diff --git a/src/components/Complex-svg.tsx b/src/components/Complex-svg.tsx
index 54e4cfc62ea8bafd2e2ac2825684bdaa28331879..e09ec6e2328861bdb40ea32ff9d2a1399a7e3d33 100644
--- a/src/components/Complex-svg.tsx
+++ b/src/components/Complex-svg.tsx
@@ -2,7 +2,7 @@ import { useNavigation } from "../utils";
export default function SVG(){
- const { goToPageWithTabAndScroll, /* , goToTextsAndOpenCollapsible */ goToPageAndScroll} = useNavigation();
+ const { goToPageWithTabAndScroll/* , goToTextsAndOpenCollapsible */ } = useNavigation();
return(
<svg
viewBox="0 0 1000 500"
diff --git a/src/components/HP-timeline.tsx b/src/components/HP-timeline.tsx
index cc21b1696465df36126031006646860bf5ff23f0..89c169560fdf34bbafa5d5789e3ed0af3cb70c25 100644
--- a/src/components/HP-timeline.tsx
+++ b/src/components/HP-timeline.tsx
@@ -5,6 +5,7 @@ import { timelinedata } from "../data/hptimelinedata";
export function HPTimeline(){
return(
<section id="Timeline" className="section">
+ <p>Feel free to scroll trough our timeline and learn more about our extensive human-centric approach!</p>
<div className="center">
</div>
<TabButtonRow data={timelinebuttonrowdata} classy="" opentype="timelinecardtabs" closing="timelinepersontabs" />
diff --git a/src/contents/Human Practices/Conclisuin.tsx b/src/contents/Human Practices/Conclisuin.tsx
index 77b826a163d6fdf8e45acff1e2d836b7b2fff27b..bca95a6aaf4360c1bae699e5d6b13aeba0aa672c 100644
--- a/src/contents/Human Practices/Conclisuin.tsx
+++ b/src/contents/Human Practices/Conclisuin.tsx
@@ -1,10 +1,12 @@
import Collapsible from "../../components/Collapsible";
import { H5, H4 } from "../../components/Headings";
import { useNavigation } from "../../utils";
+import { useTabNavigation } from "../../utils/TabNavigation";
export function HPconclusion(){
const {goToPagesAndOpenTab} = useNavigation();
const {goToPageAndScroll} = useNavigation();
+ useTabNavigation();
return(
<>
<p>Our project has evolved through a deeply collaborative and human-centered approach, integrating diverse feedback from patients, clinicians, researchers, and industry experts. These insights shaped not only the technical aspects of our gene therapy for cystic fibrosis (CF) but also our commitment to addressing real-world patient needs, ethical considerations, and the disparities in CF treatment worldwide. From <a onClick={() => goToPagesAndOpenTab('maxfirst', '')}>Max Beckmann’s</a> patient perspective to expert guidance on technical and ethical issues, each stakeholder contributed to refining our solution, ensuring it is both innovative and empathetic. Our focus on gene therapy targeting CF’s complex mutations, integrating physiotherapy, and ensuring global accessibility demonstrates our holistic and inclusive vision for this project. Importantly, the collaboration with researchers in nanoparticle stability and gene therapy, along with the development of bilingual surveys and outreach materials, highlights our efforts to make science more accessible and transparent, bridging gaps in knowledge and care. </p>
@@ -240,7 +242,20 @@ function AnalyseBharti(){
<Collapsible title="Dr. Nikhil Bharti – Expert in Primary culture " id="bhartianalyseC">
<p>Dr. Nikhil Bharti's expertise helped us overcome a major challenge with our CF cell cultures, which were consistently becoming contaminated with fungi due to their instability. He recommended a specific antibiotic composition effective against fungal infections. </p>
<p>We immediately applied this antibiotic mixture to our cell cultures, which resolved the contamination issue and allowed us to maintain stable CF cell lines. This breakthrough enabled us to proceed with testing our prime editing approach effectively. Thanks to Dr. Bharti's insights, we advanced our project to a stage where we reached the limit of in vitro testing, and the next step would be potential animal studies. </p>
-
+ <div className="row align-items-center">
+ <div className="col">
+ <figure>
+ <img src="https://static.igem.wiki/teams/5247/photos/hp/cell-culture-mucos.webp" alt="" />
+ <figcaption>Figure 1. Primary cell culture without antibiotic treatment.</figcaption>
+ </figure>
+ </div>
+ <div className="col">
+ <figure>
+ <img src="https://static.igem.wiki/teams/5247/photos/cell-culture-after-antibiotic-treatment.webp" alt=""/>
+ <figcaption>Figure 2. Primary cell culture with antibiotic treatment.</figcaption>
+ </figure>
+ </div>
+ </div>
<p><strong>Contamination challenge:</strong> Helped us address a major issue with fungal contamination in our CF cell cultures, which were unstable and prone to infection.</p>
<p><strong>Antibiotic solution:</strong> Recommended a specific antibiotic composition effective against fungal infections.</p>
@@ -273,6 +288,16 @@ function AnalyseIgnatova(){
</figure>
</div>
</div>
+<p><strong>Introduction through iGEM Hamburg:</strong> Collaborated with Prof. Ignatova, a leading expert in CF research, to deepen our understanding of the disease.</p>
+
+<p><strong>Alternative cell models:</strong> After HEK cells from Leuven proved unsuitable, we consulted Prof. Ignatova, who provided access to the CFBE41o- cell line, immortalized CF cells from a patient.</p>
+
