From 3323ff8cf5a0dd0f80356c554584c1dd33456a64 Mon Sep 17 00:00:00 2001
From: Liliana Sanfilippo <lsanfilippo@techfak.uni-bielefeld.de>
Date: Fri, 15 Nov 2024 18:24:35 +0100
Subject: [PATCH] Figures
---
src/App/App.css | 8 +-
src/components/Figures.tsx | 79 +++++++++++---
src/contents/description.tsx | 2 +
src/contents/engineering.tsx | 192 +++++++++++------------------------
src/contents/methods.tsx | 98 +++++++++++-------
src/contents/parts.tsx | 1 +
src/contents/results.tsx | 32 +++++-
7 files changed, 217 insertions(+), 195 deletions(-)
diff --git a/src/App/App.css b/src/App/App.css
index 5993e628..9ae150be 100644
--- a/src/App/App.css
+++ b/src/App/App.css
@@ -425,6 +425,9 @@ margin-bottom: 10vw !important;
.bg-lb{
background-color: var(--lightblue) !important;
}
+.bg-w{
+ background-color: white !important;
+}
.bg-d{
background-color: var(--text-primary) !important;
color: var(--ourbeige);
@@ -3887,7 +3890,7 @@ figure img{
align-items: center;
}
-.figure-wrapper-philip{
+/* .figure-wrapper-philip{
border-color: var(--darkerbeige);
border-width: 5px;
border-style: solid;
@@ -3897,12 +3900,11 @@ figure img{
margin-top: var(--big-margin) !important;
margin-bottom: var(--big-margin) !important;
padding: 0 !important;
-}
+} */
.figure-wrapper {
border-color: var(--darkerbeige);
border-width: 5px;
- background-color: white;
border-style: solid;
width: 100%;
display: flex;
diff --git a/src/components/Figures.tsx b/src/components/Figures.tsx
index 61d6e9a9..f0baf86e 100644
--- a/src/components/Figures.tsx
+++ b/src/components/Figures.tsx
@@ -6,15 +6,21 @@ interface FigureProps{
pic5?: string,
pic6?: string,
alt1: string,
- description: React.ReactNode | string,
+ description: React.ReactNode | string,
+ description2?: React.ReactNode | string,
num: string |number;
+ num2?: string |number;
+ bg?: string;
}
-export function ThreeVertical({description, num, pic1, pic2, pic3, alt1}:FigureProps){
-
+export function ThreeVertical({description, num, pic1, pic2, pic3, alt1, bg}:FigureProps){
+ let background = "";
+ if (bg){
+ background = " bg-w"
+ }
return(
- <div className="figure-wrapper">
+ <div className={"figure-wrapper" + background}>
<figure>
<img src={pic1} alt={alt1} className="responsive-image"/>
<img src={pic2} className="responsive-image"/>
@@ -26,10 +32,13 @@ export function ThreeVertical({description, num, pic1, pic2, pic3, alt1}:FigureP
)
}
-export function TwoVertical({description, num, pic1, pic2, alt1}:FigureProps){
-
+export function TwoVertical({description, num, pic1, pic2, alt1, bg}:FigureProps){
+ let background = "";
+ if (bg){
+ background = " bg-w"
+ }
return(
- <div className="figure-wrapper">
+ <div className={"figure-wrapper" + background}>
<figure>
<img src={pic1} alt={alt1} className="responsive-image"/>
@@ -42,10 +51,13 @@ export function TwoVertical({description, num, pic1, pic2, alt1}:FigureProps){
)
}
-export function TwoHorizontal({description, num, pic1, pic2, alt1}:FigureProps){
-
+export function TwoHorizontal({description, num, pic1, pic2, alt1, bg}:FigureProps){
+ let background = "";
+ if (bg){
+ background = " bg-w"
+ }
return(
- <div className="figure-wrapper">
+ <div className={"figure-wrapper" + background}>
<figure>
<div className="row align-items-center">
@@ -64,10 +76,13 @@ export function TwoHorizontal({description, num, pic1, pic2, alt1}:FigureProps){
)
}
-export function ThreeHorizontal({description, num, pic1, pic2, pic3, alt1}:FigureProps){
-
+export function ThreeHorizontal({description, num, pic1, pic2, pic3, alt1, bg}:FigureProps){
+ let background = "";
+ if (bg){
+ background = " bg-w"
+ }
return(
- <div className="figure-wrapper">
+ <div className={"figure-wrapper" + background}>
<figure>
<div className="row align-items-center">
<div className="col">
@@ -88,9 +103,13 @@ export function ThreeHorizontal({description, num, pic1, pic2, pic3, alt1}:Figur
)
}
-export function OneFigure({description, num, pic1, alt1}:FigureProps){
+export function OneFigure({description, num, pic1, alt1, bg}:FigureProps){
+ let background = "";
+ if (bg){
+ background = " bg-w"
+ }
return(
- <div className="figure-wrapper">
+ <div className={"figure-wrapper" + background}>
<figure>
<img src={pic1} alt={alt1} className="responsive-image"/>
@@ -103,4 +122,34 @@ export function OneFigure({description, num, pic1, alt1}:FigureProps){
}
+export function TwoFigureRow({description,description2, num, num2, pic1, pic2, bg}:FigureProps) {
+ let background = "";
+ if (bg){
+ background = " bg-w"
+ }
+ return(
+ <div className={"figure-wrapper" + background}>
+ <figure>
+ <div className="row align-items-center">
+ <div className="col">
+ <img src={pic1} />
+ </div>
+ <div className="col">
+ <img src={pic2}/>
+ </div>
+ </div>
+ <figcaption>
+ <div className="row align-items-center">
+ <div className="col">
+ <b>Figure {num}:</b> {description}.
