of primary cultures. It is of the utmost importance that the procedure is carried out with consideration for the rights, and particularly the well-being of the donor. The removal of cells or tissue must be medically justifiable and, moreover, ethically justifiable in every case. To this end, the potential for research use and the possible risks and burdens for the donor must be weighed against each other to ensure careful consideration. However, it is also particularly important to ensure that the donor is involved in the entire process and is able to make an informed decision. The purpose of the research, the use of the cells and possible consequences must also be made transparent at all times.
The obtaining of informed consent represents a fundamental aspect of ethical practice in the collection of primary cells. This process must encompass not only a formal consent procedure, but also the provision of comprehensive information to donors regarding the collection, utilisation and prospective future applications of the cells. The act of consent must be given freely and without undue influence, and donors must be fully aware of the consequences of their participation. Furthermore, donors must be granted the right to revoke their consent at any time without consequence. Prior to the collection of cells, a comprehensive discussion is held with the donor, during which all pertinent details are elucidated and any queries or concerns they may have, are addressed. This guarantees that the donor is adequately informed and is thus able to make an autonomous decision based on a comprehensive understanding of the procedure.
The protection of privacy and confidentiality is of paramount importance when working with primary cultures. Given that primary cultures are predominantly human tissue, they contain genetic information and other personal data that is sensitive and deserving of protection. It is therefore of great importance that the data is anonymized and kept strictly confidential in order to protect the identity of the donor.
Every person who has access to the data or samples must be obliged to comply with confidentiality standards. It must be ensured that all legal requirements for data protection are met, including compliance with data protection laws such as the GDPR in the EU.
Every person who has access to the data or samples must be obliged to comply with confidentiality standards. It must be ensured that all legal requirements for data protection are met, including compliance with data protection laws such as the <a href="https://gdpr-info.eu/">GDPR</a> in the EU.
</p>
<H4 text="Safety aspects when working with primary cultures "></H4>
<p>
......@@ -313,60 +223,566 @@ export const Safety: React.FC = () =>{
</div>
</Subesction>
<Subesction title="Consent and Guidelines" id="Bioethics3">
<LoremMedium/>
<p></p>
<p>When working with primary cultures, it is extremely important to consider the bioethical aspects of the project. To address this, we sat down with the Ethics Officer at Bielefeld University, Dr. Berens, and discussed the matter with her. From this exchange, we gained the following insights.
On the one hand, having a patient consent form is crucial, as it provides the donors of the primary cells with a sense of security, but more importantly, it gives them detailed and precise information about what will be done with the data, whether it be regular patient data or biomaterials. Additionally, it became clear that it is significantly easier for us to establish guidelines to follow. We decided to integrate this approach into our project.
As a result, we created a patient consent form for the donors of primary cells, which we also want to present as a template for future German iGEM teams. <b>However, we want to emphasize that it is not guaranteed to be comprehensive, nor does it have any legal approval</b>. We also developed a guideline, which we present as a template, on handling primary cells to ensure not only proper technical handling but also correct ethical treatment.</p>
<p></p>
<div className='row align-items-center'>
<div className='col '>
<H4 text="Patient consent form"/>
<TwoLinePDF link="https://static.igem.wiki/teams/5247/pdfs/patienteneinwilligung-mustervorlage-igem-2.pdf" name="patienteneinwilligung-mustervorlage-igem-2.pdf"/>
</div>
<div className='seperator-2 col-2'>
</div>
<div className='col '>
<H4 text="Primary Culture Safety Guideline"/>
<TwoLinePDF link="https://static.igem.wiki/teams/5247/pdfs/primary-culture-guideline.pdf" name="primary-culture-guideline.pdf"/>
</div>
</div>
</Subesction>
</Section>
<Section title="Check-Ins" id="Check-Ins">
<div>
<p>
iGEM places great emphasis on biosafety, ensuring that all projects adhere to strict safety standards. One of these measures is the iGEM White List, which includes organisms and parts that are pre-approved for use based on their safety profile. Any components or organisms not covered by this White List must be submitted as 'Check-Ins' to the iGEM Safety Committee for approval. Check-Ins are formal safety evaluations that allow the committee to assess the potential risks and ensure proper containment and handling procedures are in place. Although we used some parts and organisms that were not included on the White List, these were assessed as critical for our project and submitted as Check-Ins to the iGEM Safety Committee. Furthermore, we were in active exchange with the committee throughout the process. The Check-Ins provide a clear picture of the biosafety aspects of our project, reflecting our commitment to safety and compliance with iGEM standards.
</p>
<p>
We adhere to good laboratory practices by ensuring proper handling of materials, effective emergency procedures, and correct waste disposal methods. This commitment guarantees a safe and compliant research environment. Our project, which involved a wide range of techniques was conducted in strict compliance with safety regulations. All experiments were carried out in Prof. Dr. Kristian Müller’s laboratory at Bielefeld University, following BSL-1 standard operating procedures. Properly equipped facilities are crucial to prevent contamination, exposure, or accidental release of modified organisms, ensuring the highest level of safety in our laboratories.
Before commencing laboratory work, all participants were required to attend a mandatory safety briefing. In compliance with German regulations, each team member's participation had to be confirmed with a personal signature. The briefing, conducted by Prof. Dr. Kristian Müller must be renewed annually in accordance with §12 ArbSchG. It covered the following areas:
</p>
<ul>
<li>General laboratory safety</li>
<li>Regulations regarding hazardous and toxic substances</li>
<li>Regulations concerning biological materials</li>
<li>Regulations on genetic engineering</li>
</ul>
<p>
In addition to the general safety briefing, specific instructions for the safe operation of each device were provided. The Safety and Security Officer within the laboratory highlighted the potential hazards and necessary precautionary measures. We have focused on planning our laboratory activities to minimize risk for safer practices. This ensures not only the safe and proper use of equipment but also the generation of reliable data. To meet all safety requirements, additional safety protocols have been put in place for all targeted areas of the laboratory equipment.
</p>
<H4 text="Laboratory and safety practices"></H4>
<p>
As part of our project to develop a prime-editing complex to correct the F508del mutation in Cystic Fibrosis, we place great emphasis on safety at all stages of research. Our final construct will be tested in <a onClick={() => goToPageAndScroll ('Cell Culture3H', '/materials-methods')}> primary cultures of epithelial cells </a> obtained from nasal swabs, isolated from both patients and healthy individuals. To guarantee safety and ensure the highest level of precision and reliability of our results, we have introduced a series of carefully planned checkpoints during the experiments. These milestones allow for continuous monitoring, timely adjustments and validation at each critical stage. This ensures that potential issues are identified and addressed immediately, minimizing risk and improving the overall quality of the experimental results{/* . [link zu den Experimenten] */}. iGEM places great emphasis on biosafety, ensuring that all projects adhere to strict safety standards. One of these measures is the iGEM White List, which includes organisms and parts that are pre-approved for use based on their safety profile. Any components or organisms not covered by this White List must be submitted as 'Check-Ins' to the iGEM Safety Committee for approval. Check-Ins are formal safety evaluations that allow the committee to assess the potential risks and ensure proper containment and handling procedures are in place. Although we used some parts and organisms that were not included on the White List, these were assessed as critical for our project and submitted as Check-Ins to the iGEM Safety Committee. Furthermore, we were in active exchange with the committee throughout the process. The Check-ins provide a clear picture of the biosafety aspects of our project, reflecting our commitment to safety and compliance with iGEM standards.
The main safety measures we have implemented include:
</p>
<p>
<strong>Compliance with S1 conditions:</strong> Working in S1 laboratories ensures that only organisms in the lowest risk group are used, minimizing the risk to humans and the environment.
</p>
<p>
<strong>Sterile working practices:</strong> To avoid contamination, we have implemented strict hygiene measures, including the disinfection of work surfaces and the correct disposal of biological waste.
</p>
<p>
<strong>Controlled access:</strong> Access to laboratories was strictly regulated to ensure that only trained personnel worked with the genetically modified organisms and cell lines.
</p>
<p>
<strong>Documentation:</strong> All work steps, materials used and cell lines were carefully documented to ensure traceability and safety.
</p>
<p>
<strong>Safe handling of cell lines:</strong> The cell lines used for experiments were handled in accordance with the applicable safety regulations. This included regular checks for contamination and the safe storage and disposal of cell cultures.
</p>
<Collapsible id="Checkpek" open={false} title="Check-in for the Prime-Editing Komplex ">
<p>
<strong>Reverse transcriptase:</strong> Reverse transcriptase plays a central role in prime editing by specifically inserting the correction as DNA at the inserted nick using an RNA template provided by pegRNA. The correction of the complementary DNA strand then takes place via the natural cell repair mechanisms. This ensures an exact correction of the target sequence. We checked the reverse transcriptase to ensure it could perform precise genome editing without introducing unintended mutations. This was important to minimize the risk of off-target effects that could lead to unexpected or harmful consequences.
</p>
<p>
<strong>pegRNA (Prime Editing Guide RNA):</strong> The pegRNA is a multifunctional RNA molecule that fulfils two essential tasks. Firstly, it serves as a standard guide RNA (gRNA) that binds specifically to the target DNA and thus marks the site of editing. Secondly, it contains an RNA template that encodes the desired DNA modification. This enables the precise integration of the genetic modifications at the target site. We evaluated pegRNA for its ability to specifically target and modified the intended DNA sequence. Ensuring its specificity was crucial to avoid the potential disruption of other genes.
</p>
<p>
<strong>Nickase nCas9, CasX, Fanzor (SpuFz1):</strong> These modified nucleases are designed to cut only one strand of DNA. This leads to controlled and precise editing of the genome, as cutting only one strand minimizes the risk of unwanted double-strand breaks. CasX and Fanzor offer smaller alternatives to Cas9, which is particularly advantageous for use in cells or organisms where space and efficiency requirements in terms of the transport system are an issue. Fanzor, being a newly introduced endonuclease, was particularly scrutinized in our project to ensure its safety and effectiveness in different cellular contexts.
This prime-editing complex thus represents a precise and efficient method for gene editing. By combining these components, genetic modifications can be performed with minimal side effects
</p>
</Collapsible>
<Collapsible id="Checkcloning" open={false} title="Check-in for Cloning">
<p>
For our cloning experiments and the development of our prime editing complexes, we have amplified various plasmids in <i>E. coli</i> K-12 strains (DH5α,10-Beta). When working with microbial strains such as <i>E. coli</i> K-12 strains, it's important to consider potential risks associated with their use, even though they are generally regarded as safe in laboratory settings. All experiments were performed under strict S1 conditions, following all relevant safety protocols. Below you will find an overview of the <i>E. coli</i> K-12 strains for our cloning experiments, submitted by us as a check-In and the specific safety measures:
</p>
<p>
<strong><i>E. coli K-12</i> strains (DH5α, 10-Beta):</strong> Although these strains are non-pathogenic and have been modified to minimize the risk of spreading antibiotic resistance, there remains a low risk of horizontal gene transfer, where genetic material could be transferred to other microorganisms, potentially leading to the spread of resistance genes or other traits. If accidentally released into the environment, <i>E. coli</i> K-12 strains could potentially interact with native microbial communities. While they are typically outcompeted in natural environments, there's a remote possibility of ecological disruption, particularly in microenvironments where they could find a niche.While these strains are non-virulent, they still pose a minimal risk to humans, particularly immunocompromised individuals, through accidental ingestion or inhalation in a laboratory setting.
</p>
<p>
We submitted the yeast strain <i>Pichia pastoris</i> (SMD1163) for the protein expression of Fanzor.