+<p><strong>Cell acquisition and cultivation:</strong> Obtained the CFBE41o- cells with permission from Prof. Karl Kunzelmann, successfully cultivated them in our lab, though they required extended acclimation.</p>
+
+<p><strong>Future testing plans:</strong> Plan to use these cells for patch-clamp experiments to validate our prime editing approach.</p>
+
+<p><strong>Broadened perspective on gene therapy:</strong> Discussions with Prof. Ignatova introduced us to alternative therapeutic strategies, such as recoding tRNAs, expanding our understanding of potential treatments for CF.</p>
+
</Collapsible>
)
@@ -339,12 +364,23 @@ function AnalyseMattijs(){
</figure>
</div>
</div>
+<p><strong>Consultation because of test system:</strong> HEK cells with corresponding CFTR mutation derived from Leuven were successfully cultured in our lab for testing purposes.</p>
+
+<p><strong>Understanding test cell lines:</strong> Explained the system behind the test cell lines, enabling us to adapt pegRNA engineering for our Prime Editing complex.</p>
+
+<p><strong>Integration of TevoPreQ1:</strong> Helped us incorporate the structural motif TevoPreQ1 into the pegRNA, significantly enhancing the efficiency of the Prime Editor.</p>
+
+<p><strong>Critical improvement:</strong> This enhancement was successfully tested and demonstrated in our results, marking a major advancement in our project.</p>
+
+<p><strong>Switching testing systems: </strong>After patch clamp measurements revealed that HEK cells were unsuitable for further experiments, we adjusted our system and switched to Ignatova cells from Hamburg for continued testing.</p>
+
+
</Collapsible>
)
}
function AnalyseMichaela(){
return(
- <Collapsible title="Michaela Bienert – Scientific Sales Representative at Stemcell Technologies" id="michaelaanalyseC">
+ <Collapsible title="Dr. Michaela Bienert – Scientific Sales Representative at Stemcell Technologies" id="michaelaanalyseC">
<p>Through hands-on experience with Michaela Bienert and Julie Watson, we gained valuable insights into the cultivation methods of Air-Liquid Interface (ALI) and apical-out organoids. We evaluated the advantages and limitations of different culturing techniques, which enabled us to make informed decisions about their implementation in our research. With the protocols and resources provided, we successfully generated ALI cultures and organoids from primary cells of CF patients and healthy control donors, which we were able to test throughout the course of our project. </p>
<div className="row align-items-center">
<div className="col">
@@ -354,8 +390,7 @@ function AnalyseMichaela(){
</div>
<div className="col gif-wrapper">
<figure>
- <img src="https://video.igem.org/w/5Ux9MfiKnLYjEwSDW3VzUQ" alt=""/>
-
+ <iframe title="Apical Out Organoids vs. Air Liquid Culture" width="560" height="315" src="https://video.igem.org/videos/embed/27ba6dab-ac52-4259-9a0e-153c9e626804?loop=1&autoplay=1&muted=1&title=0" frameBorder="0" allowFullScreen={true} sandbox="allow-same-origin allow-scripts allow-popups allow-forms"></iframe>
</figure>
</div>
</div>
@@ -458,14 +493,20 @@ function AnalyseMoor(){
<p>We gained valuable insights into the unique properties of chitosan, a cationic polymer with significant potential to stabilize RNA in our lipid nanoparticle (LNP) formulations. Chitosan offers robust protection against RNases and exhibits heat stability, making it suitable for processing methods like spray drying. Additionally, its mucoadhesive properties enable optimal choice as LNP component. </p>
<p>A critical insight was the necessity for chitosan to be in an acidic environment (pH 4-6) to maintain its positive charge, which is essential for effective RNA interaction. While it cannot replace PEG due to its hydrophilic nature, chitosan is ideal for forming RNA-chitosan complexes, which can then be encapsulated within LNPs. This approach significantly enhances RNA stability during spray drying, a method we intend to further test in collaboration with <a href="https://rnhale.com/">Rnhale</a>. </p>
<p> In terms of implementation, Benjamin educated us on the chemical and structural properties of chitosan, reinforcing our approach to improve stability, particularly against heat, in our LNP formulations. He provided guidance on formulating chitosan-RNA complexes and developed a protocol for integrating them into our LNP formulation without affecting the charge of the nanoparticles. Additionally, he supplied us with chitosan in various sizes, enabling us to test different chitosan complexes for optimal results. </p>
- <div className="row">
- <div className="col">
- <figure>
- <img src="https://static.igem.wiki/teams/5247/integrated-human-practices/chitosan.webp" style={{height:"50%", width:"auto"}} alt=""/>
-
- </figure>
- </div>
+ <div className="row align-items-center">
+ <div className="col">
+ <figure>
+ <img src="https://static.igem.wiki/teams/5247/integrated-human-practices/chitosan.webp" alt="" />
+ </figure>
+ </div>
+ <div className="col">
+ <figure>
+ <img src="//TODO (ISI)" alt=""/>
+
+ </figure>
+ </div>
</div>
+
<p><strong>Chitosan properties:</strong> Provided valuable insights into chitosan, a cationic polymer with strong potential to stabilize RNA in lipid nanoparticle (LNP) formulations due to its robust protection against RNases and heat stability.</p>