+ </div>
+ <div className="col">
+ <b>Figure {num2}:</b> {description2}.
+ </div>
+ </div>
+ </figcaption>
+ </figure>
+ </div>
+ )
+}
diff --git a/src/contents/description.tsx b/src/contents/description.tsx
index 3d2893ca..b0b3dc9f 100644
--- a/src/contents/description.tsx
+++ b/src/contents/description.tsx
@@ -64,6 +64,7 @@ export function Description() {
<OneFigure
pic1="https://static.igem.wiki/teams/5247/project-description/lung-ephitel-biorender.png"
num={1}
+ bg="white"
description="Lung ephitelium of human with correct CFTR expression (left) and Cystic Fibrosis (right)"
alt1="Lung ephitelium of human with correct CFTR expression (left) and Cystic Fibrosis (right)."
/>
@@ -79,6 +80,7 @@ export function Description() {
pic1="https://static.igem.wiki/teams/5247/charts-maps/cfper10-000.png"
alt1=""
num="?"
+ bg="white"
description="Philip, mach hier eine Description rein"
/>
diff --git a/src/contents/engineering.tsx b/src/contents/engineering.tsx
index 53e1f4f6..3a1d4c05 100644
--- a/src/contents/engineering.tsx
+++ b/src/contents/engineering.tsx
@@ -16,7 +16,7 @@ import EngPegsources from "../sources/eng-peg-sources";
import EngNicksources from "../sources/eng-nickases-sources";
import EngDelsources from "../sources/eng-delivery-sources";
import { TwoLinePDF, PDF } from "../components/Pdfs";
-import { OneFigure } from "../components/Figures";
+import { OneFigure, TwoFigureRow } from "../components/Figures";
export function Engineering() {
@@ -28,7 +28,7 @@ export function Engineering() {
<>
<div className="row mt-4">
<div className="col">
- <br/> <br/> <br/>
+ <br/> <br/>
<div id="tab-our-cycle" className="enginneeringtab" style={{display: "block"}}>
<section > <br id="obenengineering"/>
<div className="eng-box box" >
@@ -725,28 +725,16 @@ export function Engineering() {
<p>
Based on the function of this prototypical Cas9 nickase, we assumed that SpuFz1 would operate similarly, with two active centers—RuvC and TNB—each cutting one DNA strand. Following this logic, we hypothesized that by deactivating the TNB domain, which contains a zinc finger motif (Fig. 2) crucial for DNA coordination, we could convert SpuFz1 into a nickase. To test this, we aimed to replace the cysteine residues involved in zinc ion coordination within the TNB domain with alanine, thereby impairing its DNA-binding ability and producing a SpuFz1 nickase that cuts only one strand. At that time, we believed both domains in SpuFz1 were directly responsible for DNA cleavage, and our strategy was based on this assumption.
</p>
- <div className="figure-wrapper-philip">
- <figure>
- <div className="row align-items-center">
- <div className="col">
- <img src="https://static.igem.wiki/teams/5247/engineering-cycle/spufz-wt-3d-model.webp"/>
- </div>
- <div className="col">
- <img src="https://static.igem.wiki/teams/5247/engineering-cycle/spufz-wt-3d-model-zinc-finger.webp"/>
- </div>
- </div>
- <figcaption>
- <div className="row align-items-center">
- <div className="col">
- <b>Figure 1:</b> Schematic illustration of SpuFz1 (PDB code: 8GKH) visualized in ChimeraX
- </div>
- <div className="col">
- <b>Figure 2:</b> Close up of the zinc finger motif of SpuFz1 (PDB code: 8GKH) visualized in ChimeraX - in the middle of the image the zinc ion of the motif can be seen, which is coordinated by 4 surrounding cysteine residues
- </div>
- </div>
- </figcaption>
- </figure>
- </div>
+ <TwoFigureRow
+ pic1="https://static.igem.wiki/teams/5247/engineering-cycle/spufz-wt-3d-model.webp"
+ description="Schematic illustration of SpuFz1 (PDB code: 8GKH) visualized in ChimeraX"
+ alt1="Schematic illustration of SpuFz1 (PDB code: 8GKH) visualized in ChimeraX"
+ num={1}
+ num2={2}
+ description2="Close up of the zinc finger motif of SpuFz1 (PDB code: 8GKH) visualized in ChimeraX - in the middle of the image the zinc ion of the motif can be seen, which is coordinated by 4 surrounding cysteine residues"
+ pic2="https://static.igem.wiki/teams/5247/engineering-cycle/spufz-wt-3d-model-zinc-finger.webp"
+ />
+
<H4 text="Build" id="text"/>
<p>
Using the protein visualization software ChimeraX, we carefully examined the structure of SpuFz1 to identify the key cysteine residues responsible for coordinating the zinc ion. With this insight, we designed our nickase candidates by modifying the wild-type sequence, specifically substituting these cysteines with alanine, to disrupt the zinc ion coordination and potentially alter the protein's function.