</p>
<p>
<strong><i>Pichia pastoris</i> (SMD1163):</strong> <i>Pichia pastoris</i> (SMD1163) is a widely used yeast strain for the expression of recombinant proteins. It is characterized by a methanol-inducible expression system (AOX1 promoter) and high cell growth rates, which makes it ideal for industrial applications. The strain can be easily genetically manipulated and can perform post-translational modifications, which supports correct protein production.
When working with <i>Pichia pastoris</i> (SMD1163), various safety-relevant aspects must be observed. Although the organism is considered non-pathogenic and biologically safe (S1), skin contact and aerosol formation should be avoided to minimize the risk of infection or allergic reactions. When using genetically modified strains, it is important to follow the relevant GMO guidelines to prevent uncontrolled release. In addition, handling chemicals such as methanol requires special precautions as they are toxic and highly flammable. The disposal of cell cultures and waste must also be carried out in accordance with biosafety regulations, especially in the case of genetically modified organisms.
</p>
</Collapsible>
<Collapsible id="CheckcellLines" open={false} title="Check-in for Testing in cell lines">
<p>
In our project, we paid attention to safety at every step, especially when working with specific <a onClick={() => goToPageAndScroll ('cell-culture', '/materials-methods')}> cell lines </a> . All experiments were performed under strict S1 conditions, following all relevant safety protocols. Given the sensitivity of the human cell lines we used, we placed great emphasis on controlled and well-designed workflows. All transfections were performed in our own transfection laboratory to ensure a high level of safety and compliance. Below you will find an overview of the cell lines submitted by us as a checkin and the specific safety measures:
</p>
<p>
<strong>HEK293 cell line: </strong>HEK 293 (Human Embryonic Kidney 293) cells are an immortal cell line originally derived from the kidney cells of a human embryo. They are characterized by their fast division rate and high transfection efficiency, which makes them a popular model in biomedical research. For our studies, the basic HEK293 cells were provided to us by the Cellular and Molecular Biotechnology Group at Bielefeld University, headed by Prof. Dr. Kristian Müller. Prof. Dr. Müller is also one of the Principal Investigators of our team. We use this cell line in our proof-of-concept studies and for testing the Prime Editing Guide pegRNA (pegRNA) to evaluate the efficiency and functionality of our constructs.
</p>
<p>
<strong>HEK293T-3HA-CFTR cell line: </strong>The HEK293T-3HA-CFTR cell line is based on HEK293T cells expressing an additional tsA1609 allele of the SV40 large T antigen. This allele enables the replication of vectors containing the SV40 origin of replication. In addition to the native CFTR gene, which is not expressed in HEK cells, the HEK293T-3HA-CFTR cell line carries another copy of the CFTR gene embedded in an expression cassette. This cassette contains a CMV promoter, which is derived from the human cytomegalovirus and is frequently used for the overexpression of genes in human cells. In addition, the cassette contains a puromycin resistance gene that is co-expressed with CFTR, allowing continuous selection of CFTR-expressing cells.
</p>
<p>
<strong>HEK293T-3HA-F508del-CFTR cell line:</strong> The HEK293T-3HA-F508del-CFTR cell line is a modified HEK293T cell line that carries the F508del mutation in the CFTR gene, which is responsible for the most common mutation in Cystic Fibrosis. This mutation leads to a defective CFTR protein that impairs the normal function of the chloride channel. The cell line is therefore ideal for studying the effects of this mutation and for evaluating potential therapies for Cystic Fibrosis.
</p>
<p>
<strong>CFBE41o- cell line:</strong> The CFBE41o- cell line, derived from the bronchial epithelial cells of a Cystic Fibrosis patient, is homozygous for the F508del-CFTR mutation and was essential for our Cystic Fibrosis research. A reduced CFTR expression level is present. The cell line carries the CFTR defect and can therefore represent a patient with CF. The cell line is used to test our mechanism. These cells were immortalized with a replication-defective plasmid that retains their physiological properties.
</p>
<p>
When working with the HEK293T and CFBE41o- cell lines, it’s important to consider the minimal risks associated with their use. While not harmful on their own, the genetic modifications in HEK293T cells require careful handling to prevent accidental release or exposure. These cells, engineered to overexpress CFTR, including the F508del mutation, necessitate strict safety measures like regular monitoring and proper waste disposal to comply with S1 laboratory standards. Similarly, CFBE41o- cells, due to their genetic modifications and disease relevance, require careful handling to avoid cross-contamination and ensure biosafety.
</p>
<p>
<strong>Human nasal epithelial cells (hNECs):</strong> Human nasal epithelial cells (hNECs) were harvested using a nasal brush, a minimally invasive procedure, and cultured in air-liquid interface (ALI) cultures to model the airway epithelium. Human nasal epithelial cells (hNECs) were obtained using a nasal brush, a minimally invasive technique, and then cultured in air-liquid interface (ALI) cultures to model the airway epithelium. Using these primary cultures, derived from donors with airway diseases such as Cystic Fibrosis, we were able to simulate the in vivo conditions of such diseases.
Due to the sensitive nature of these primary human cells, we performed all experiments with hNECs in our S2 laboratory, where increased safety precautions were taken. This included strict safety controls, safe handling of samples and proper disposal of materials after testing. In particular, the hNECs underwent HHH (Triple H: HIV, HCV and HBV) testing to ensure that no contamination occurred during sample collection or experimentation. These tests included sterility testing, viability assessments and contamination testing to ensure the safety and integrity of both the samples and the laboratory environment. After a negative HHH test, the primary cultures can be treated as S1. In addition, the nasal epithelial cells were handled with the utmost care during collection, ensuring that all procedures were performed under sterile conditions to avoid any risk of contaminationFor this purpose, the intensive examination of ethical questions was fundamental and a constant companion of our project. The numerous results from the interviews in the areas of: Ethics, storage and training in the handling of samples have been summarized in a guideline for patient consent for Germany and are intended to provide iGEM teams with the scope, critical examination and observance of iGEM rules, international and national guidelines.
</p>
</Collapsible>
<Collapsible id="CheckDelivery" open={false} title="Check-in for Delivery">
<p>
Our finished construct is designed to be delivered into the lung via an inhaler using lipid nanoparticles (LNPs). To be more spezific a selective organ-targeting (SORT)- LNPs were developed to deliver mRNA specifically to the lung, with special measures taken to increase biocompatibility and safety. Since the LNP composition is very specific and also differs from other formulas, we submitted the LNP as a checkin:
</p>
<p>
<strong>LNP:</strong> These LNPs are then taken up by epithelial cells through endocytosis, releasing the construct into the cytosol. We carefully evaluated the potential risks, including unintended immune responses and the need for precise dosing to minimize side effects. In addition, we have conducted an in-depth analysis of the dual-use potential of our technology. Dual-use refers to the possibility that scientific advances can be used for both civilian and military purposes. Therefore, we have implemented strict safety protocols and ethical guidelines to ensure that our technology is used exclusively for peaceful and therapeutic applications.
</p>
</Collapsible>
</div>
</Section>
<Section title="Our Lab" id="Our Lab">
<p>
As part of our laboratory activities for our <PreCyse/> project, we worked in various laboratories. For general lab work and cloning experiments, you can find some pictures of our laboratories below:
</p>
<H4 text="Our Cloning Lab"></H4>
<p>
Our Cloning-laboratory is divided into different work areas to ensure that the experiments run smoothly and efficiently. These include the gel station, the PCR station, the transformation section and the measurement area. Each area is specially equipped for the respective method, and the corresponding experiments were carried out exclusively in the designated stations. In this way, we ensure that our work is carried out under optimal conditions and with the greatest possible precision.
<OneFigure
pic1="https://static.igem.wiki/teams/5247/photos/biosafety/kollage/new/img-2041.jpeg"
num={3}
description="Photo-gallery of laboratory. A: Key lock. B: Key-locked door. C: Alarm plan. D: Emergeny button for electriotion stop. E: Emergency telephone. F: First aid kit, cardiac defibrillaton and emergency exit and fire alarm plan. G: Wash bin with emergency eye wash. H: Emergency shower. I: Lockable cabinets for chemical storage"
alt1="Photo-gallery of laboratory. A: Key lock. B: Key-locked door. C: Alarm plan. D: Emergeny button for electriotion stop. E: Emergency telephone. F: First aid kit, cardiac defibrillaton and emergency exit and fire alarm plan. G: Wash bin with emergency eye wash. H: Emergency shower. I: Lockable cabinets for chemical storage"
/>
<OneFigure
pic1="https://static.igem.wiki/teams/5247/photos/biosafety/kollage/new/img-2037.jpeg"
num={4}
description="Photo-gallery of S1 laboratory. A: Autoclave. B: Refrigerator with chemicals. C: Weighing room with chemical storage. D: Clean bench work space with vortex, pipettes, heat block and bench top centrifuge. E: pH electrode in fume hood. F: Ice machine. G: Fire distinguisher and S1 waste. H: Fume hood with liquid waste"
alt1="Photo-gallery of S1 laboratory. A: Autoclave. B: Refrigerator with chemicals. C: Weighing room with chemical storage. D: Clean bench work space with vortex, pipettes, heat block and bench top centrifuge. E: pH electrode in fume hood. F: Ice machine. G: Fire distinguisher and S1 waste. H: Fume hood with liquid waste."
/>
</p>
<H4 text="Our Cell Culture Lab "></H4>
<p>
In our cell culture laboratory, we work under sterile conditions to ensure optimal growth conditions for human cell lines. Among other things, we carry out transfections in order to introduce genetic material into cells and investigate their behavior. Strict protocols and state-of-the-art technology ensure the precision and reproducibility of our experiments.
</p>
<OneFigure
pic1="https://static.igem.wiki/teams/5247/photos/biosafety/kollage/new/img-2040.jpeg"
num={5}
description="Photo-gallery of laboratory and chemical storage. A: Safety cabinets. B: Incubator. C: Safety cabinet"
alt1="Photo-gallery of laboratory and chemical storage. A: Safety cabinets. B: Incubator. C: Safety cabinet"
/>
<p>
In our S2 laboratory, the harvested nasal epithelial cells that serve as primary cultures undergo a comprehensive HHH test to ensure their safety and suitability for further experiments. This test is crucial to ensure that we can subsequently work safely with these cells in the S1 range without the risk of contamination or unwanted release of biological material.
</p>
<OneFigure
pic1="https://static.igem.wiki/teams/5247/photos/biosafety/kollage/new/img-2042.jpeg"
num={6}
description="Photo-gallery of S2 laboratory. A: Door of S2 lab with S2 sign. B: Emergency shower and fire distinguisher. C: Clean bench with centrifuge. D: Incubator. E: Safety cabinet. F: Emergeny telephone. G: S2 lab coat with S2 sign. H: Microscope. I: Autoclave"
alt1="Photo-gallery of S2 laboratory. A: Door of S2 lab with S2 sign. B: Emergency shower and fire distinguisher. C: Clean bench with centrifuge. D: Incubator. E: Safety cabinet. F: Emergeny telephone. G: S2 lab coat with S2 sign. H: Microscope. I: Autoclave."
/>
</Section>
<Section title="References" id="References">
<ol>
{/*<!-- Citation num 1--> */}
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<span property="schema:author" typeof="schema:Organisation">
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<ol>{/*<!-- Citation num 1--> */}
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<span property="schema:author" typeof="schema:Person">
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<span property="schema:Name"> Beaulieu, J.</span>
<span property="schema:Name"> Suthananthan, A.</span>
<span property="schema:Name"> Lehnertz, B.</span>
<span property="schema:Name"> Sauvageau, G.</span>
<span property="schema:Name"> Sheppard, H. M.</span>
<span property="schema:Name"> Knapp, D. J. H. F.</span>
</span>
<span property="schema:name">&nbsp;
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progenitor cells
</span>.