<p><strong>Mucoadhesive properties:</strong> Highlighted chitosan's mucoadhesive characteristics, making it an ideal component for LNPs in terms of RNA delivery.</p>
diff --git a/src/contents/Human Practices/Feedback.tsx b/src/contents/Human Practices/Feedback.tsx
index 24cbbd7958fc164094da661b80edb7d41a8b4acd..63b853ac72256eb9c9b1b6cadc80397a17e281fa 100644
--- a/src/contents/Human Practices/Feedback.tsx
+++ b/src/contents/Human Practices/Feedback.tsx
@@ -5,9 +5,11 @@ import { Collapsible } from "../../components/Collapsible";
import PreCyse from '../../components/precyse';
import { HPconTabs } from './Conclisuin';
import { useNavigation } from '../../utils';
+import { useTabNavigation } from '../../utils/TabNavigation';
export function HPFeedback(){
const {goToPageWithTabAndScroll} = useNavigation();
+ useTabNavigation();
return(
<div>
<p>Through our project, the insights and feedback from various stakeholders and experts played a crucial role in shaping and refining our approach. We actively integrated their input into the design, execution, and public engagement aspects of our work, ensuring a human-centered, scientifically sound solution. Below, we highlight key contributors and how their feedback impacted the project's development across multiple phases. </p>
diff --git a/src/contents/Human Practices/Further Engagement/Collaborations.tsx b/src/contents/Human Practices/Further Engagement/Collaborations.tsx
index 5e27dd6721ac9a6b058693c2ae2c88c6dbc283b2..e83196abb5f50ae6fdbee3cc94511809e20bf94a 100644
--- a/src/contents/Human Practices/Further Engagement/Collaborations.tsx
+++ b/src/contents/Human Practices/Further Engagement/Collaborations.tsx
@@ -2,10 +2,12 @@ import { ButtonOne } from "../../../components/Buttons";
import { H5, H4 } from "../../../components/Headings";
import { PDF } from "../../../components/Pdfs";
import { useNavigation } from "../../../utils";
+import { useTabNavigation } from "../../../utils/TabNavigation";
export function HPCollabs(){
const {goToPagesAndOpenTab} = useNavigation();
+ useTabNavigation();
return (
<div className="col">
<div className="row align-items-center" style={{marginTop: "5vh", marginBottom: "5vh"}}>
diff --git a/src/contents/Human Practices/Further Engagement/Education.tsx b/src/contents/Human Practices/Further Engagement/Education.tsx
index 5775ac3161eb084191d3ee66de6f9f342c1c65fd..453ab5cd5c7df3e2b77c5221d1361059f1539416 100644
--- a/src/contents/Human Practices/Further Engagement/Education.tsx
+++ b/src/contents/Human Practices/Further Engagement/Education.tsx
@@ -5,11 +5,13 @@ import { TabScrollLink } from "../../../components/Link";
import PreCyse from "../../../components/precyse";
import EduSources from "../../../sources/education-souces";
import { useNavigation } from "../../../utils";
+import { useTabNavigation } from "../../../utils/TabNavigation";
export function HPEducation(){
const {goToPageAndScroll} = useNavigation();
+ useTabNavigation();
return(
<div className="col">
<div className="row align-items-center" style={{marginTop: "5vh", marginBottom: "5vh"}}>
diff --git a/src/contents/Human Practices/Further Engagement/Entrepreneurship.tsx b/src/contents/Human Practices/Further Engagement/Entrepreneurship.tsx
index 10348a2e4504f871ee127a836a532637c7242c91..c26294af67122e2135bcb9f574b6d25b456bd38a 100644
--- a/src/contents/Human Practices/Further Engagement/Entrepreneurship.tsx
+++ b/src/contents/Human Practices/Further Engagement/Entrepreneurship.tsx
@@ -1,10 +1,11 @@
import { ButtonOneWithScroll } from "../../../components/Buttons";
import { H4, H5 } from "../../../components/Headings";
+import { useTabNavigation } from "../../../utils/TabNavigation";
export function HPEntrepreneur(){
-
- return(
+ useTabNavigation();
+ return(
<div className="col">
<div className="row align-items-center" style={{marginTop: "5vh", marginBottom: "5vh"}}>
<div className="col">
diff --git a/src/contents/Human Practices/Further Engagement/FurtherEngagement.tsx b/src/contents/Human Practices/Further Engagement/FurtherEngagement.tsx
index fb80f18ef1859418d10ba38f4100e62d7d0026ab..f6f385b26cf79c5258cfa46763fa70c9e1cb2f9c 100644
--- a/src/contents/Human Practices/Further Engagement/FurtherEngagement.tsx
+++ b/src/contents/Human Practices/Further Engagement/FurtherEngagement.tsx
@@ -1,4 +1,5 @@
import { Section, Subesction } from "../../../components/sections";
+import { useTabNavigation } from "../../../utils/TabNavigation";
import { HPCollabs } from "./Collaborations";
import { HPEducation } from "./Education";
import { HPEntrepreneur } from "./Entrepreneurship";
@@ -6,7 +7,7 @@ import { HPOutreach } from "./Outreach";
import { HPPartnerships } from "./Partnerships";
export function HPFurtherEngagement(){
-
+ useTabNavigation();
return(
<Section title="Further Engagement" id="Further Engagement">
<Subesction title="Education" id="Further Engagement1">
diff --git a/src/contents/Human Practices/Further Engagement/Outreach.tsx b/src/contents/Human Practices/Further Engagement/Outreach.tsx
index c78d48ba20e975b0bb39802f8d838e854c797433..50125f43772aed8d538521e10cad21e07f385586 100644
--- a/src/contents/Human Practices/Further Engagement/Outreach.tsx
+++ b/src/contents/Human Practices/Further Engagement/Outreach.tsx
@@ -1,9 +1,11 @@
import { ButtonOne } from "../../../components/Buttons";