@@ -819,28 +807,15 @@ export function Engineering() {
<p>
We used AlphaFold to model the 3D structures of our zinc finger mutation candidates. After generating these models, we used ChimeraX to perform a structural overlay comparison between the native SpuFz1 protein and the mutated versions (Fig. 3). This comparison revealed significant differences, particularly in the TNB domain, indicating that the zinc finger plays a crucial structural role (Fig. 4).
</p>
- <div className="figure-wrapper-philip">
- <figure>
- <div className="row align-items-center">
- <div className="col">
- <img src="https://static.igem.wiki/teams/5247/engineering-cycle/spufz-wt-vs-zf-nikase.webp"/>
- </div>
- <div className="col">
- <img src="https://static.igem.wiki/teams/5247/engineering-cycle/spufz-wt-vs-zf-nikase-zinc-finger.webp"/>
- </div>
- </div>
- <figcaption>
- <div className="row align-items-center">
- <div className="col">
- <b>Figure 3:</b> Structural overlay of wildtype SpuFz1 (color: Lilac) (PDB code: 8GKH) and modeled zinc-finger mutation candidate (color: orange) visualized in ChimeraX – the yellow circle shows the location of the zinc-finger. A structural deviation of both proteins locally is evident.
- </div>
- <div className="col">
- <b>Figure 4:</b> Close-up of the zinc finger motif of the structural overlay - the zinc finger appears to be structurally significant: there are strong structural differences locally
- </div>
- </div>
- </figcaption>
- </figure>
- </div>
+ <TwoFigureRow
+ pic1="https://static.igem.wiki/teams/5247/engineering-cycle/spufz-wt-vs-zf-nikase.webp"
+ pic2="https://static.igem.wiki/teams/5247/engineering-cycle/spufz-wt-vs-zf-nikase-zinc-finger.webp"
+ num={3}
+ num2={4}
+ alt1="Structural overlay of wildtype SpuFz1 (color: Lilac) (PDB code: 8GKH) and modeled zinc-finger mutation candidate (color: orange) visualized in ChimeraX – the yellow circle shows the location of the zinc-finger. A structural deviation of both proteins locally is evident"
+ description="Structural overlay of wildtype SpuFz1 (color: Lilac) (PDB code: 8GKH) and modeled zinc-finger mutation candidate (color: orange) visualized in ChimeraX – the yellow circle shows the location of the zinc-finger. A structural deviation of both proteins locally is evident"
+ description2="Close-up of the zinc finger motif of the structural overlay - the zinc finger appears to be structurally significant: there are strong structural differences locally"
+ />
<H4 text="Learn" id="learn-head"/>
<p>
From this analysis, we concluded that the zinc finger mutation is not a suitable candidate for generating a nickase, as altering this domain would likely compromise the structural integrity of SpuFz1. Prof. Hammer suggested that instead of focusing on SpuFz1, we explore other endonucleases with similar mechanisms. His recommendation was to identify endonucleases that are structurally comparable to SpuFz1 and analyze the strategies used to convert these into nickases. We would then apply these same strategies to our selected endonucleases, adapting them for our purposes.
@@ -857,28 +832,15 @@ export function Engineering() {
<p>
In addition to these insights, we noticed a significant phylogenetic relationship between Fanzor endonucleases, like SpuFz1, and Cas12 endonucleases<TabScrollLink tab="tab-nickase" num="1" scrollId="desc-1"/>. This connection was crucial, as Cas12 proteins have a similar cutting mechanism to Fanzor proteins, utilizing a single active site for cleavage while coordinating both DNA strands. More importantly, we identified a precedent in the literature where a Cas12a endonuclease was successfully converted into a nickase by substituting a single amino acid in the TNB domain<TabScrollLink tab="tab-nickase" num="6" scrollId="desc-6"/> (Fig. 5 and 6). This provided us with a clear model strategy to follow, as this targeted mutation allowed the endonuclease to selectively cut only one DNA strand, effectively converting it into a nickase.