<i property="schema:publisher" typeof="schema:Organization"> eLife</i>
<b property="issueNumber" typeof="PublicationIssue"> 12,</b>
&nbsp;(<time property="schema:datePublished" datatype="xsd:gYear" dateTime=" 2024">2024</time>).
<a className="doi" href="https://doi.org/10.7554/eLife.91288"> doi: 10.7554/eLife.91288</a>
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<li typeof="schema:ScolarlyArticle" role="doc-biblioentry" property="schema:citation" id="desc-2">
<span property="schema:author" typeof="schema:Person">
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<span property="schema:Name"> Everette, K. A.</span>
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<span property="schema:Name"> Anzalone, A. V.</span>
<span property="schema:Name"> An, M.</span>
<span property="schema:Name"> et al.</span>
</span>
<span property="schema:name">&nbsp;Engineered pegRNAs improve prime editing efficiency</span>.
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<a className="doi" href="https://doi.org/10.1038/s41587-021-01039-7"> doi: 10.1038/s41587-021-01039-7</a>
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<li typeof="schema:ScolarlyArticle" role="doc-biblioentry" property="schema:citation" id="desc-3">
<span property="schema:author" typeof="schema:Person">
<span property="schema:Name"> Doench, J. G.</span>
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<span property="schema:Name"> White, N.</span>
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<span property="schema:Name"> Wei, T.</span>
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<span property="schema:Name"> Ibrahim, M.</span>
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(<time property="schema:datePublished" datatype="xsd:gYear" dateTime=" 2022">2022</time>).
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<span property="schema:author" typeof="schema:Person">
<span property="schema:Name"> Jiang, A. Y.</span>
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<span property="schema:Name"> Oladimeji, F. A.</span>
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<b property="issueNumber" typeof="PublicationIssue"> 19</b>
,&nbsp;<span property="schema:pageBegin"> 364</span>-<span property="schema:pageEnd">375</span>&nbsp;
(<time property="schema:datePublished" datatype="xsd:gYear" dateTime=" 2024">2024</time>).
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<li typeof="schema:ScolarlyArticle" role="doc-biblioentry" property="schema:citation" id="desc-10">
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<span property="schema:Name"> Vilà-González, M.</span>
<span property="schema:Name"> Pinte, L.</span>
<span property="schema:Name"> Fradique, R.</span>
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<span property="schema:Name"> Paris, K.</span>
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<span property="schema:author" typeof="schema:Person">
<span property="schema:Name"> Wickiser, J. K.</span>
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<span property="schema:Name"> Cohen, J.</span>
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,&nbsp;<span property="schema:pageBegin"> 219</span>-<span property="schema:pageEnd">228</span>&nbsp;
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<span property="schema:author" typeof="schema:Person">
<span property="schema:Name"> Doudna, J. A.</span>
<span property="schema:Name"> Charpentier, E.</span>
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,&nbsp;<span property="schema:pageBegin"> 144</span>-<span property="schema:pageEnd">156</span>&nbsp;
(<time property="schema:datePublished" datatype="xsd:gYear" dateTime=" 2020">2020</time>).
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<span property="schema:Name"> Shwartz, M.</span>
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&nbsp;(<time property="schema:datePublished" datatype="xsd:gYear" dateTime=" 2019">2019</time>).
<a className="doi" href="https://doi.org/10.22323/2.18040202"> doi: 10.22323/2.18040202</a>
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<span property="schema:Name"> Chadwick, R. F.</span>
</span>
<span property="schema:name">&nbsp;Encyclopedia of applied ethics.</span>
<i property="schema:publisher" typeof="schema:Organization">&nbsp;Academic Press</i>
&nbsp;(<time property="schema:datePublished" datatype="xsd:gYear" dateTime="2012">2012</time>).
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{/*<!-- Citation num 2--> */}
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<span property="schema:author" typeof="schema:Person">
<span property="schema:Name"> Rubeis, G.</span>;
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<span property="schema:name">&nbsp;Risks and benefits of human germline genome editing: An ethical analysis. </span>
<span property="schema:name">&nbsp;Risks and benefits of human germline genome editing: An ethical analysis</span>.
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<b property="issueNumber" typeof="PublicationIssue"> 10</b>
,&nbsp;<span property="schema:pageBegin"> 133</span>-<span property="schema:pageEnd">141</span>&nbsp;
(<time property="schema:datePublished" datatype="xsd:gYear" dateTime=" 2018">2018</time>).
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<span property="schema:Name"> Ansah, E.</span>
<span property="schema:Name"> Ansah, E. O.</span>
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<span property="schema:Name"> Pugh, J.</span>
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{/*<!-- Citation num 20--> */}
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</li>
</ol>
</ol>
</Section>
</>
);
......
src/contents/team.tsx
View file @ 3fd292fd
......@@ -15,6 +15,7 @@ export function Team() {
<br/>
<br/>
<iframe title="Bielefeld-CeBiTec: Sitcom Team Presentation (2024) [English]" width="100%" height="600px" src="https://video.igem.org/videos/embed/e40d89a0-2461-421c-a75c-92b3b238e0f8" frameBorder="0" allowFullScreen={true} sandbox="allow-same-origin allow-scripts allow-popups allow-forms"></iframe>
<Section title="Members" id="members">
{teambriefe}
</Section>
......@@ -28,6 +29,7 @@ export function Team() {
<Section title="Principal Investigators" id="PIS">
{pibriefe}
</Section>
<img src="https://static.igem.wiki/teams/5247/photos/team-photos/gruppe-v.webp" alt="Team Bielefeld in lab coats and V-Formation"/>
<BackUp/>
</>
);
......@@ -36,7 +38,7 @@ export function Team() {
function createSteckbriefe(data: Array<SteckbriefInterface>){
let briefe = []
let briefe: JSX.Element[] = []
for (let index = 0; index < data.length; index++) {
let thename = "" + data[index].vorname + data[index].nachname;
// Conditional head of
......@@ -47,7 +49,10 @@ function createSteckbriefe(data: Array<SteckbriefInterface>){
// Jobs
var jobs = "";
for (let i = 0; i < data[index].igemjob.length; i++) {
if (i + 1 == data[index].igemjob.length ) {
if (data[index].igemjob.length == 1) {
jobs += data[index].igemjob[i]
}
else if (i + 1 == data[index].igemjob.length ) {
jobs += " and " + data[index].igemjob[i];
}
else if(i + 2 == data[index].igemjob.length){
......@@ -59,7 +64,7 @@ function createSteckbriefe(data: Array<SteckbriefInterface>){
}
}
// Funfacts
var funfacts = [];
var funfacts: JSX.Element[] = [];
for (let i = 0; i < data[index].funfacts.length; i++) {
funfacts.push(<li key={`f${i}`}> {data[index].funfacts[i]} </li>);
}
......@@ -80,7 +85,7 @@ function createSteckbriefe(data: Array<SteckbriefInterface>){
}
}
// Whyigem
var why = [];
var why: JSX.Element[] = [];
for (let i = 0; i < data[index].whyigem.length; i++) {
why.push(<p>{data[index].whyigem[i]}</p>);
}
......@@ -90,21 +95,21 @@ function createSteckbriefe(data: Array<SteckbriefInterface>){
title = data[index].title!;
}
// challenges
let challs = [];
let challs: JSX.Element[] = [];
for (let i = 0; i < data[index].biggestchallenge.length; i++) {
challs.push(<li key={`d${i}`}>{data[index].biggestchallenge[i]}</li>);
}
// bestparts
let bests = [];
let bests: JSX.Element[] = [];
for (let i = 0; i < data[index].bestpart.length; i++) {
bests.push(<li key={`e${i}`}>{data[index].bestpart[i]}</li>);
}
let frontbriefclass = "frontbrief frontbrief"+thename;
let backbriefclass = "backbrief backbrief"+thename;
let picture = <div className="col-2 lnp center"><img src={data[index].zweitfoto} style={{display: "none"}} className={"img team-img "+backbriefclass}/><img src={data[index].hauptfoto} className={"img team-img "+frontbriefclass}></img></div>
let picture = <div className="col-2 lnp center"><img src={data[index].zweitfoto} style={{display: "none"}} className={"img team-img side-margins-auto "+backbriefclass}/><img src={data[index].hauptfoto} className={"img team-img "+frontbriefclass}></img></div>
let namerow = <div className="row"><div className="team-name"> {title} {data[index].vorname} {data[index].nachname} <span className="pronouns"> ({data[index].pronouns}) </span> </div> <div className="col"> <a href={data[index].linkedinurl}> <img className="team-socials" src="https://static.igem.wiki/teams/5247/design/icons/linkedin.png" /> </a></div> </div>;
let frontparagraph = <div className={"row " + frontbriefclass}> <h6>Why I took part in iGEM</h6> {why} </div> ;
let frontparagraph = <div className={"row " + frontbriefclass}> <h6>Why I took part in iGEM ?</h6> {why} </div> ;
let facts = <div className={frontbriefclass}><div className=""> <b>Age:</b> {data[index].age} </div> <br/> {headof} <div> <b>Part of:</b> {jobs}</div> <br/> <div className=""> <b>Major:</b> {data[index].studiengang} </div> <br/> <div className=""> <b>Scientific interests:</b> {data[index].scientificinterests} </div> </div>;
let backbutton = <div className={backbriefclass} style={{display: "none"}}> <div className="parent-button"><button onClick={flipBack(thename)} className="frontbutton">Click me</button></div></div>
let funfactlist = <div className={backbriefclass} style={{display: "none"}}><b>Funfacts: </b><ul> {funfacts}</ul></div>
......@@ -128,7 +133,7 @@ function createSteckbriefe(data: Array<SteckbriefInterface>){
<div className="col-4 brieffacts">
<br/><br/> {funfactlist} {facts}
</div>
</div> <div className="row" style={{marginTop: "1rem", marginBottom: "1rem"}}> <div className="col-2"> {frontbutton} {backbutton}</div> <div className="col"><details className={frontbriefclass} ><summary> <b>Personal motivation and challenges</b> </summary><div> {details}</div></details></div> </div>
</div> <div className="row steckbriefbuttonrow" style={{marginTop: "1rem", marginBottom: "1rem"}}> <div className="col-2"> {frontbutton} {backbutton}</div> <div className="col"><details className={frontbriefclass} ><summary> <b>Personal motivation and challenges</b> </summary><div> {details}</div></details></div> </div>
</div>
let whole = <div className={"steckbrief-box"} id={thename}> {hole} </div>;
briefe.push(whole);
......@@ -137,7 +142,7 @@ function createSteckbriefe(data: Array<SteckbriefInterface>){
}
function createPiSteckbriefe(data: Array<SteckbriefInterface>){
let briefe = []
let briefe: JSX.Element[] = []
for (let index = 0; index < data.length; index++) {
let thename = "" + data[index].vorname + data[index].nachname;
// Conditional head of
......@@ -156,7 +161,7 @@ function createPiSteckbriefe(data: Array<SteckbriefInterface>){
}
}
// Funfacts
var funfacts = [];
var funfacts: JSX.Element[] = [];
for (let i = 0; i < data[index].funfacts.length; i++) {
funfacts.push(<li key={`a${i}`}> {data[index].funfacts[i]} </li>);
}
......@@ -175,7 +180,7 @@ function createPiSteckbriefe(data: Array<SteckbriefInterface>){
}
}
// Whyigem
var why = [];
var why: JSX.Element[] = [];
for (let i = 0; i < data[index].whyigem.length; i++) {
why.push(<p>{data[index].whyigem[i]}</p>);
}
......@@ -185,19 +190,19 @@ function createPiSteckbriefe(data: Array<SteckbriefInterface>){
title = data[index].title!;
}
// challenges
let challs = [];
let challs: JSX.Element[] = [];
for (let i = 0; i < data[index].biggestchallenge.length; i++) {
challs.push(<li key={`b${i}`}>{data[index].biggestchallenge[i]}</li>);
}
// bestparts
let bests = [];
let bests: JSX.Element[] = [];
for (let i = 0; i < data[index].bestpart.length; i++) {
bests.push(<li key={`c${i}`}>{data[index].bestpart[i]}</li>);
}
let frontbriefclass = "frontbrief frontbrief"+thename;
let backbriefclass = "backbrief backbrief"+thename;
let picture = <div className="col-2 lnp center"><img src={data[index].zweitfoto} style={{display: "none"}} className={"img team-img "+backbriefclass}/><img src={data[index].hauptfoto} className={"img team-img "+frontbriefclass}></img></div>
let picture = <div className="col-2 lnp center"><img src={data[index].zweitfoto} style={{display: "none"}} className={"img team-img side-margins-auto "+backbriefclass}/><img src={data[index].hauptfoto} className={"img team-img "+frontbriefclass}></img></div>
let namerow = <div className="row"><div className="team-name"> {title} {data[index].vorname} {data[index].nachname} <span className="pronouns"> ({data[index].pronouns}) </span> </div> <div className="col"> <a href={data[index].linkedinurl}> <img className="team-socials" src="https://static.igem.wiki/teams/5247/design/icons/linkedin.png" /> </a></div> </div>;
let frontparagraph = <div className={"row " + frontbriefclass}> <h6>Why I took part in iGEM</h6> {why} </div> ;
let facts = <div className={frontbriefclass}><div className=""> <b>Age:</b> {data[index].age} </div> <br/> {headof} <div> <b>Affiliation:</b> {jobs}</div> <br/> <div className=""> <b>Regular Job:</b> {data[index].studiengang} </div> <br/> <div className=""> <b>Scientific interests:</b> {data[index].scientificinterests} </div> </div>;
......@@ -223,7 +228,7 @@ function createPiSteckbriefe(data: Array<SteckbriefInterface>){
<div className="col-4 brieffacts">
<br/><br/> {funfactlist} {facts}
</div>
</div> <div className="row" style={{marginTop: "1rem", marginBottom: "1rem"}}> <div className="col-2"> {frontbutton} {backbutton}</div> <div className="col"><details className={frontbriefclass} ><summary> <b>Personal motivation and challenges</b> </summary><div> {details}</div></details></div> </div>
</div> <div className="row steckbriefbuttonrow" style={{marginTop: "1rem", marginBottom: "1rem"}}> <div className="col-2"> {frontbutton} {backbutton}</div> <div className="col"><details className={frontbriefclass} ><summary> <b>Personal motivation and challenges</b> </summary><div> {details}</div></details></div> </div>
</div>
let whole = <div className={"steckbrief-box"} id={thename}> {hole} </div>;
briefe.push(whole);
......