import { H4 } from "../../../components/Headings";
+import { useTabNavigation } from "../../../utils/TabNavigation";
import { useNavigation } from "../../../utils/useNavigation";
export function HPOutreach(){
const { goToPagesAndOpenTab } = useNavigation();
+ useTabNavigation();
return(
<div className="col">
<div className="row align-items-center" style={{marginTop: "5vh", marginBottom: "5vh"}}>
diff --git a/src/contents/Human Practices/Further Engagement/Partnerships.tsx b/src/contents/Human Practices/Further Engagement/Partnerships.tsx
index eebbd67333316d42cecd5622dc02baf51746c201..5dd64741a43a1bb8db8b278204a58ecf5c86464a 100644
--- a/src/contents/Human Practices/Further Engagement/Partnerships.tsx
+++ b/src/contents/Human Practices/Further Engagement/Partnerships.tsx
@@ -1,7 +1,8 @@
import { H4 } from "../../../components/Headings";
+import { useTabNavigation } from "../../../utils/TabNavigation";
export function HPPartnerships(){
-
+ useTabNavigation();
return(
<div className="col">
<H4 text="CF Vests"></H4>
diff --git a/src/contents/Human Practices/Further Engagement/SupMaterial.tsx b/src/contents/Human Practices/Further Engagement/SupMaterial.tsx
index 59b8ac76da1d77bc5790b7e3ffd5ff6e41fc16b7..2a5f20df31b36d9bf4ba2b68c4ea2ceabe76bf77 100644
--- a/src/contents/Human Practices/Further Engagement/SupMaterial.tsx
+++ b/src/contents/Human Practices/Further Engagement/SupMaterial.tsx
@@ -1,8 +1,9 @@
import { DownloadLink } from "../../../components/Buttons";
import { Section } from "../../../components/sections";
+import { useTabNavigation } from "../../../utils/TabNavigation";
export function HPSupplement(){
-
+ useTabNavigation();
return(
<Section title="Supplementary Material" id="Supplementary Material">
<div className="row">
diff --git a/src/contents/Human Practices/HP-abstract.tsx b/src/contents/Human Practices/HP-abstract.tsx
index 05063eb97f1f7e2088c504ed029e06bfa6f4fd90..37b79cfd6bd737c841dd2e86a173a8250c58b3f0 100644
--- a/src/contents/Human Practices/HP-abstract.tsx
+++ b/src/contents/Human Practices/HP-abstract.tsx
@@ -1,21 +1,15 @@
import { Section } from "../../components/sections"
+import { useTabNavigation } from "../../utils/TabNavigation";
export function HPAbstract(){
-
+ useTabNavigation();
return(
<Section title="Abstract" id="Abstract">
- <p>As the iGEM Bielefeld-CeBiTec team, our project aimed to develop a gene therapy for cystic fibrosis (CF) with a human-centered approach.
- From the beginning, we engaged extensively with CF patients, clinicians, regulatory bodies, and researchers to ensure that our solution
- addressed real-world needs. We established continuous feedback loops to refine and evolve our project at every stage, incorporating insights
- from diverse stakeholders to create a responsible, impactful, and ethically sound therapy. Our mission was not just to develop a technical
- solution but to create one that aligns with societal values and patient expectations. </p>
- <p>Our integrated Human Practices approach was not just an addition to our project—it was the foundation on which every decision was built. By actively
- engaging with CF patients, healthcare professionals, and regulatory experts, we ensured that their feedback directly shaped our
- project, from design to implementation. Our patient-centered, globally focused approach not only addresses disparities in CF treatment but also sets
- a new standard for accessibility and ethical reflection. We extensively documented our methodologies, including CF cell culture protocols and stakeholder
- engagement processes, to empower future teams to build upon our success. Our thoughtful integration of diverse stakeholder insights, combined with expert
- guidance in regulatory compliance, nanoparticle research, and gene therapy, ensures that our project is responsible, scientifically sound, and globally
- impactful. </p>
+ <p>As the iGEM Bielefeld-CeBiTec team, we adopted a human-centered approach and delivered an extensive framework of <strong>eight reglementations</strong> for future iGEM teams, establishing <strong>eight frameworks</strong> that were validated through our project. Our methodology was shaped by over <strong>80 interviews with stakeholders and institutions</strong> , creating a comprehensive and multidimensional perspective on the complex challenge of gene therapy for cystic fibrosis (CF). Through <strong>33 key interviews</strong> , we navigated <strong>seven thematic areas</strong> simultaneously, incorporating <strong>five iterative feedback loops</strong> to ensure the project’s refinement at every stage.
+ </p>
+ <p> Our approach offers future iGEM teams and the synthetic biology community a valuable blueprint for responsible and ethical project development. By actively engaging with CF patients, clinicians, researchers, and regulatory bodies, we ensured that real-world needs and societal values were central to our project's evolution. Our efforts culminated in a thoughtful, scientifically sound solution, setting new standards for accessibility, patient focus, and regulatory compliance. Future teams can leverage our documented methodologies to further their own impact, ensuring that innovation remains aligned with the needs of those it serves.