</p>
- <div className="figure-wrapper-philip">
- <figure>
- <div className="row align-items-center">
- <div className="col">
- <img src="https://static.igem.wiki/teams/5247/engineering-cycle/cas12-nikase.webp"/>
- </div>
- <div className="col">
- <img src="https://static.igem.wiki/teams/5247/engineering-cycle/cas12-nikase-close-up.webp"/>
- </div>
- </div>
- <figcaption>
- <div className="row align-items-center">
- <div className="col">
- <b>Figure 5:</b> Schematic representation of Cas12a (PDB code: 8SFH) visualized in ChimeraX - the yellow circle highlights the position of arginine (R) (1226th amino acid in the primary structure) which, when replaced by an alanine (A), converts the Cas12a endonuclease into an nCas12a nickase
- </div>
- <div className="col">
- <b>Figure 6:</b> Close-up of Cas12a (PDB code: 8SFH) - arginine (R) (1226th amino acid in the primary structure) is colored purple
- </div>
- </div>
- </figcaption>
- </figure>
- </div>
+ <TwoFigureRow
+ pic1="https://static.igem.wiki/teams/5247/engineering-cycle/cas12-nikase.webp"
+ pic2="https://static.igem.wiki/teams/5247/engineering-cycle/cas12-nikase-close-up.webp"
+ num={5}
+ num2={6}
+ alt1="Schematic representation of Cas12a (PDB code: 8SFH) visualized in ChimeraX - the yellow circle highlights the position of arginine (R) (1226th amino acid in the primary structure) which, when replaced by an alanine (A), converts the Cas12a endonuclease into an nCas12a nickase"
+ description="Schematic representation of Cas12a (PDB code: 8SFH) visualized in ChimeraX - the yellow circle highlights the position of arginine (R) (1226th amino acid in the primary structure) which, when replaced by an alanine (A), converts the Cas12a endonuclease into an nCas12a nickase"
+ description2="Close-up of Cas12a (PDB code: 8SFH) - arginine (R) (1226th amino acid in the primary structure) is colored purple"
+ />
<p>
Based on these findings, two key decisions emerged. First, recognizing the structural and mechanistic similarities between Fanzor and Cas12 endonucleases, we decided to explore CasX—a smaller Cas12-related endonuclease—as an additional candidate in our project. CasX shares many of the advantages of SpuFz1, such as a more compact structure compared to Cas9, making it ideal for applications requiring smaller editing systems. Secondly, we resolved to adapt the mutation strategy used to convert Cas12a into a nickase for both CasX and SpuFz1. By applying these learnings, we aimed to generate effective nickases from these endonucleases to further optimize the Prime Editing complex.
@@ -900,28 +862,15 @@ export function Engineering() {
<p>
Structurally, we observed that arginine 1226 protrudes from the NUC domain of AsCas12a and is oriented toward the RuvC domain (Fig. 8). This positioning led us to hypothesize that the arginine helps coordinate the DNA strand as it moves into the RuvC domain for cutting. Based on this observation, we speculated that the mutation of arginine 1226 disrupts this coordination, preventing the full double-strand cut and effectively converting AsCas12a into a nickase.
</p>
- <div className="figure-wrapper-philip">
- <figure>
- <div className="row align-items-center">
- <div className="col">
- <img src="https://static.igem.wiki/teams/5247/engineering-cycle/ascas12a-nuc-domain.webp"/>
- </div>
- <div className="col">
- <img src="https://static.igem.wiki/teams/5247/engineering-cycle/ascas12a-nuc-domain-close-up.webp"/>
- </div>
- </div>
- <figcaption>
- <div className="row align-items-center">
- <div className="col">
- <b>Figure 7:</b> AsCas12a (PDB code: 8SFH) visualized in ChimeraX. The NUC domain (TNB) is colored purple and is attached to the RuvC domain. The DNA strand is colored orange.
- </div>
- <div className="col">
- <b>Figure 8:</b> Close-up of NUC domain (colored purple) of AsCas12a(PDB code: 8SFH) - the arginines (R) (1226th amino acid in the primary structure) is colored yellow. Its positively charged side chain is oriented towards the RuvC domain, as well as the DNA strand fixated in the RuvC domain.
- </div>
- </div>
- </figcaption>
- </figure>
- </div>
+ <TwoFigureRow
+ pic1="https://static.igem.wiki/teams/5247/engineering-cycle/ascas12a-nuc-domain.webp"
+ pic2="https://static.igem.wiki/teams/5247/engineering-cycle/ascas12a-nuc-domain-close-up.webp"
+ num={7}
+ num2={8}
+ description2="Close-up of NUC domain (colored purple) of AsCas12a(PDB code: 8SFH) - the arginines (R) (1226th amino acid in the primary structure) is colored yellow. Its positively charged side chain is oriented towards the RuvC domain, as well as the DNA strand fixated in the RuvC domain"
+ description="AsCas12a (PDB code: 8SFH) visualized in ChimeraX. The NUC domain (TNB) is colored purple and is attached to the RuvC domain. The DNA strand is colored orange"
+ alt1="AsCas12a (PDB code: 8SFH) visualized in ChimeraX. The NUC domain (TNB) is colored purple and is attached to the RuvC domain. The DNA strand is colored orange"
+ />
<p>
We then applied this structural insight to SpuFz1 and CasX, searching for positively charged amino acids with long side chains, similar to arginine 1226, that were positioned at the interface between the NUC and RuvC domains. We specifically looked for amino acids that protruded from the NUC domain and oriented toward the RuvC domain, mirroring the structural role of arginine 1226 in AsCas12a. These amino acids became our mutational targets, allowing us to design a strategy to convert SpuFz1 and CasX into nickases by disrupting their ability to make double-strand cuts, while preserving their functionality for single-strand cuts. The amino acids we identified in SpuFz1 are the 564th and the 568th arginine located in its NUC domain. For CasX we identified the 904th arginine as a promising candidate.