src/data/drug-data.tsx
View file @ 3fd292fd
import { SupScrollLink } from "../components/ScrollLink";
export interface DrugDatensatz {
name: string;
picture: string;
introduction: string;
introduction: string | React.ReactNode;
examples: Array<example>;
}
interface example{
title: string,
text: Array<string> | Array<React.ReactNode>;
......@@ -32,50 +34,66 @@ export const drugdata: (Array<DrugDatensatz>) = [
{
//gibt 4 Modulator Beispiele
name: "Modulators",
picture: "...",
introduction: "CFTR modulators represent a significant advancement in CF treatment since they are small molecules improving the function of the defective CFTR protein in a mutation-specific way, which helps restore chloride ion transport across cell membranes. Notable pharmaceutical agents include Trikafta®, Symdeko®, Orkambi® and Kalydeco® [1]. These medications have been demonstrated to significantly improve lung function and reduce pulmonary exacerbations. However, they are expensive and may cause side effects such as liver enzyme elevations and cataracts in pediatric patients [2]. Furthermore, they are not suitable for all CF patients since only mutations which produce a CFTR channel can be supported by CFTR modulators, not those mutations which lead to a missing CFTR channel (knock out) [1], e.g. stop-mutations including p.Arg553Ter or p.Gly542Ter [3]. ",
picture: "https://static.igem.wiki/teams/5247/scientific-figures/modulators.svg",
introduction: <>CFTR modulators represent a significant advancement in CF treatment since they are small molecules improving the function of
the defective CFTR protein in a mutation-specific way, which helps restore chloride ion transport across cell membranes. Notable
pharmaceutical agents include Trikafta®, Symdeko®, Orkambi® and Kalydeco®<SupScrollLink label="48"/>{/* ehem74 */}.
These medications have been demonstrated to significantly improve lung function and reduce pulmonary exacerbations. However, they are
expensive and may cause side effects such as liver enzyme elevations and cataracts in pediatric patients<SupScrollLink label="49"/>{/* ehem75 */}.
Furthermore, they are not suitable for all CF patients since only mutations which produce a CFTR channel can be supported by CFTR
modulators, not those mutations which lead to a missing CFTR channel (knock out) <SupScrollLink label="48"/>{/* ehem74 */}, e.g.
stop-mutations including p.Arg553Ter or p.Gly542Ter<SupScrollLink label="50"/>{/* ehem76 */}. </>,
examples: [
{
title: "Trikafta", //quelle 4
text: ["Active ingredient(s): Combination of elexacaftor/tezacaftor/ivacaftor","Indications: For CF patients aged 2 years and older with at least one F508del mutation = 85 % of CF patients","Mechanism: Elexacaftor and tezacaftor act as correctors on misfolded CFTR and permit delivery to the cell surface, thereby improving the channel density at the plasma membrane, while ivacaftor as a potentiator acts on CFTR channels that have reached the cell surface and increase the gating and conductance of ions [5]","Administration: Oral tablets","Approval: Approved by the EMA in 2020 "]
text: [<>Active ingredient(s): Combination of elexacaftor/tezacaftor/ivacaftor","Indications: For CF patients aged 2 years and older
with at least one F508del mutation = 85 % of CF patients","Mechanism: Elexacaftor and tezacaftor act as correctors on misfolded CFTR
and permit delivery to the cell surface, thereby improving the channel density at the plasma membrane, while ivacaftor as a potentiator
acts on CFTR channels that have reached the cell surface and increase the gating and conductance of ions<SupScrollLink label="51"/>{/*ehem77*/}","Administration: Oral tablets</>,"Approval: Approved by the EMA in 2020 "]
},
{
title: "Symdeko", //quelle 1
text: ["Active ingredient(s): Combination of tezacaftor and ivacaftor","Indications: For CF patients aged 6 years and older with specific mutations in combination with F508del or with two copies of F508del mutation", "Mechanism: Tezacaftor acts as a corrector on misfolded CFTR and permit delivery to the cell surface, thereby improving the channel density at the plasma membrane, while ivacaftor as a potentiator acts on CFTR channels that have reached the cell surface and increase the gating and conductance of ions [5]", "Administration: Oral tablets", "Approval: Approved by the EMA in 2018"]
text: ["Active ingredient(s): Combination of tezacaftor and ivacaftor","Indications: For CF patients aged 6 years and older with specific mutations in combination with F508del or with two copies of F508del mutation",
<>Mechanism: Tezacaftor acts as a corrector on misfolded CFTR and permit delivery to the cell surface, thereby improving the channel
density at the plasma membrane, while ivacaftor as a potentiator acts on CFTR channels that have reached the cell surface and increase
the gating and conductance of ions<SupScrollLink label="51"/>{/*ehem77*/} </>, "Administration: Oral tablets", "Approval: Approved by the EMA in 2018"]
},
{
title: "Orkambi", //quelle 6
text: ["Active ingredient(s): Combination of lumacaftor and ivacaftor", "Indications: For CF patients aged 1 year and older with two copies of the F508del mutation","Mechanism: Lumacaftor acts as a corrector on misfolded CFTR and permit delivery to the cell surface, thereby improving the channel density at the plasma membrane, while ivacaftor as a potentiator act on CFTR channels that have reached the cell surface and increase the gating and conductance of ions [5]","Administration: Oral tablets","Approval: Approved by the EMA in 2015"]
text: ["Active ingredient(s): Combination of lumacaftor and ivacaftor", "Indications: For CF patients aged 1 year and older with two copies of the F508del mutation",<>Mechanism: Lumacaftor acts as a corrector on misfolded CFTR and permit delivery to the cell surface, thereby improving the channel density at the plasma membrane, while ivacaftor as a potentiator act on CFTR channels that have reached the cell surface and increase the gating and conductance of ions<SupScrollLink label="51"/>{/*ehem77*/}</>,"Administration: Oral tablets","Approval: Approved by the EMA in 2015"]
},
{
title: "Kalydeco", //quelle 7
text: ["Active ingredient(s): Ivacaftor","Indications: For CF patients aged 4 months and older with a gating mutation in the CFTR gene (excluding F508del)","Mechanism: Ivacaftor as a potentiator acts on CFTR channels that have reached the cell surface and increase the gating and conductance of ions [5]","Administration: Oral tablets","Approval: Approved by the EMA in July 2012"]
text: ["Active ingredient(s): Ivacaftor","Indications: For CF patients aged 4 months and older with a gating mutation in the CFTR gene (excluding F508del)",<>Mechanism: Ivacaftor as a potentiator acts on CFTR channels that have reached the cell surface and increase the gating and conductance of ions<SupScrollLink label="51"/>{/*ehem77*/}</>,"Administration: Oral tablets","Approval: Approved by the EMA in July 2012"]
},
]
},
{
name: "Mucolytics and inhalation", //gibt 2 Inhalation Beispiele
picture: "...",
introduction: "Mucolytics help thin and loosen the mucus in the lungs, making it easier to cough up and clear the airways. These therapies are typically administered via wet or dry inhalation, providing direct delivery to the lungs. In the case of wet inhalation, the medication is inhaled as an aqueous solution and nebulized, while in the case of dry inhalation, the medication is inhaled as a powder. [1] Key Therapies include mannitol, Pulmozyme® and hypertonic saline.",
picture: "https://static.igem.wiki/teams/5247/scientific-figures/mucolytics.svg",
introduction: <>Mucolytics help thin and loosen the mucus in the lungs, making it easier to cough up and clear the airways. These therapies are
typically administered via wet or dry inhalation, providing direct delivery to the lungs. In the case of wet inhalation, the medication
is inhaled as an aqueous solution and nebulized, while in the case of dry inhalation, the medication is inhaled as a powder<SupScrollLink label="48"/>{/* ehem74 */}.