+ </p>
+
</Section>
)
}
\ No newline at end of file
diff --git a/src/contents/Human Practices/IHP.tsx b/src/contents/Human Practices/IHP.tsx
index 70c4144b0f083e9d9272864ead147f933c0a190e..18894d91400e5ae2528d9d4bcfacbc874fc85330 100644
--- a/src/contents/Human Practices/IHP.tsx
+++ b/src/contents/Human Practices/IHP.tsx
@@ -4,6 +4,7 @@ import { HPTimeline } from "../../components/HP-timeline";
import PreCyse from "../../components/precyse";
import { Section, Subesction } from "../../components/sections";
import { useNavigation } from "../../utils";
+import { useTabNavigation } from "../../utils/TabNavigation";
import { HPconclusion } from "./Conclisuin";
import { HPFeedback } from "./Feedback";
import { HP3new } from "./HP svgs/hp3";
@@ -14,7 +15,7 @@ import { HPUnderstanding } from "./HP svgs/understanding";
export function HPIntegrated(){
const { goToPageWithTabAndScroll} = useNavigation();
-
+ useTabNavigation();
return(
<Section title="Integrated Human Practices" id="Integrated Human Practices">
<Subesction title="Framework" id="Integrated Human Practices1">
diff --git a/src/contents/Human Practices/Introduction.tsx b/src/contents/Human Practices/Introduction.tsx
index 56f15e26f69c6cfcc87f515af6e4959f2af4b866..cb75083b7e1fd1c91ad1f8663ee0e0733c943ead 100644
--- a/src/contents/Human Practices/Introduction.tsx
+++ b/src/contents/Human Practices/Introduction.tsx
@@ -4,10 +4,12 @@ import PreCyse from "../../components/precyse";
import { BlockQuoteB } from "../../components/Quotes";
import { Section } from "../../components/sections";
import { useNavigation } from "../../utils";
+import { useTabNavigation } from "../../utils/TabNavigation";
export function HPIntroduction(){
const {goToPageAndScroll} = useNavigation();
+ useTabNavigation();
return(
<Section title="Introduction" id="Introduction">
<H5 text="- Connecting our project to real life -"/>
diff --git a/src/contents/Human Practices/Overwiev.tsx b/src/contents/Human Practices/Overwiev.tsx
index 45ee29b36a1da08bfd22aa4f0ddae6458754cc44..c6d64814f2460d0ba77342901dd1907860e599e0 100644
--- a/src/contents/Human Practices/Overwiev.tsx
+++ b/src/contents/Human Practices/Overwiev.tsx
@@ -1,20 +1,23 @@
import { BlockQuoteB } from "../../components/Quotes";
+import { useTabNavigation } from "../../utils/TabNavigation";
export function HPOverview(){
-
+ useTabNavigation();
return(
<div className="col">
<section id="OverviewH">
<h2 id="Overview"></h2>
<img src="https://static.igem.wiki/teams/5247/photos/overview-ihp-final-final-min.webp" alt="Stakeholder Overview"></img>
- <figcaption>Figure 1. We </figcaption>
+ <strong>Figure: Visualization of the Human-Centric Approach by iGEM Bielefeld-CeBiTec 2024</strong><p>Our project has been supported by over 80 individuals and institutions, enhancing our integrative human practices. We are deeply grateful for the invaluable feedback and the collective effort of everyone who has contributed to our vision of safe and precise gene therapy through PreCyse. Thank you for your support!
+
+ </p>
+
<span id="hp-quote"><BlockQuoteB
text="Human Practices is the study of how your work affects the world, and how the world affects your work."
cite="- Peter Carr, Director of Judging"
/> </span>
- <img id="overview-stakeholders" src="https://static.igem.wiki/teams/5247/photos/overview-ihp-final-final-min.webp" alt="Stakeholder Overview"></img>
<p>In the development of our project, we embraced a holistic and human-centric approach, putting people at the core of our efforts. Our goal was to foster interdisciplinary collaboration and open dialogue, ensuring that diverse perspectives shaped the trajectory of our work. Over the course of this journey, we have engaged with more than 80 individuals and institutions, who have significantly influenced our project.
The strength of our approach lies in its interdisciplinary nature. We have brought together representatives from a wide range of categories, each playing a vital role in shaping the direction and success of our initiative. These categories include: </p>
<ol>
diff --git a/src/contents/description.tsx b/src/contents/description.tsx
index e98c7653a82a77b5a4d633038ff9f60665bb9bdf..6d930233f709b61d8f9a6891b7797a4be3608233 100644
--- a/src/contents/description.tsx
+++ b/src/contents/description.tsx
@@ -24,7 +24,7 @@ export function Description() {
<div className="row mt-4">
<div className="col">
<Section title="Abstract" id="Abstract">
- <p id="obenindescription">We are proud to present <PreCyse/>, a next-generation Prime Editing technology, as innovative gene therapy approach for cystic fibrosis
+ <p id="obenindescription">We are proud to present <PreCyse/>, a next-generation Prime Editing technology, as innovative gene therapy approach for cystic fibrosis (CF)
specifically targeting the most common mutation <b>F508del</b> of the CFTR gene. Cystic fibrosis is a severe disorder that primarily affects the lungs
and for which only short-term symptomatic treatments exist. PreCyse aims to provide long-term relief by delivering a small genetic payload with speed
and precision. Our approach integrates <b>PrimeGuide</b>, a highly optimized Prime Editing system, with <b>AirBuddy</b>, a novel lipid nanoparticle
diff --git a/src/contents/results.tsx b/src/contents/results.tsx
index 2b7c0c76518266d3581a0ffe15366b9aa7fb6340..ea76fcfe1951db6040c7cd1ac657657376a2378b 100644
--- a/src/contents/results.tsx
+++ b/src/contents/results.tsx
@@ -170,25 +170,25 @@ export function Results() {
<figure>
<img src="https://static.igem.wiki/teams/5247/delivery/results/rna-gel-final.png"/>
<figcaption>
- <b>Figure 1. </b>
+ <b>Figure 16. </b>
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.