</p>
@@ -931,51 +880,26 @@ export function Engineering() {
<p>
We structurally analyzed CasX and SpuFz1, as well as the known AsCas12a nickase, using Chimera. Our objective was to understand why the specific amino acid substitution converted AsCas12a into a nickase. We then identified analogous amino acids in SpuFz1 (Fig. 7 and Fig. 8) and CasX (Fig. 9 and Fig. 10) that might play a similar role, allowing us to design new mutation candidates for our project. After designing these new mutation candidates, we modeled them using AlphaFold to predict their 3D structures and assess their potential effectiveness.
</p>
- <div className="figure-wrapper-philip">
- <figure>
- <div className="row align-items-center">
- <div className="col">
- <img src="https://static.igem.wiki/teams/5247/engineering-cycle/casx-nikase.webp"/>
- </div>
- <div className="col">
- <img src="https://static.igem.wiki/teams/5247/engineering-cycle/casx-nikase-close-up.webp"/>
- </div>
- </div>
- <figcaption>
- <div className="row align-items-center">
- <div className="col">
- <b>Figure 9:</b> Schematic representation of PlmCasX (PDB code: 7WAZ) in ChimeraX - the yellow circle highlights the position of arginine (R) (904th amino acid in the primary structure) and glutamine (Q) (907th amino acid in the primary structure), which, when replaced by an alanine (A), convert the endonuclease into a nickase, according to our hypothesis
- </div>
- <div className="col">
- <b>Figure 10:</b> Close-up of PlmCasX (PDB code: 7WAZ) - arginine (R) (904th amino acid in the primary structure) and glutamine (Q) (907th amino acid in the primary structure) are purple in color
- </div>
- </div>
- </figcaption>
- </figure>
- </div>
+ <TwoFigureRow
+ pic1="https://static.igem.wiki/teams/5247/engineering-cycle/casx-nikase.webp"
+ pic2="https://static.igem.wiki/teams/5247/engineering-cycle/casx-nikase-close-up.webp"
+ num={9}
+ num2={10}
+ description2="Close-up of PlmCasX (PDB code: 7WAZ) - arginine (R) (904th amino acid in the primary structure) and glutamine (Q) (907th amino acid in the primary structure) are purple in color"
+ alt1="Schematic representation of PlmCasX (PDB code: 7WAZ) in ChimeraX - the yellow circle highlights the position of arginine (R) (904th amino acid in the primary structure) and glutamine (Q) (907th amino acid in the primary structure), which, when replaced by an alanine (A), convert the endonuclease into a nickase, according to our hypothesis"
+ description="Schematic representation of PlmCasX (PDB code: 7WAZ) in ChimeraX - the yellow circle highlights the position of arginine (R) (904th amino acid in the primary structure) and glutamine (Q) (907th amino acid in the primary structure), which, when replaced by an alanine (A), convert the endonuclease into a nickase, according to our hypothesis"
+ />
+
+ <TwoFigureRow
+ pic1="https://static.igem.wiki/teams/5247/engineering-cycle/spufz-nikase.webp"
+ pic2="https://static.igem.wiki/teams/5247/engineering-cycle/spufz-nikase-close-up.webp"
+ num={11}
+ num2={12}
+ description2="Close-up of SpuFz1 (PDB code: 8GKH) - the two arginines (R) (564th and 568th amino acid in the primary structure) are purple in color"
+ alt1="Schematic representation of SpuFz1 (PDB code: 8GKH) in ChimeraX - the yellow circle highlights the position of the two arginines (R) (564th and 568th amino acid in the primary structure), which, when replaced by an alanine (A), transform the endonuclease into a nickase according to our hypothesis"
+ description="Schematic representation of SpuFz1 (PDB code: 8GKH) in ChimeraX - the yellow circle highlights the position of the two arginines (R) (564th and 568th amino acid in the primary structure), which, when replaced by an alanine (A), transform the endonuclease into a nickase according to our hypothesis"
+ />
- <div className="figure-wrapper-philip">
- <figure>
- <div className="row align-items-center">
- <div className="col">
- <img src="https://static.igem.wiki/teams/5247/engineering-cycle/spufz-nikase.webp"/>
- </div>
- <div className="col">
- <img src="https://static.igem.wiki/teams/5247/engineering-cycle/spufz-nikase-close-up.webp"/>
- </div>
- </div>
- <figcaption>
- <div className="row align-items-center">
- <div className="col">
- <b>Figure 11:</b> Schematic representation of SpuFz1 (PDB code: 8GKH) in ChimeraX - the yellow circle highlights the position of the two arginines (R) (564th and 568th amino acid in the primary structure), which, when replaced by an alanine (A), transform the endonuclease into a nickase according to our hypothesis
- </div>
- <div className="col">
- <b>Figure 12:</b> Close-up of SpuFz1 (PDB code: 8GKH) - the two arginines (R) (564th and 568th amino acid in the primary structure) are purple in color
- </div>
- </div>
- </figcaption>
- </figure>
- </div>
<H4 text="Test" id="test-head"/>
<p>
To validate our approach, we conducted an interview with <a onClick={() => goToPagesAndOpenTab('saito', '/human-practices')}> Makoto Saito </a>, the lead author of the main Fanzor paper. Given his expertise, there was no better person to consult on this topic. We presented our project and our strategy for creating nickases, and he found our approach to be very plausible. He confirmed that the zinc finger mutation is likely structurally critical and agreed that our new strategy, based on the precedent with AsCas12a, was more promising. This conversation gave us confidence that we were on a good track.