Key Therapies include mannitol, Pulmozyme® and hypertonic saline.</>,
examples: [
{
title: "Pulmozyme", //quelle 8
text: ["Active ingredient(s): Dornase alfa as mucolytic enzyme","Indications: For CF patients aged 5 years and older","Mechanism: breaks up and thins mucus via DNase activity","Administration: Inhalation via nebulizer, once or twice daily","Approval: Approved by the FDA in 1993 [9]"]
text: ["Active ingredient(s): Dornase alfa as mucolytic enzyme","Indications: For CF patients aged 5 years and older","Mechanism: breaks up and thins mucus via DNase activity","Administration: Inhalation via nebulizer, once or twice daily","Approval: Approved by the FDA in 1993"]
},
{
title: "Hypertonic saline",//quelle 10
text: ["Active ingredient(s): Osmotic agent sodium chloride (3%, 3.5%, 7%)","Indications: For CF patients aged 6 years and older","Mechanism: Draws water into the airways, hydrating the mucus and improving clearance","Administration: Inhalation via nebulizer, used twice daily","Approval: no official approval by EMA or FDA available, but widely used for several decades [11]","Price: low-cost [11]"]
text: ["Active ingredient(s): Osmotic agent sodium chloride (3%, 3.5%, 7%)","Indications: For CF patients aged 6 years and older","Mechanism: Draws water into the airways, hydrating the mucus and improving clearance","Administration: Inhalation via nebulizer, used twice daily","Approval: no official approval by EMA or FDA available, but widely used for several decades","Price: low-cost"]
},
]
},
{
name: "Antibiotics", //gibt 2 AB Beispiele
picture: "...",
introduction: "Antibiotics are crucial for treating bacterial infections in CF patients since the mucus serves as an optimal environment for pathogens. A huge variety of antibiotics can be inhaled, oral, or intravenous, depending on the severity and kind of the infection [12]. Commercially available medications include TOBI® and CAYSTON®. A major problem associated with the application of antibiotics is the fact that long-term use can lead to antibiotic resistance and potential side effects like kidney damage and hearing loss [12].",
picture: "https://static.igem.wiki/teams/5247/scientific-figures/antibiotics.svg",
introduction: <>Antibiotics are crucial for treating bacterial infections in CF patients since the mucus serves as an optimal environment for pathogens. A huge variety of antibiotics can be inhaled, oral, or intravenous, depending on the severity and kind of the infection<SupScrollLink label="52"/>{/*ehem12*/}. Commercially available medications include TOBI® and CAYSTON®. A major problem associated with the application of antibiotics is the fact that long-term use can lead to antibiotic resistance and potential side effects like kidney damage and hearing loss<SupScrollLink label="52"/>{/*ehem12*/}.</>,
examples: [
{
title: "TOBI", //quelle 13 + Pseudo muss kursiv
text: ["Active ingredient(s): Tobramycin",<>Indications: For CF patients aged 6 years and older with <i> Pseudomonas aeruginosa </i> infections </>,"Mechanism: Aminoglycoside antibiotic disrupts bacterial protein synthesis, leading to the death of the pathogen","Administration: Inhalation of antibiotic via nebulizer, typically taken twice daily in 28-day cycles","Approval: Approved by the EMA in 2011 [14]"]
text: ["Active ingredient(s): Tobramycin",<>Indications: For CF patients aged 6 years and older with <i> Pseudomonas aeruginosa </i> infections </>,<>Mechanism: Aminoglycoside antibiotic disrupts bacterial protein synthesis, leading to the death of the pathogen","Administration: Inhalation of antibiotic via nebulizer, typically taken twice daily in 28-day cycles","Approval: Approved by the EMA in 2011<SupScrollLink label="53"/>{/*ehem14*/}</>]
},
{
title: "CAYSTON", //quelle 15 + Pseudo muss kursiv
......@@ -85,8 +103,11 @@ export const drugdata: (Array<DrugDatensatz>) = [
},
{
name: "Digestive enzymes and diet", //ein beispiel
picture: "...",
introduction: "The digestive process is impaired in 80% of patients with cystic fibrosis (CF), as a result of pancreatic insufficiency, which in turn leads to difficulties in digesting food and absorbing nutrients. Enzyme supplements like Creon® are therefore essential [16]. Moreover CF patients are also advised to eat a balanced and energy-rich diet, as the increased work of breathing and increased coughing, as well as infections, fever and diarrhoea, consume more energy than a healthy person. [1] It is also an option for patients to use nutritional supplements. Electrolyte preparations are also used in this context to compensate for the increased need for fluids and the required salts.",
picture: "https://static.igem.wiki/teams/5247/scientific-figures/diet.svg",
introduction: <> The digestive process is impaired in 80% of patients with Cystic Fibrosis (CF), as a result of pancreatic insufficiency, which in
turn leads to difficulties in digesting food and absorbing nutrients. Enzyme supplements like Creon® are therefore essential<SupScrollLink label="54"/>{/* ehem16 */}.
Moreover CF patients are also advised to eat a balanced and energy-rich diet, as the increased work of breathing and increased coughing, as well as infections,
fever and diarrhoea, consume more energy than a healthy person<SupScrollLink label="48"/>{/* ehem74 */}. It is also an option for patients to use nutritional supplements. Electrolyte preparations are also used in this context to compensate for the increased need for fluids and the required salts.</>,
examples: [
{
title: "Creon", //quelle 17
......
src/data/hptimelinedata.tsx
View file @ 3fd292fd
Source diff could not be displayed: it is too large. Options to address this: view the blob.
src/data/legend.html
View file @ 3fd292fd
......@@ -3,7 +3,7 @@
<rect width="170" height="13" style="fill: url(&quot;#g-gdqp&quot;); shape-rendering: crispEdges;">
</rect>
<g class="labels" aria-hidden="true" transform="translate(0,16)">
<text class="min" transform="translate(0,0)" style="text-anchor: start; dominant-baseline: hanging; fill: black; ">
<text class="min" transform="translate(0,0)" style="text-anchor: start; dominant-baseline: hanging; fill: var(--offblack); ">
<tspan x="0" dy="0">0</tspan>
</text>
<text class="max" transform="translate(170,0)">
......
src/data/parts.ts
View file @ 3fd292fd
export interface Part{
partname: string,
registrycode: number,
registrycode: string,
description: string,
length: number,
type: PartType,
length?: number,
type: string,
url: string
}
type PartType = "DNA" | "Protein";
/*
Vorlage:
......@@ -24,22 +24,234 @@ Vorlage:
export const BasicParts: Array<Part> = [
{
partname: "Beispiel",
registrycode: 0,
description: "Beispiel Description",
length: 0,
partname: "n1SpuFz1",
registrycode: "BBa_K5247101",
description: "nickase",
length: 1917,
type: "DNA",
url: "https://parts.igem.org/Part:BBa_K5247101"
},
{
partname: "n2SpuFz1",
registrycode: "BBa_K5247102",
description: "nickase",
length: 1917,
type: "DNA",
url: "https://parts.igem.org/Part:BBa_K5247102"
},
{
partname: "n3SpuFz1",
registrycode: "BBa_K5247103",
description: "nickase",
length: 1917,
type: "DNA",
url: "https://parts.igem.org/Part:BBa_K5247103"
},
{
partname: "n4SpuFz1",
registrycode: "BBa_K5247104",
description: "nickase",
length: 1917,
type: "DNA",
url: "https://parts.igem.org/Part:BBa_K5247104"
},
{
partname: "n1CasX",
registrycode: "BBa_K5247105",
description: "nickase",
length: 2958,
type: "DNA",
url: "https://parts.igem.org/Part:BBa_K5247105"
},{
partname: "n2CasX",
registrycode: "BBa_K5247106",
description: "nickase",
length: 2958,
type: "DNA",
url: "https://parts.igem.org/Part:BBa_K5247106"
},
{
partname: "n3CasX",
registrycode: "BBa_K5247107",
description: "nickase",
length: 2958,
type: "DNA",
url: "https://parts.igem.org/Part:BBa_K5247107"
},
{
partname: "pegRNA_PEAR_01",
registrycode: "BBa_K5247136",
description: "pegRNA",
length: 153,
type: "DNA",
url: "....."
url: "https://parts.igem.org/Part:BBa_K5247136"
},
{
partname: "pegRNA_PEAR_02",
registrycode: "BBa_K5247110",
description: "pegRNA",
length: 153,
type: "DNA",
url: "https://parts.igem.org/Part:BBa_K5247110"
},
{
partname: "pegRNA_PEAR_03",
registrycode: "BBa_K5247111",
description: "pegRNA",
length: 183,
type: "DNA",
url: "https://parts.igem.org/Part:BBa_K5247111"
},
{
partname: "pegRNA_PEAR_04",
registrycode: "BBa_K5247112",
description: "pegRNA",
length: 183,
type: "DNA",
url: "https://parts.igem.org/Part:BBa_K5247112"
},
{
partname: "pegRNA_PEAR_05",
registrycode: "BBa_K5247113",
description: "pegRNA",
length: 184,
type: "DNA",
url: "https://parts.igem.org/Part:BBa_K5247113"
},
{
partname: "pegRNA_PEAR_06",
registrycode: "BBa_K5247114",
description: "pegRNA",
length: 184,
type: "DNA",
url: "https://parts.igem.org/Part:BBa_K5247114"
},
{
partname: "pegRNA_PEAR_07",
registrycode: "BBa_K5247115",
description: "pegRNA",
length: 186,
type: "DNA",
url: "https://parts.igem.org/Part:BBa_K5247115"
},
]
export const CompositeParts: Array<Part> = [
{
partname: "Beispiel",
registrycode: 0,
description: "Beispiel Description",
length: 0,
partname: "pegRNA_PEAR_08",
registrycode: "BBa_K5247116",
description: "pegRNA",
length: 186,
type: "DNA",
url: "....."