</figcaption>
</figure>
</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 lipid nanoparticles (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 X). This mRNA was subsequently used in further LNP formulations with RNA.</p>
+ <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 lipid nanoparticles (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"/>
<div className="row align-items-center">
<div className="col">
- <p>Next, we formulated the LNPs using the Cayman LipidLaunchâ„¢ 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 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 X).</p>
+ <p>Next, we formulated the LNPs using the Cayman LipidLaunchâ„¢ 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 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">
<figure>
<img src="https://static.igem.wiki/teams/5247/delivery/results/caymanlnpblue.webp"/>
<figcaption>
- <b>Figure X. </b>
+ <b>Figure 17. </b>
Cayman LNP Formation indicated by blue color and turbidity. Mini DNA = Minicircle DNA from PlasmidFactory.
</figcaption>
</figure>
@@ -196,52 +196,52 @@ export function Results() {
</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 technical positive control and LNP without cargo.</p>
- <p>24 h, 48 h and 72 h post-transfection, we observed in the conditions with Lipofectamine alone, or combined with DNA or RNA, no fluorescence, indicating unsuccessful transfection. 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, clear fluorescence was observed, indicating successful transfection and expression of our eYFP reporter under this condition (figure X). However, a strong background fluorescence from the OptiMEM medium was observed, complicating the analysis.</p>
+ <p>Until 72 h 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>
<figure>
- <img src="Insert URL here"/>
+ <img src="https://static.igem.wiki/teams/5247/delivery/results/precyse/cayman.png"/>
<figcaption>
- <b>Figure X. </b>
- Figure X Fluorescence microscopic images of transfected HEK293 cells at 20x magnification after 72 h post-transfection with different Cayman LNP formulations recorded with Leica DMI6000 B.
+ <b>Figure 18. </b>
+ Overlay of phase contrast and fluorescence microscopic images of transfected HEK293 cells at 20x magnification after 72 h post-transfection with different Cayman LNP formulations recorded with Leica DMI6000 B. For Lipofectamine (Lipo) + Minicircle DNA (Mini DNA) only the fluorescence image is shown.
</figcaption>
</figure>
<H5 text="SEM"/>
<div className="row align-items-center">
<div className="col">
- <p>Scanning Electron Microscopy (SEM) (Phenom ProX, Thermo Fisher) was employed by us to examine the morphology and surface characteristics of Cayman LNPs. The SEM images revealed that the LNPs displayed a generally spherical morphology with a relatively smooth surface (Figure X). 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>
+ <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">
<figure>
<img src="https://static.igem.wiki/teams/5247/delivery/results/screenshot-2024-10-01-200629.png" alt="CayREM" style={{maxHeight: "200pt"}}/>
<figcaption>
- <b>Figure X.</b>
+ <b>Figure 19.</b>
SEM image of Cayman LNPs (10,000x magnification) with Topography mode. </figcaption>
</figure>
</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"/>
- <H5 text="Transfection"/>
- <p>Fluorescence microscopy with the Leica DMI6000 B microscope at 20x magnification was 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 24 h, 48 h, and 72 h post-transfection.</p>
<div className="row align-items-center">
<div className="col">
<figure>
<img src="https://static.igem.wiki/teams/5247/delivery/results/whatsapp-image-2024-09-24-at-12-57-59.jpeg"/>
<figcaption>
- <b>Figure X. </b>
+ <b>Figure 20. </b>
Turbidity after components of the Corden LNP have been pipetted together indicates particle formation. </figcaption>
</figure>
</div>
<div className="col">
- <p>During the preparation of the LNPs, the solution became turbid and bluish, indicating successful nanoparticle formation (Figure X). This was further confirmed by cryo-EM 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>
+ <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 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>
- <p>Quantitatively, none of the LNP-treated samples showed significant fluorescence, indicating a failure in transfection. The lack of fluorescence in all experimental groups, except the positive control, suggests either insufficient uptake of the LNPs by the cells or a failure in expression of the YFP reporter. </p>
+ <H5 text="Transfection"/>
+ <p>Fluorescence microscopy with the Leica DMI6000 B microscope at 20x magnification was by performed 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 24 h, 48 h, and 72 h post-transfection.</p>
+ <p>Quantitatively, after 72 h 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 as the positive control, 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>
<figure>
- <img src="Insert URL here"/>
+ <img src="https://static.igem.wiki/teams/5247/delivery/results/precyse/corden.png"/>
<figcaption>
- <b>Figure X. </b>
- Fluorescence microscopic images of transfected HEK293 cells at 20x magnification after 48 h post-transfection with different Corden LNP formulations recorded with Leica DMI6000 B.
+ <b>Figure 21. </b>
+ Overlay of phase contrast and fluorescence microscopic images of transfected HEK293 cells at 20x magnification after 72 h post-transfection with different Corden LNP formulations recorded with Leica DMI6000 B.
</figcaption>
</figure>
<H5 text="Cryo-EM"/>
@@ -251,52 +251,53 @@ export function Results() {
<figure>
<img src="https://static.igem.wiki/teams/5247/delivery/results/corden-lnp.jpg"/>
<figcaption>
- <b>Figure X. </b>
+ <b>Figure 22. </b>
Cryo-EM image of Corden LNPs.
</figcaption>
</figure>
</div>
<div className="col">
- <p>The images reveal the presence of spherical LNP structures with an approximate size of 100 nm (Figure X). 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>
+ <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"/>
+ <H4 text="SORT LNP"/>
<H5 text="Transfection"/>
- <p>Text :D</p>
+ <p>Already at 48 h 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 for our DNA choice and RNA synthesis.</p>
<figure>
- <img src="Insert URL here"/>
+ <img src="https://static.igem.wiki/teams/5247/delivery/results/precyse/sort.png"/>
<figcaption>
- <b>Figure X. </b>
- Description here
+ <b>Figure 23. </b>
+ Overlay of phase contrast and fluorescence microscopic images of transfected HEK293 cells at 20x magnification after 48 h post-transfection with different SORT LNP formulations recorded with Leica DMI6000 B.