diff --git a/src/contents/methods.tsx b/src/contents/methods.tsx
index 485667b8..4632d27f 100644
--- a/src/contents/methods.tsx
+++ b/src/contents/methods.tsx
@@ -3,6 +3,7 @@ import { useTabNavigation } from "../utils/TabNavigation";
import {H4} from "../components/Headings";
import MethodSources from "../sources/methods-sources";
import { useNavigation } from "../utils";
+import { OneFigure } from "../components/Figures";
export function Methods() {
@@ -20,17 +21,22 @@ export function Methods() {
</Subesction>
<Subesction title="Principles of the patch clamp technique" id="Patch Clamp2">
<p>Patch clamp recording involves the use of a glass micropipette which is manufactured from a glass capillary through the use of a Micropipette Puller. The micropipette is then filled with an electrolyte solution, which is subsequently brought into contact with the cell membrane. By applying gentle suction, a high-resistance seal called giga seal is formed between the pipette tip and the membrane patch. This enables the measurement of ionic currents with minimal noise interference [3]. <strong>Whole-Cell Configuration</strong> records currents from the entire cell by rupturing the membrane patch, accessing the intracellular environment, and is useful for analysing overall ion channel activity and cellular responses. <strong>Single-Channel Recording</strong> measures currents through individual ion channels without rupturing the membrane, enabling high-resolution study of channel conductance, gating, and selectivity [2].</p>
+ <div className="figure-wrapper">
<figure>
<iframe title="Bielefeld-CeBiTec: Patch Clamp Measurement (2024)" width="560" height="315" src="https://video.igem.org/videos/embed/0d948e57-5997-430a-a2df-815b71a2fc67?autoplay=1" frameBorder="0" allowFullScreen={true} sandbox="allow-same-origin allow-scripts allow-popups allow-forms"></iframe>
<figcaption> <b>Figure 1. </b> Microscopic recording of micropipette sealing of a HEK293 cell. </figcaption>
- </figure>
+ </figure>
+ </div>
<p>The success of patch clamp experiments heavily depends on the composition of the solutions used. Typically, two main types of solutions are employed: The <strong>Pipette Solution</strong> in the micropipette mimics the intracellular environments, while the <strong>Bath Solution</strong> surrounds the cell and usually contains components that replicate the extracellular environment. Both solutions are meticulously designed to reflect the physiological conditions under which the cells operate, thereby ensuring that the measurements accurately reflect ion channel activity in a natural setting [2].</p>
- <figure>
- <img src="https://static.igem.wiki/teams/5247/photos/for-wiki-texts/meth-patch-clamp/bild-meth-patch-clamp.png" alt="Patch clamp setup"/>
- <figcaption><b>Figure 2. </b> Patch clamp setup.</figcaption>
- </figure>
+ <OneFigure
+ pic1="https://static.igem.wiki/teams/5247/photos/for-wiki-texts/meth-patch-clamp/bild-meth-patch-clamp.png"
+ alt1="Patch clamp setup"
+ description="Patch clamp setup"
+ num={2}
+ />
+
</Subesction>
<Subesction title="Application in CFTR gene prime editing validation" id="Patch Clamp3">
<p>In our ongoing research project focusing on the treatment of cystic fibrosis, our patch clamp measurements, performed in collaboration with Dr. Oliver Dräger from the Cellular Neurophysiology working group at Bielefeld University, serve as a powerful validation tool for the assessment of the functional correction of the CFTR gene, particularly the common F508del mutation, via prime editing. The patch clamp technique can be employed in this context to measure the resulting chloride ion channel activity which is altered by the mutation [4]. Whole-Cell recordings were performed to assess whether the corrected CFTR channels function similarly to those in healthy cells. If the chloride ion currents in the edited cells approach levels of healthy cells, this would strongly suggest successful gene editing and validate the functionality of our therapeutic approach.</p>
@@ -40,30 +46,43 @@ export function Methods() {
<Section title="Cell Culture" id="Cell Culture">
<Subesction title="HEK293 and HEK293T cell lines" id="Cell Culture1">
<p>For testing our prime editing approach, we needed an easy-to-handle cell line with a measurable high expression of CFTR and the CFTR F508del mutation. When talking to Mattijs Bulcaen from the Laboratory of Molecular Virology and Gene Therapy at KU Leuven, he recommended to use HEK293T cell lines overexpressing CFTR they had used. HEK293 cells are a very common immortalized human cell line derived from the kidneys of a female embryo. They are particularly suited to research due to their convenient handling and transfection properties. Basic HEK293 cells were provided to us by the Cellular and Molecular Biotechnology working group at Bielefeld University led by Prof. Dr. Kristian Müller, who is also one of the Principal Investigators of our team. HEK293T cells express an additional tsA1609 allele of the SV40 large T-antigen, allowing for replication of vectors containing the SV40 origin of replication[5]. Besides the native CFTR gene, which is not expressed in HEK cells, the HEK293T cell lines used in Leuven carry another copy of the gene embedded in an expression cassette. The cassette includes a CMV promoter, which is a standard promoter used for gene overexpression in human cells derived from the human Cytomegalovirus[6], as well as a puromycin resistance co-expressed with the CFTR allowing for continuous selection of CFTR expressing cells. The whole construct was stably inserted into the genome using lentiviral transduction[7][8]. </p>