url: "https://parts.igem.org/Part:BBa_K5247116"
},
{
partname: "pegRNA_PEAR_09",
registrycode: "BBa_K5247117",
description: "pegRNA",
length: 187,
type: "DNA",
url: "https://parts.igem.org/Part:BBa_K5247117"
},
{
partname: "pegRNA_PEAR_10",
registrycode: "BBa_K5247118",
description: "pegRNA",
length: 187,
type: "DNA",
url: "https://parts.igem.org/Part:BBa_K5247118"
},
{
partname: "pegRNA_PEAR_11",
registrycode: "BBa_K5247119",
description: "pegRNA",
length: 189,
type: "DNA",
url: "https://parts.igem.org/Part:BBa_K5247119"
},
{
partname: "pegRNA_PEAR_12",
registrycode: "BBa_K5247120",
description: "pegRNA",
length: 189,
type: "DNA",
url: "https://parts.igem.org/Part:BBa_K5247120"
},
{
partname: "pegRNA_PEAR_13",
registrycode: "BBa_K5247121",
description: "pegRNA",
length: 190,
type: "DNA",
url: "https://parts.igem.org/Part:BBa_K5247121"
},
{
partname: "pegRNA_PEAR_14",
registrycode: "BBa_K5247122",
description: "pegRNA",
length: 190,
type: "DNA",
url: "https://parts.igem.org/Part:BBa_K5247122"
},
{
partname: "pegRNA_CFTR01",
registrycode: "BBa_K5247123",
description: "pegRNA",
length: 183,
type: "DNA",
url: "https://parts.igem.org/Part:BBa_K5247123"
},
{
partname: "pegRNA_CFTR02",
registrycode: "BBa_K5247124",
description: "pegRNA",
length: 184,
type: "DNA",
url: "https://parts.igem.org/Part:BBa_K5247124"
},
{
partname: "pegRNA_CFTR03",
registrycode: "BBa_K5247125",
description: "pegRNA",
length: 186,
type: "DNA",
url: "https://parts.igem.org/Part:BBa_K5247125"
},
{
partname: "pegRNA_CFTR04",
registrycode: "BBa_K5247126",
description: "pegRNA",
length: 186,
type: "DNA",
url: "https://parts.igem.org/Part:BBa_K5247126"
},
{
partname: "pegRNA_CFTR05",
registrycode: "BBa_K5247127",
description: "pegRNA",
length: 189,
type: "DNA",
url: "https://parts.igem.org/Part:BBa_K5247127"
},
{
partname: "PE_CO-mini RT",
registrycode: "BBa_K5247227",
description: "RT",
length: 1410,
type: "DNA",
url: "https://parts.igem.org/Part:BBa_K5247227"
},
{
partname: "PE6c RT",
registrycode: "BBa_K5247228",
description: "RT",
length: 1548,
type: "DNA",
url: "https://parts.igem.org/Part:BBa_K5247227"
},
{
partname: "PEAR_CFTR",
registrycode: "BBa_K5247135",
description: "DNA fragment for CFTR-specific pegRNA screening",
length: 1511,
type: "DNA",
url: "https://parts.igem.org/Part:BBa_K5247135"
},
]
\ No newline at end of file
src/data/results-table.tsx 0 → 100644
View file @ 3fd292fd
export interface Res{
nr: string,
silent: string,
rtt: string,
pbs: string,
q1: string,
}
export const headercols = [
"Nr.", "Silent Edits", "RTT-Lengths", "PBS-Length", "trevopreQ1"
]
export const resultdata: Res[] = [
{
nr: "1",
silent: "no",
rtt: "33",
pbs: "17",
q1: "no",
},
{
nr: "2",
silent: "yes",
rtt: "33",
pbs: "17",
q1: "no",
},
{
nr: "3",
silent: "no",
rtt: "27",
pbs: "16",
q1: "yes",
},
{
nr: "4",
silent: "yes",
rtt: "27",
pbs: "16",
q1: "yes",
},
{
nr: "5",
silent: "no",
rtt: "27",
pbs: "17",
q1: "yes",
},
{
nr: "6",
silent: "yes",
rtt: "27",
pbs: "17",
q1: "yes",
},
{
nr: "7",
silent: "no",
rtt: "30",
pbs: "16",
q1: "yes",
},
{
nr: "8",
silent: "yes",
rtt: "30",
pbs: "16",
q1: "yes",
},
{
nr: "9",
silent: "no",
rtt: "30",
pbs: "17",
q1: "yes",
},
{
nr: "10",
silent: "yes",
rtt: "30",
pbs: "17",
q1: "yes",
},
{
nr: "11",
silent: "no",
rtt: "33",
pbs: "16",
q1: "yes",
},
{
nr: "12",
silent: "yes",
rtt: "33",
pbs: "16",
q1: "yes",
},
{
nr: "13",
silent: "no",
rtt: "33",
pbs: "16",
q1: "yes",
},
{
nr: "14",
silent: "yes",
rtt: "33",
pbs: "16",
q1: "yes",
}
]
\ No newline at end of file
src/data/steckbriefe.ts
View file @ 3fd292fd
......@@ -34,7 +34,7 @@ Legende:
Anmerkung:
Eine Liste darf auch nur ein Element haben, das ist okay. Also z.B. ["hallo"]
Und wenn du Daten noch nicht hast ist das vorgehen "XXX" einzutragen sehr gut, denn dann kann man später per
Und wenn du Daten noch nicht hast ist das vorgehen "" einzutragen sehr gut, denn dann kann man später per
Textsuche die fehlenden Stellen heraussuchen (Daumen hoch).
*/
......@@ -57,9 +57,9 @@ Vorlage Datensatz:
bestpart: [ "", ""],
biggestchallenge: [ "", ""],
funfacts: [ "", ""],
favlabmusic: "XXX",
islands: "XXX",
onechange: "XXX",
favlabmusic: "",
islands: "",
onechange: "",
hobbies: [ "", ""],
scientificinterests: [ "", ""],
......@@ -75,7 +75,7 @@ export const teammembers: Array<SteckbriefInterface> = [
hauptfoto: "https://static.igem.wiki/teams/5247/photos/team-photos/anna-1-5-11zon.webp",
zweitfoto: "https://static.igem.wiki/teams/5247/photos/team-photos/anna-2-6-11zon.webp",
pronouns: "she/her",
onechange: "XXX",
onechange: "",
studiengang: "M.Sc. Molecular Cell Biology",
headof: "Wet lab",
igemjob: ["Delivery", "Creativity", "Sponsoring"],
......@@ -97,7 +97,6 @@ export const teammembers: Array<SteckbriefInterface> = [
"Expanding my skills in the lab",
"The competition with fellow students",
"Telling myself 'There must be stupider people than me' and 'Shit happens'",
"Crying",
],
biggestchallenge: [
">12-hour shift in the lab",
......@@ -105,7 +104,8 @@ export const teammembers: Array<SteckbriefInterface> = [
],
funfacts: [
"Proud Lab Mom - They see me rollin' with my E-Scooter",
"I would describe myself as a creative mind and food lover",
"Always humming and with a song in my head in the lab",
"Peitsche and Zuckerbrot is my motto",
],
favlabmusic: "Techno workout playlist from Lisa",
islands: [
......@@ -124,12 +124,12 @@ export const teammembers: Array<SteckbriefInterface> = [
},
{
title: "B.Sc.",
title: "M.Sc.",
vorname: "Asal Sahami",
nachname: "Moghaddam",
age: "24",
favlabmusic: "Music by Malte Marten",
linkedinurl: "https://www.linkedin.com/in/ asal-sahami-moghaddam-665302315",
linkedinurl: "https://www.linkedin.com/in/asal-sahami-moghaddam-665302315",
hauptfoto: "https://static.igem.wiki/teams/5247/photos/team-photos/asal-1-7-11zon.webp",
zweitfoto: "https://static.igem.wiki/teams/5247/photos/team-photos/asal-2-8-11zon.webp",
pronouns: "she/her",
......@@ -200,7 +200,7 @@ export const teammembers: Array<SteckbriefInterface> = [
"More people in science",
],
hobbies: [
"TDiving",
"Diving",
"swimming",
"hiking",
],
......@@ -221,7 +221,7 @@ export const teammembers: Array<SteckbriefInterface> = [
igemjob: ["Wet lab", "Delivery", "Creativity"],
whyigem: [
"It's great to be part of a project from the formation of the idea until the final results, so you can contribute with your work but also gain new experiences.",
"Because I want to be part of a dedicated team of young scientists and follow a project from the idea to the final result with all its' challenges.",
"Because I want to be part of a dedicated team of young scientists and follow a project from the idea to the final result with all it's challenges.",
],
bestpart: [
"Learning new techniques in the lab",
......@@ -301,8 +301,8 @@ export const teammembers: Array<SteckbriefInterface> = [
vorname: "Kathleen",
nachname: "Susat",
age: "23",
onechange: "XXX",
linkedinurl: "https://www.linkedin.com/in/anna-lena-b-a488102a5",
onechange: "",
linkedinurl: "https://www.linkedin.com/in/kathleen-susat-261979301",
hauptfoto: "https://static.igem.wiki/teams/5247/photos/team-photos/kathleen-1-3-11zon.webp",
zweitfoto: "https://static.igem.wiki/teams/5247/photos/team-photos/kathleen-2-4-11zon.webp",
pronouns: "she/her",
......@@ -349,7 +349,7 @@ export const teammembers: Array<SteckbriefInterface> = [
pronouns: "she/her",
studiengang: "M.Sc. Molecular Biotechnology",
headof: "Human Practice",
igemjob: ["Wet lab", "Delivery", "Mechanism", "Public Outreach"],
igemjob: ["Wet lab", "Delivery", "Mechanism", "Public Outreach", "Wiki"],
whyigem: [
"I wanted to be part of an incredible team implementing a large and significant project together.",
"iGEM is for me an opportunity to actively shape the next steps in biotechnology and thus make a contribution that goes beyond the competition.",
......@@ -357,11 +357,11 @@ export const teammembers: Array<SteckbriefInterface> = [
],
bestpart: [
"Learning a lot about scientific research, handling stress and working in the lab",
"Working in a team, implementing a large and significant project together to achieve our goal",
"Learning a lot about scientific research, handling stress and working in the lab.",
"Working in a team, implementing a large and significant project together to achieve our goal.",
],
biggestchallenge: [
"The biggest challenge is time management, beacuse it is quite difficult to manage all the different aspects of the project"
"The biggest challenge is time management, beacuse it is quite difficult to manage all the different aspects of the project."
],
funfacts: [
"I tend to say yes to everything",
......@@ -400,27 +400,26 @@ export const teammembers: Array<SteckbriefInterface> = [
headof: "Wiki",
igemjob: [ "Human Practices", "Public Engagement"],
whyigem: [
"I am very curious and like projects.",
"I am curious and prefer practical learning experiences.",
],
islands: "XXX",
islands: "",
bestpart: [
"Actually seeing results of what you are doing",
],
biggestchallenge: [
"The biggest challenge is time management. It is quite difficult to manage all the different aspects of the project",
"The biggest challenge for me was time and resource management. Not only for myself but especially to ernforce deadlines and distribute tasks for the team.", "I am also not quite sure anymore what my hobbies were before iGEM."
],
funfacts: [
"My roommates don't let me do experiments in the kitchen anymore",
"I just have a serious obsession with isopods and efficiency",
"Doing projects is fun",
],
favlabmusic: "Classical crossover, Rock and Baroque Pop",
"Gifted at creating tasteful postcards",
"Without me there would be no wiki and we would be disqualified,so my team calls me 'Wiki-Hero'"
],
favlabmusic: "Classical crossover, Rock and Baroque Pop - also bagpipe music",
onechange: [
"Make researchers put ALL their data into very detailed databases, that would make looking up data so efficient. You too, faunisticians and anatomists. I'm looking at you"
],
hobbies: [
"Painting & Drawing",
"Maintaining a website",
"Reading",
"Keeping isopods",
],
scientificinterests: "Terrestrial Isopods, Databases, Phylogenetics and Machine Learning",
......@@ -431,12 +430,12 @@ export const teammembers: Array<SteckbriefInterface> = [
vorname: "Lisa",
nachname: "Wiesner",
age: "28",
linkedinurl: "XXX",
linkedinurl: "https://www.linkedin.com/in/lisa-wiesner-8a008b340/",
hauptfoto: "https://static.igem.wiki/teams/5247/photos/team-photos/lisa-1-3-11zon.webp",
zweitfoto: "https://static.igem.wiki/teams/5247/photos/team-photos/lisa-2-4-11zon.webp",
pronouns: "she/her",
studiengang: "M.Sc. Molecular Cell Biology",
igemjob: ["Delivery", "Creativity"],
igemjob: ["Delivery", "Creativity", "Wet lab", "Wiki"],
whyigem: [
"My motivation for iGEM comes from the excitement of tackling real world problems through innovative synthetic biology solutions, alongside a global community of passionate scientists.",
"The opportunity to showcase our work at the iGEM Giant Jamboree and potentially make a meaningful impact on the world.",
......@@ -463,8 +462,8 @@ export const teammembers: Array<SteckbriefInterface> = [
],
hobbies: [
"Agility",
"softball",
"climbing",
"Softball",
"Climbing",
"basically everything outdoors",
],
scientificinterests: "Cell Biology, Genetic Engineering and Biomedicine",
......@@ -475,7 +474,7 @@ export const teammembers: Array<SteckbriefInterface> = [
nachname: "Lenger",
age: "21",
linkedinurl: "https://www.linkedin.com/in/malte-lenger-08j2003",
hauptfoto: "https://static.igem.wiki/teams/5247/photos/team-photos/malte-1-2-11zon.webp",
hauptfoto: "https://static.igem.wiki/teams/5247/photos/team-photos/malte-neu-besser.webp",
zweitfoto: "https://static.igem.wiki/teams/5247/photos/team-photos/malte-2-1-11zon.webp",
pronouns: "he/him",
studiengang: "B.Sc. Molecular Biotechnology",
......@@ -487,17 +486,19 @@ export const teammembers: Array<SteckbriefInterface> = [
"my strong interests in science, as well as the idea of my future life as a scientist.",
],
bestpart: [
"My favourite part of iGEM was/is meeting new people as well as working and researching with them. It's also the new insights you gain: both into working methods, for example in the lab, and into the field of work in general",
"My favourite part of iGEM was/is meeting new people, especially my team members, and working/researching with them. It's also the new insights you gain: both into working methods, for example in the lab, and into the field of work in general",
"When something you've designed in silicio actually works out as intended in the lab",
],
biggestchallenge: [
"The biggest challenge at iGEM during the semester was balancing the project and my own university tasks. And in general, it was to remain self-confident, both with some setbacks and with activities where you had a lot of responsibility but had never done them yourself before",
"The biggest challenge at iGEM during the semester was balancing the project and my own university tasks",
"It was also challenging to remain self-confident, both with some setbacks and with activities where you had a lot of responsibility but had never done them yourself before",
],
funfacts: [
"I like meeting up with people, especially if you go out in the city in the evening and meet up for a drink or something like that",
"I also like travelling, although at the end of my life I would like to be able to say that I have seen most of the world",
"But last but not least I am very interested in sports, with my favourite sports being football, basketball, skiing and tennis. However, I am very broad-minded when it comes to sports, so I actually watch every sport if none of the above are on TV. For example, during the Olympic Games this year, I might spend a whole afternoon just watching the swimming competitions."