</figcaption>
</figure>
+ <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 cytometries analysis 72 h 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>
+ <p>We performed flow cytometry analysis 72 h 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">
<figure>
<img src="https://static.igem.wiki/teams/5247/delivery/results/sortlnp-facs.png" alt="SORTFACS" style={{maxHeight: "200pt"}}/>
<figcaption>
- <b>Figure X. </b>
+ <b>Figure 24. </b>
Percentage of fluorescent cells (FITC-A+) performed 72 h post-transfection of SORT LNP in HEK293. Mean +/- SEM for n=3. For statistics one-way ANOVA was performed.
</figcaption>
</figure>
</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 a). 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>
+ <p>The SORT LNP-transfected sample carrying Minicircle DNA exhibited a significant increase in fluorescence compared to the lipofectamine transfection of Minicircle DNA, with approximately 14 times more fluorescent cells compared to the lipofectamine-transfected sample (Figure 24). This substantial difference indicates that the transfection efficiency with LNPs is markedly higher than with lipofectamine, demonstrating the superior performance of our LNP formulation in delivering nucleic acids to HEK cells.</p>
</div>
</div>
<H5 text="Zeta Potential"/>
<div className="row align-items-center">
<div className="col">
- <p>We measured both the particle size distribution and the Zeta potential using the Nanotrack Wave II. We could assume that the particles exhibit a polarized Zeta potential, which is sufficient to provide electrostatic stabilization, thereby preventing aggregation and maintaining particle stability. For effective targeting of lung cells which have negatively charged surfaces, a negative polarity is desirable meaning the LNP is positively charged, so there can be electrostatic attraction to lung epithelial cells. We were able to show that our SORT LNP has these properties regardless of the load. Furthermore we could <a href="https://chem.libretexts.org/Bookshelves/Analytical_Chemistry/Physical_Methods_in_Chemistry_and_Nano_Science_(Barron)/02%3A_Physical_and_Thermal_Analysis/2.05%3A_Zeta_Potential_Analysis" title="StabZeta" >determine the stability via the Zeta potential</a>. In detail the mean of the Zeta potential lays at 16.2 mV for the SORT LNP with Minicircle DNA as cargo, indicating incipient stability, at 59.45 mV for the SORT LNP with pcDNA 3.1 eYFP as cargo, indicating good stability and at 88.22 mV for the SORT LNP without cargo indicating excellent stability (Figure z). 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>
+ <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">
<figure>
<img src="https://static.igem.wiki/teams/5247/delivery/results/sort-zeta.webp"/>
<figcaption>
- <b>Figure X. </b>
+ <b>Figure 25. </b>
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. </figcaption>
</figure>
</div>
@@ -306,25 +307,25 @@ export function Results() {
<figure>
<img src="https://static.igem.wiki/teams/5247/delivery/results/screenshot-2024-10-01-204938.png"/>
<figcaption>
- <b>Figure X. </b>
+ <b>Figure 26. </b>
Size distribution for the SORT LNP with different cargos weighted by scattering intensity measured with Nanotrack Wave II.
</figcaption>
</figure>
</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 d). 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>
+ <p>The size distribution for all three samples shows a predominantly monomodal, yet broad, distribution with diameters ranging between 50 nm and 700 nm, with the peak of the distribution lying between 150 nm and 200 nm (Figure 26). SORT LNPs without DNA exhibited larger radii, with a peak around 300 nm. The SORT LNP containing Minicircle DNA suggests the presence of larger aggregates with diameters exceeding 1 µm. The likely reason for this variable particle size distribution, despite loading with different types of DNA, could be attributed to the manufacturing method. Since the LNPs were not produced using an extruder but rather via dialysis, this is highly plausible.</p>
</div>
</div>
<H5 text="Cryo-EM"/>
<div className="row align-items-center">
<div className="col">
- <p>Cryo-EM analysis was also performed of SORT LNPs by us with the same JEOL JEM-2200FS microscope at 200kV, allowing visualization of LNPs in their native hydrated state. The images show spherical LNP structures around 100 nm, with some larger aggregates also present (data not shown). 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 X). The low sample concentration likely contributed to the limited number of visible particles.</p>
+ <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 (data not shown). 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">
<figure>
<img src="https://static.igem.wiki/teams/5247/delivery/results/sortcryoem.png"/>
<figcaption>
- <b>Figure X. </b>
+ <b>Figure 27. </b>
Cryo-EM image of SORT LNPs. The different colored outlines indicate different size populations of LNPs.
</figcaption>
</figure>
@@ -338,13 +339,13 @@ export function Results() {
<figure>
<img src="https://static.igem.wiki/teams/5247/delivery/results/sort-dls.webp"/>
<figcaption>
- <b>Figure X. </b>
+ <b>Figure 28. </b>
Results for hydrodynamic radius determination by DLS Measurements for our SORT LNP, indicating a radius of approximately 100 nm.