- <figure>
- <img src="https://static.igem.wiki/teams/5247/photos/for-wiki-texts/meth-used-cells/mikroskopie-hek293t.png" alt="Phase contrast image of HEK293T at 20x magnification"/>
- <figcaption> <b>Figure 3. </b>Phase contrast image of HEK293T at 20x magnification.</figcaption>
- </figure>
+ <OneFigure
+ pic1="https://static.igem.wiki/teams/5247/photos/for-wiki-texts/meth-used-cells/mikroskopie-hek293t.png"
+ alt1="Phase contrast image of HEK293T at 20x magnification"
+ description="Phase contrast image of HEK293T at 20x magnification"
+ num={3}
+ bg="white"
+ />
</Subesction>
<Subesction title="CFBE41o- cell line" id="Cell Culture2">
<p>The CFBE41o- cell line, derived from bronchial epithelial cells of a one-year-old cystic fibrosis patient, serves as a vital model for studying cystic fibrosis. These cells closely mimic the physiological environment of the airway epithelium, allowing for more accurate studies on how CFTR mutations affect cell function and response to treatments. They were immortalized through calcium-phosphate-mediated transfection using a replication-defective pSVori plasmid that carries the simian virus 40 large T-antigen (SV40-LT). The plasmid's defective origin of replication prevents viral propagation, thus preserving essential physiological characteristics of the cells while enabling them to develop differentiated morphologies. CFBE41o- cells are homozygous for the F508del CFTR mutation [9]. We are happy we got this cell line with permission from <a onClick={() => goToPagesAndOpenTab('ignatova', '/human-practices')}>Prof. Dr. Ignatova</a>, who is leader of a working group at the Institute for Biochemistry and Molecular Biology of Hamburg University and an iGEM supporter since a long time [10]. </p>
</Subesction>
<Subesction title="Human nasal epithelial cells (hNECs)" id="Cell Culture3">
<p>Human nasal epithelial cells were obtained by nasal brushing, a minimally invasive method. These cells function/act as primary cultures. Cultivated in air-liquid interface (ALI) cultures and apical-out airway organoids (AOAO), they serve as a suitable model to visualise the functional epithelium of the airways in a differentiated form. The <i>in vivo</i> aspects of an airway disease, such as CF, can be modelled using donors with those airway diseases [11]. This model is therefore particularly suitable for testing our prime editing complex. </p>
- <div className="row">
- <div className="col">
- <figure>
- <iframe title="Bielefeld-CeBiTec: ALI cell culture (2024) [English]" width="560" height="315" src="https://video.igem.org/videos/embed/ff557f5a-94be-45e6-90ca-0affa14423e3?autoplay=1&muted=1" frameBorder="0" allowFullScreen={true} sandbox="allow-same-origin allow-scripts allow-popups allow-forms"></iframe>
- <figcaption> <b>Figure 4. </b> ALI cultures of hNECs: The active cilia beat frequency of differentiated human nasal epithelial cells (hNECs) in air-liquid interface (ALI) culture is visible. This ciliary movement is crucial for mucociliary transport, which contributes to the clearance of particles and pathogens in the respiratory tract. </figcaption>
- </figure>
+ <div className="figure-wrapper">
+ <figure>
+ <div className="row align-items-center">
+ <div className="col">
+ <iframe title="Bielefeld-CeBiTec: ALI cell culture (2024) [English]" width="500" height="315" src="https://video.igem.org/videos/embed/ff557f5a-94be-45e6-90ca-0affa14423e3?autoplay=1&muted=1" frameBorder="0" allowFullScreen={true} sandbox="allow-same-origin allow-scripts allow-popups allow-forms"></iframe>
+
+ </div>
+ <div className="col">
+ <iframe title="Bielefeld-CeBiTec: AOAO cell culture (2024) [English]" width="500" height="315" src="https://video.igem.org/videos/embed/058d83cf-ab09-476e-9ab2-30cd114fbc0c?autoplay=1&muted=1" frameBorder="0" allowFullScreen={true} sandbox="allow-same-origin allow-scripts allow-popups allow-forms"></iframe>
+
+ </div>
</div>
- <div className="col">
- <figure>
- <iframe title="Bielefeld-CeBiTec: AOAO cell culture (2024) [English]" width="560" height="315" src="https://video.igem.org/videos/embed/058d83cf-ab09-476e-9ab2-30cd114fbc0c?autoplay=1&muted=1" frameBorder="0" allowFullScreen={true} sandbox="allow-same-origin allow-scripts allow-popups allow-forms"></iframe>
- <figcaption> <b>Figure 5. </b> Apical-Out Airway Organoid (AOAO) culture: Visible apical-out airway organoids in action. These 3D structures, which mimic the airway epithelium, allow detailed study of cellular processes such as mucociliary transport and secretory activities, in which cilia and vesicles play a key role. </figcaption>
- </figure>
+ <figcaption>
+ <div className="row align-items-center">
+ <div className="col">
+ <b>Figure 4:</b> ALI cultures of hNECs: The active cilia beat frequency of differentiated human nasal epithelial cells (hNECs) in air-liquid interface (ALI) culture is visible. This ciliary movement is crucial for mucociliary transport, which contributes to the clearance of particles and pathogens in the respiratory tract.