"I may have spent some whole weekends just watching sports and designing pegRNAs",
"My family still doesn't know what exactly this is all about",
"One of my goals is to have been to every continent once in my life",
],
favlabmusic: "My favorite music in the lab is the music I usually listen to. This mainly includes hip-hop and American rap, especially artists like Travis Scott, Gunna, Metro Boomin and Reezy",
favlabmusic: "My favorite music in the lab is the music I usually listen to. This mainly includes hip-hop and American rap, especially artists like Travis Scott, Gunna, Metro Boomin, Luciano and Reezy",
islands: [
"a football",
"sun protection",
......@@ -509,9 +510,10 @@ export const teammembers: Array<SteckbriefInterface> = [
hobbies: [
"Football",
"Basketball",
"Skiing",
"Partying",
"Meeting friends",
"Traveling",
"Meeting friends",
],
scientificinterests: "Medicine, especially cancer therapy or gene therapy in general",
},
......@@ -586,7 +588,7 @@ export const teammembers: Array<SteckbriefInterface> = [
"The biggest challenge definitely is to handle the neverending amount of tasks, most of which are completely new challenges you have to learn by yourself",
],
favlabmusic: "Technoo",
funfacts: ["I am to uncreative to submit a fun fact"],
funfacts: ["I have been, no joke, dreaming of primers lately"],
islands: [
"a saw",
"a metal pot",
......@@ -610,16 +612,15 @@ export const teammembers: Array<SteckbriefInterface> = [
vorname: "Vera",
nachname: "Köhler",
age: "26",
linkedinurl: "https://www.linkedin.com/in/vera-k%C3%B6hler-50ba35336/",
hauptfoto: "https://static.igem.wiki/teams/5247/photos/team-photos/vera-1-3-11zon.webp",
zweitfoto: "https://static.igem.wiki/teams/5247/photos/team-photos/vera-2-4-11zon.webp",
pronouns: "she/her",
studiengang: "M.Sc. Genome Based Systems Biology",
headof: "Delivery",
igemjob: ["Wet lab", "Human Practice", "Public Outreach", "Creativity"],
igemjob: ["Wet lab", "Delivery", "Human Practice", "Public Outreach", "Creativity", "Wiki"],
whyigem: [
"I am motivated by the idea of working together as a team on a creative project to provide a valuable solution for the world",
"The possibility to uncover a trivial slice of knowledge & a really good cup of coffee",
"To test my limits and expand various laboratory and soft skills",
"I'm excited by the chance to collaborate as a team on a creative project that makes a difference in the world, while discovering new knowledge, enjoying a great cup of coffee, and pushing my boundaries to grow both technically and personally.",
],
bestpart: [
"The amazing Team and the funny, but also frustrating moments we lived through",
......@@ -628,7 +629,7 @@ export const teammembers: Array<SteckbriefInterface> = [
"Completing ten different emergency tasks at the same time without losing your head",
],
funfacts: [
"As a child I thought I would be arrested if I ate in the car, so I hid my snack as soon as we passed another car. I wonder what my parents had drilled into me",
"My favourite algae is Volvox",
],
favlabmusic: "Upbeat fun songs like Unwritten, C'est la bourgeoisie or Feminenomenom to keep me motivated",
islands: [
......@@ -637,61 +638,18 @@ export const teammembers: Array<SteckbriefInterface> = [
"instant ramen",
],
onechange: [
"Eliminating the bias that science is for old white men with crazy hair",
"That women in science get the recognition they deserve for their achievements",
"That women in science get the recognition they deserve for their achievements.",
],
hobbies: [
"Yoga",
"reading",
"game nights with friends",
"ourdoor Sports",
"creating Banger Spotify playlists",
"ourdoor sports",
"creating banger Spotify playlists",
],
scientificinterests: "Algae & Microbiology, OMICs and Systems Biology",
},
{
vorname: "Vincent Carl",
nachname: "Stöckl",
age: "20",
hauptfoto: "https://static.igem.wiki/teams/5247/photos/team-photos/vincent-1-1-11zon.webp",
zweitfoto: "https://static.igem.wiki/teams/5247/photos/team-photos/vincent-2-2-11zon.webp",
pronouns: "he/him",
studiengang: "B.Sc. Molecular Biotechnology",
igemjob: ["Sponsoring"],
whyigem: [
"a chance to work as a real scientist as well as being part of a team working at the cutting edge of biotechnology",
"this is a unique opportunity to become an active part of a team of (student) scientits who are collaborating in a high profile scientific project with studenta ranging from 3rd semester to the one's who are currently involved in their master's",
],
bestpart: [
"The challenge of trying new things and working as a real scientist",
],
biggestchallenge: [
"balancing iGEM and university work",
],
funfacts: [
"no fun facts",
],
favlabmusic: "Techno workout playlist from Lisa",
islands: [
"a pocket knive",
"sunscreen",
"water",
],
onechange: [
"Make it more accesseble to people from all walks of life",
],
hobbies: [
"Medieval Reenactment",
"sword Fighting",
"gun club",
"friends",
"painting",
"drawing",
],
scientificinterests: "Phytomining and Remediation and Synthetic Organs/ Limbs",
},
]
export const advisors: Array<SteckbriefInterface> = [
......@@ -699,13 +657,14 @@ export const advisors: Array<SteckbriefInterface> = [
title: "B.Sc.",
vorname: "Felicitas",
nachname: "Zimmer",
hauptfoto: "https://static.igem.wiki/teams/5247/placeholders/placehilderperson.jpeg",
hauptfoto: "https://static.igem.wiki/teams/5247/photos/team-photos/felicitas-1-3-11zon.webp",
zweitfoto: "https://static.igem.wiki/teams/5247/photos/team-photos/felicitas-2-4-11zon.webp",
pronouns: "she/her",
studiengang: "M.Sc. Molecular Cell Biology",
igemjob: ["Advisor"],
whyigem: ["Trying to change the world step by step.","The idea of being able to change something and thus help people."],
bestpart: ["XXX"],
biggestchallenge: "XXX",
bestpart: [""],
biggestchallenge: "",
funfacts: [
"I do not know any fun facts about me",
],
......@@ -725,19 +684,17 @@ export const advisors: Array<SteckbriefInterface> = [
title: "M.Sc.",
vorname: "Lucas",
nachname: "Krause",
linkedinurl: "XXX",
hauptfoto: "https://static.igem.wiki/teams/5247/placeholders/placehilderperson.jpeg",
linkedinurl: "",
hauptfoto: "https://static.igem.wiki/teams/5247/photos/team-photos/lukas-1-min.webp",
zweitfoto: "https://static.igem.wiki/teams/5247/photos/team-photos/lukas-2-min-min-1.webp",
pronouns: "he/him",
studiengang: "PhD Molecular Biotechnology",
igemjob: ["Advisor"],
whyigem: ["Passing on knowledge to the current iGEMers and being along for the ride of an exciting project"],
bestpart: ["XXX"],
bestpart: [""],
biggestchallenge: "Staying organized throughout lab work, deadlines and wiki texts",
funfacts: [
"no fun facts",
"XXX",
"XXX",
"XXX"
"no fun facts"
],
favlabmusic: "Electric Callboy, Wolfmother, RHCP",
age: "27",
......@@ -811,9 +768,9 @@ export const advisors: Array<SteckbriefInterface> = [
"I can cook and eat really spicy Indian food without breaking a sweat, but here’s where I might lose some friends",
"I think the Harry Potter movies are totally overrated",
],
favlabmusic: "XXX",
favlabmusic: "",
islands: [
"XXX",
"",
],
onechange: "Remove the heavy fees charged by many top scientific journals for publishing research. In my opinion, thus fees create inherent inequality in the world of science, limiting opportunities for researchers with fewer resources. Moreover, publically funded research should be accessible to the public without paying large amounts of money",
hobbies: [
......