</figcaption>
</figure>
</div>
<div className="col">
- <p>The results showed a hydrodynamic diameter of SORT LNPs yielding an average radius of approximately 100 nm (Figure X). 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>
+ <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"/>
@@ -353,15 +354,86 @@ export function Results() {
<figure>
<img src="https://static.igem.wiki/teams/5247/fanzor/sort-mtt.webp"/>
<figcaption>
- <b>Figure X. </b>
+ <b>Figure 29. </b>
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. </figcaption>
</figure>
</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 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="SORT LNP Chitosan-RNA Complex"/>
+ <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.</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>
+ <H4 text="Flow cytometry"/>
+ <p>We tested two different chitosan and mRNA concentrations (always used both in the same concentrations), 50 ng/µl and 500 ng/µl, in combination with our LNP to assess their impact on transfection efficiency. Interestingly, both concentrations resulted in similarly high levels of transfection, as evidenced by the fluorescence microscopy images, further validation is necessary to confirm these results.</p>
+ <div className="row align-items-center">
+ <div className="col">
+ <figure>
+ <img src="https://static.igem.wiki/teams/5247/delivery/results/precyse/lnp-chito-rna500-t24.webp"/>
+ <figcaption>
+ <b>Figure 30. </b>
+ Overlay of phase contrast and fluorescence microscopic images of with SORT + chitosan 500 transfected CFBE41o- cells at 20x magnification after 48 h post-transfection recorded with Leica DMI6000 B.
+ </figcaption>
+ </figure>
+ </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>
+ <div className="row align-items-center">
+ <div className="col">
+ <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">
+ <figure>
+ <img src="https://static.igem.wiki/teams/5247/delivery/results/precyse/lnpchito-rna50-t24.webp"/>
+ <figcaption>
+ <b>Figure 31. </b>
+ Overlay of phase contrast and fluorescence microscopic images of with SORT + chitosan 50 transfected CFBE41o- cells at 20x magnification after 48 h post-transfection recorded with Leica DMI6000 B.
+ </figcaption>
+ </figure>
+ </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>
+ <div className="row align-items-center">
+ <div className="col">
+ <figure>
+ <img src="https://static.igem.wiki/teams/5247/delivery/results/precyse/chito-t24-1.jpeg"/>
+ <figcaption>
+ <b>Figure 32. </b>
+ Overlay of phase contrast and fluorescence microscopic images of with SORT transfected CFBE41o- cells at 20x magnification after 48 h post-transfection recorded with Leica DMI6000 B.
+ </figcaption>
+ </figure>
+ </div>
+ <div className="col">
+ <figure>
+ <img src="https://static.igem.wiki/teams/5247/delivery/results/precyse/lnp-leer-t24.webp"/>
+ <figcaption>
+ <b>Figure 33. </b>
+ Overlay of phase contrast and fluorescence microscopic images of with chitosan transfected CFBE41o- cells at 20x magnification after 48 h post-transfection recorded with Leica DMI6000 B.
+ </figcaption>
+ </figure>
+ </div>
+ </div>
+ <div className="row align-items-center">
+ <div className="col">
+ <figure>
+ <img src="https://static.igem.wiki/teams/5247/delivery/results/precyse/ntc-t24.webp"/>
+ <figcaption>
+ <b>Figure 34. </b>
+ Overlay of phase contrast and fluorescence microscopic images of untransfected CFBE41o- cells at 20x magnification after 48 h post-transfection recorded with Leica DMI6000 B.
+ </figcaption>
+ </figure>
+ </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="PreCyse" id="Results4">
<H4 text="Goals"/>
@@ -371,6 +443,7 @@ export function Results() {
<H4 text="Conclusion"/>
<p>text</p>
</Subesction >
+
<Subesction title="Patch Clamp" id="Results5">
<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"/>
@@ -379,25 +452,25 @@ export function Results() {
<figure>
<img src="https://static.igem.wiki/teams/5247/photos/results/patchclamp/pc1.webp" alt="PC1" style={{maxHeight: "300pt"}}/>
<figcaption>
- <b>Figure 1. </b>
+ <b>Figure 35. </b>
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.
</figcaption>
</figure>
</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 1). 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, impairing chloride ion transport through the CFTR channel.</p>
+ <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 35). 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, 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 2). 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>
+ <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 36). 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'>
<figure>
<img src="https://static.igem.wiki/teams/5247/photos/results/patchclamp/pc2.webp" alt="PC1" style={{maxHeight: "300pt"}}/>
<figcaption>
- <b>Figure 2. </b>
+ <b>Figure 36. </b>
Repeated validation of current density measurements in HEK293T CFTR WT and HEK293T CFTR-F508del, showing consistent results with the initial experiment. For statistics one-way ANOYA was performed.
</figcaption>
</figure>
diff --git a/src/contents/safety.tsx b/src/contents/safety.tsx
index a0a2e925acac0118f32f4ec925d423ed3e321816..15c47f010a478cd8edf68ebdb30bc74502cec049 100644
--- a/src/contents/safety.tsx
+++ b/src/contents/safety.tsx
@@ -1,18 +1,19 @@
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";
// message for test commit.
export const Safety: React.FC = () =>{
const {goToPageAndScroll, goToPageWithTabAndScroll, goToPagesAndOpenTab} = useNavigation();
-
+ useTabNavigation();
return (
<>
@@ -215,7 +216,19 @@ export const Safety: React.FC = () =>{
</div>
</Subesction>
<Subesction title="Consent and Guidelines" id="Bioethics3">
- <LoremMedium/>
+ <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">
diff --git a/src/data/steckbriefe.ts b/src/data/steckbriefe.ts
index b6c96bfcca1b843d2ecc8a2894eb5078f68d61db..13ff00ed7dcd2cd246038ef4db0573513bc89da5 100644
--- a/src/data/steckbriefe.ts
+++ b/src/data/steckbriefe.ts
@@ -627,7 +627,7 @@ 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: [