src/data/stroke.svg deleted 100644 → 0
View file @ f16b822c
<svg width="100" height="64" fill="none" xmlns="http://www.w3.org/2000/svg">
<g clip-path="url(#a)">
<path d="M-17 30.5C-1 22 72-4 54 13 37.9 28.2-2.5 57.5 16 55.5s72-29 104-40" stroke="#850F78" stroke-width="10"/>
</g>
<defs>
<clipPath id="a">
<path fill="#fff" d="M0 0h100v64H0z"/>
</clipPath>
</defs>
</svg>
src/data/symptom-data.tsx
View file @ 3fd292fd
import { SupScrollLink } from "../components/ScrollLink";
export interface SymptomDatensatz {
name: string;
......@@ -5,57 +6,73 @@ export interface SymptomDatensatz {
introduction: Array<string> | Array<React.ReactNode>;
}
//Bilder+Quellen müssen noch angepasst werden
//da kommen jetzt folgende Organe: Pancreas, Intestines, Liver, Sexual glands, Lungs, Skeletal System, Skin, Nose, Brain/Mental Health
export const symptomdata: (Array<SymptomDatensatz>) = [
{//da kommen jetzt folgende Organe: Pancreas, Intestines, Liver, Sexual glands, Lungs, Skeletal System, Skin, Nose, Brain/Mental Health , Bilder müssen noch angepasst werden
{
name: "Pancreas",
picture: "https://static.igem.wiki/teams/5247/scientific-figures/organs-together-normal.svg",
introduction: [<>Located behind the stomach in the back abdominal cavity [1]</>,"Responsible for neutralizing stomach acid, production of hormones (like Insulin) and digestion enzymes [1]","Clogging caused by CF, so that products of the pancreatic gland can not be distributed [2]","pancreatic insufficiency due to CF can lead to malnutrition [3], digestive problems and abdominal pain [4], CF-associated diabetes [5]","Treatment options include nutritional therapy and supplementation of pancreatic enzymes [6]",]
picture: "https://static.igem.wiki/teams/5247/scientific-figures/pancreas.svg",
introduction: [<>Located behind the stomach in the back abdominal cavity</>,<>Responsible for neutralizing stomach acid,
production of hormones (like Insulin) and digestion enzymes</>,<>Clogging caused by CF, so that products of the pancreatic
gland can not be distributed <SupScrollLink label="17"/>{/* ehem75 */}</>,<>Pancreatic insufficiency due to CF can lead to malnutrition
<SupScrollLink label="18"/>{/* ehem76 */}, digestive problems and abdominal pain, CF-associated diabetes <SupScrollLink label="19"/>{/* ehem78 */}
</>,<>Treatment options include nutritional therapy and supplementation of pancreatic enzymes <SupScrollLink label="20"/>{/* ehem79 */}</>]
},
{
name: "Intestines",
picture: "https://static.igem.wiki/teams/5247/scientific-figures/lungs.svg",
introduction: ["located in the abdominal cavity and extends from the stomach to the anus","Responsible for nutrient absorption, water reabsorption, and waste elimination [7]","CF can cause blocking of the intestines, preventing the normal movement of food and absorption of nutrients [8]"," Intestinal blockage due to CF can lead to malabsorption of nutrients, and conditions like meconium ileus in newborns or distal intestinal obstruction syndrome (DIOS) in adults [8]","Treatment options include the use of laxatives, enzyme supplementation, feeding tubes, and in severe cases, surgical intervention or intestinal transplantation [8]",]
picture: "https://static.igem.wiki/teams/5247/scientific-figures/largeintestine.svg",
introduction: [<>Located in the abdominal cavity and extends from the stomach to the anus</>,
<>Responsible for nutrient absorption, water reabsorption, and waste elimination <SupScrollLink label="21"/>{/* ehem80 */}</>,
<>CF can cause blocking of the intestines, preventing the normal movement of food and absorption of nutrients
<SupScrollLink label="22"/>{/*ehem81*/}</>,<>Intestinal blockage due to CF can lead to malabsorption of nutrients, and conditions like meconium ileus in newborns or distal intestinal obstruction syndrome (DIOS) in adults<SupScrollLink label="22"/>{/*ehem81*/}</>,<>Treatment options include the use of laxatives, enzyme supplementation, feeding tubes, and in severe cases, surgical intervention or intestinal transplantation<SupScrollLink label="22"/>{/*ehem81*/}</>]
},
{
name: "Liver",
picture: "https://static.igem.wiki/teams/5247/scientific-figures/pancreas.svg",
introduction: ["Located directly below the diaphragm near the pancreatic gland and stomach [9]","Responsible for detoxification, bile production for enhancement of digestion, protein synthesis for blood clotting and immune functions, nutrient processing and storage [9]","CF affects the normal secretion and function of bile- it becomes stickier [10]","Symptoms caused by CF include bruising, nose bleeds, Inflammation, more frequent chest infections, low body weight, liver cirrhosis, lethargy [10]","Treatment options include nutritional therapy, modulators, UDCA, liver transplantation [11]",]
picture: "https://static.igem.wiki/teams/5247/scientific-figures/liver.svg",
introduction: [<>Located directly below the diaphragm near the pancreatic gland and stomach<SupScrollLink label="23"/>{/*ehem82*/}</>,<>Responsible for detoxification, bile production for enhancement of digestion, protein synthesis for blood clotting and immune functions, nutrient processing and storage<SupScrollLink label="23"/>{/*ehem82*/}</>,<>CF affects the normal secretion and function of bile- it becomes stickier<SupScrollLink label="24"/>{/*ehem83*/}</>,<>Symptoms caused by CF include bruising, nose bleeds, Inflammation, more frequent chest infections, low body weight, liver cirrhosis, lethargy<SupScrollLink label="24"/>{/*ehem83*/}</>,<>Treatment options include nutritional therapy, modulators, UDCA, liver transplantation<SupScrollLink label="25"/>{/*ehem84*/}</>]
},
{
name: "Sexual glands",
picture: "https://static.igem.wiki/teams/5247/scientific-figures/liver.svg",
introduction: ["Located in pelvic area [12]","Responsible for production of sperm (male)/ production of oocytes and as site for embryo development (female) [13]","CF causes thick mucus to block reproductive ducts (both), affects sperm transport (male) [14] and cervical mucus density (female) [15]","This can lead in CF context to reduced fertility or even infertility [14][15]",<> <i> In vitro </i> fertilization can be an option for CF patients [16] </>,]
picture: "https://static.igem.wiki/teams/5247/scientific-figures/glands.svg",
introduction: [<>Located in pelvic area</>,<>Responsible for production of sperm (male)/ production of oocytes and as site for embryo development (female)</>,<>CF causes thick mucus to block reproductive ducts (both), affects sperm transport (male)<SupScrollLink label="26"/>{/*ehem87*/} and cervical mucus density (female)<SupScrollLink label="26"/>{/*ehem88*/}</>,<>This can lead in context of CF to reduced fertility or even infertility<SupScrollLink label="26"/>{/*ehem87*/}<SupScrollLink label="88"/></>,<> <i> In vitro </i> fertilization can be an option for CF patients<SupScrollLink label="26"/>{/*ehem89*/} </>]
},
{
name: "Lungs",
picture: "https://static.igem.wiki/teams/5247/scientific-figures/brain.svg",
introduction: ["Located in the ribcage [17]","Responsible for breathing – aspiration of life-giving oxygen and deposition of carbondioxide [17]","CF causes the thickening of mucus on top of the respiratory epithelium, serving as nutrition ground as for pathogens and impairing the movement of cilia so that the mucus can not be cart away [18]",<>Symptoms caused by CF include shortness of breath, persistent cough [19], lung infections (mainly caused by <i> Pseudomonas aeruginosa </i>, <i> Staphylococcus aureus </i> and <i> Burkholderia cepacia </i>) [20], bronchiectasis [21] </>,"Treatment options include respiratory physiotherapy (e.g. bronchiodilators), sports, inhalation, antibiotics against pathogens, lung transplantation [22]",]
name: "Lungs",
picture: "https://static.igem.wiki/teams/5247/scientific-figures/lungs.svg",
introduction: [<>Located in the ribcage</>,<>Responsible for breathing – aspiration of life-giving oxygen
and deposition of carbondioxide</>,<>CF causes the thickening of mucus on top of the respiratory epithelium,
serving as nutrition ground as for pathogens and impairing the movement of cilia so that the mucus can not be cart away <SupScrollLink label="27"/>
</>,<>Symptoms caused by CF include shortness of breath, persistent cough, lung infections (mainly caused by <i> Pseudomonas aeruginosa
</i>, <i> Staphylococcus aureus </i> and <i> Burkholderia cepacia </i>), bronchiectasis<SupScrollLink label="28"/><sup>,</sup><SupScrollLink label="29"/></>
,<>Treatment options include respiratory physiotherapy (e.g. bronchiodilators), sports, inhalation, antibiotics against
pathogens, lung transplantation<SupScrollLink label="30"/></>]
},
{
name: "Skeletal System",
picture: "https://static.igem.wiki/teams/5247/scientific-figures/heart.svg",
introduction: ["Located throughout the entire body","Responsible for structuration of the body, protection of organs, mineral storage, blood cell production [23]","CF affects the skeletal system by reducing bone mineral density [24]","This can lead in CF context to osteoporosis, spinal fractures, kyphosis, scoliosis [24]","Treatment options include uptake of biophosphonates to increase bone density, vitamin D for maintenance of calcium levels needed for mineralization [24]"],
picture: "https://static.igem.wiki/teams/5247/scientific-figures/bones.svg",
introduction: [<>Located throughout the entire body</>,
<>Responsible for structuration of the body, protection of organs, mineral storage, blood cell production</>,<>CF affects the skeletal system by reducing bone mineral density <SupScrollLink label="31"/>{/*ehem24*/}</>,<>This can lead in context of CF to osteoporosis, spinal fractures, kyphosis, scoliosis <SupScrollLink label="31"/>{/*ehem24*/}</>,<>Treatment options include uptake of biophosphonates to increase bone density, vitamin D for maintenance of calcium levels needed for mineralization <SupScrollLink label="31"/>{/*ehem24*/}</>]
},
{
name: "Skin",
picture: "https://static.igem.wiki/teams/5247/scientific-figures/largeintestine.svg",
introduction: ["Located on the surface of the entire body","Responsible for barrier function, immunological defense, regulation of homeostasis, sensory functions [25]","CF affects CFTR channels of sweat glands, which leads to salt deposits on the skin [26]","This can lead in CF context to skin irritation (rash or dermatitis) [26]","Prevention via drinking a lot and a rather salty diet possible [27]"],
},
// {
// name: "guts",
// picture: "https://static.igem.wiki/teams/5247/scientific-figures/pregnancy.svg",
// introduction: "",
// subsections: [
// {
// title: "",
// text: ["string"]
// },
// {
// title: "",
// text: ["string"]
// },
// ]
// }
picture: "https://static.igem.wiki/teams/5247/scientific-figures/skin.svg",
introduction: [<>Located on the surface of the entire body</>,<>Responsible for barrier function, immunological defense,
regulation of homeostasis, sensory functions<SupScrollLink label="32"/></>,
<>CF affects CFTR channels of sweat glands, which leads to salt deposits on the skin</>,
<>This can lead in context of CF to skin irritation (rash or dermatitis)<SupScrollLink label="33"/></>,
<>Prevention via drinking a lot and a rather salty diet possible<SupScrollLink label="33"/></>]
},
{
name: "Nasal mucosa and nose",
picture: "https://static.igem.wiki/teams/5247/scientific-figures/nose.svg",
introduction: [<>Responsible for smell perception, breathing, filtration and cleaning, and serving as a resonance chamber for the
voice</>,<>CF affects thickness of nasal secretions, which cannot drain well<SupScrollLink label="34"/>{/*ehem26*/}</>,<>This can lead in context of CF to nasal congestion, pressure headaches, sinusitis, inflammation of nasal polyps<SupScrollLink label="34"/>{/*ehem26*/}</>,<>Therapy ranges from nasal sprays to surgical removal of nasal polyps<SupScrollLink label="34"/>{/*ehem26*/}</>]
},
{
name: "Brain",
picture: "https://static.igem.wiki/teams/5247/scientific-figures/brain.svg",
introduction: [<>Located inside the skull</>,<>Responsible for cognitive functions, movement coordination, and control of vital
functions</>,<>CF affects mental health due to psychological stress and social isolation<SupScrollLink label="35"/></>,
<>This can lead in context of CF to physical illnesses like depression and anxiety disorders<SupScrollLink label="36"/></>,<>Treatment options include psychotherapy, antidepressants, support groups and sports [31]</>]
}
]
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<svg width="1080" height="4000" fill="none" xmlns="test-namespace">
<path d="M 50 50 C 500 -50 1000 100 1870 50 C 1820 220 2024 528 1870 590 C 1351 678 118 451 54 561 C 3 672 12 985 55 985 C 586 1090 1342 898 1881 1038 C 1994 1194 1986 1568 1890 1681 C 1751 1803 281 1481 168 1646 C 81 1794 21 1977 168 2142 C 499 2246 1403 2325 1081 2142
" stroke="#850F78" stroke-width="10"/>
</svg>
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export const timelinepersontabs = [
]
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src/headers/attribution-h.tsx
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......@@ -3,7 +3,7 @@ import HeaderBox from "../components/HeaderBox";
export function ATTH() {
return (
<HeaderBox title="Attributions">
<HeaderBox title="Attributions" id="atthead">
</HeaderBox>
);
}
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src/headers/cont-h.tsx
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......@@ -3,7 +3,7 @@ import HeaderBox from "../components/HeaderBox";
export function CONTH() {
return (
<HeaderBox title="Contribution">
<HeaderBox title="Contribution" id="conhead">
</HeaderBox>
);
......
src/headers/des-h.tsx deleted 100644 → 0
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export function DESH() {
return (
<HeaderBox title="Design">
</HeaderBox>
);
}
import HeaderBox from "../components/HeaderBox";
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