+ </div>
+ <div className="col">
+ <b>Figure 5:</b> Apical-Out Airway Organoid (AOAO) culture: Visible apical-out airway organoids in action. These 3D structures, which mimic the airway epithelium, allow detailed study of cellular processes such as mucociliary transport and secretory activities, in which cilia and vesicles play a key role..
+ </div>
+ </div>
+ </figcaption>
+ </figure>
</div>
- </div>
</Subesction>
@@ -73,15 +92,15 @@ export function Methods() {
<H4 text="Assessing the Safety of Our LNPs "></H4>
<p>Ensuring the safety and thorough characterization of our LNPs was a central part of our project, as these particles are intended for use in biological systems. We implemented a comprehensive range of assays and techniques to assess their biosafety and physical properties, ensuring their suitability for applications such as drug delivery and gene therapy. Below is an overview of the key steps we took in our assessment.</p>
<H4 text="MTT Assay"></H4>
+
<div className='row align-items-center'>
<div className='col'>
- <figure>
- <img src="https://static.igem.wiki/teams/5247/integrated-human-practices/mttassay.webp" alt="PC1" style={{maxHeight: "200pt"}}/>
- <figcaption>
- <b>Figure 6. </b>
- MTT Assay: Formation of purple formazan crystals by living cells.
- </figcaption>
- </figure>
+ <OneFigure
+ pic1="https://static.igem.wiki/teams/5247/integrated-human-practices/mttassay.webp"
+ alt1="PC1"
+ description="MTT Assay: Formation of purple formazan crystals by living cells"
+ num={6}
+ />
</div>
<div className='col'>
<p>To evaluate the cytotoxicity of our LNPs, we conducted an MTT assay, which measures the metabolic activity of cells. This assay is based on the ability of living cells to reduce MTT, a yellow tetrazolium salt, into purple formazan crystals through NAD(P)H-dependent enzymes. Cells were treated with various concentrations of LNPs, and after dissolving the formazan crystals with DMSO, we measured absorbance. Higher absorbance values indicate greater cell viability. Our results showed no significant reduction in cell viability across all LNP concentrations, demonstrating that the LNPs did not induce cytotoxic effects. This finding is crucial for ensuring that the LNPs are safe for biological use, supporting their potential in clinical applications such as drug delivery and gene therapy. Overall, the MTT assay provided strong evidence of the biocompatibility of our LNPs. </p>
@@ -105,10 +124,12 @@ export function Methods() {
Overall, the combination of PCS, DLS, and zeta potential measurements provided a comprehensive characterization of the LNPs, confirming their hydrodynamic properties, stability, and suitability for drug delivery applications. </p>
<div className='row align-items-center'>
<div className='col'>
- <figure>
- <img src="https://static.igem.wiki/teams/5247/photos/lab/dls-methods.webp"/>
- <figcaption> <b>Figure 7. </b>Dynamic Light Scattering (DLS) measurement set-up.</figcaption>
- </figure>
+ <OneFigure
+ pic1="https://static.igem.wiki/teams/5247/photos/lab/dls-methods.webp"
+ alt1="Dynamic Light Scattering (DLS) measurement set-up"
+ description="Dynamic Light Scattering (DLS) measurement set-up"
+ num={7}
+ />
</div>
</div>
<H4 text="SEM and Cryo-EM for Structural Analysis"></H4>
@@ -119,13 +140,12 @@ export function Methods() {
In addition to Cryo-EM, we employed scanning electron microscopy (SEM) to further characterize the morphology and surface structure of the LNPs. SEM provided high-resolution images that confirmed the spherical shape and uniformity of the LNPs.</p>
</div>
<div className='col'>
- <figure>
- <img src="https://static.igem.wiki/teams/5247/delivery/plasmatem.webp" alt="PC1" style={{maxHeight: "200pt"}}/>
- <figcaption>
- <b>Figure 8. </b>
- Sample preparation for SEM: Sputtering in Argon plasma.
- </figcaption>
- </figure>
+ <OneFigure
+ pic1="https://static.igem.wiki/teams/5247/delivery/plasmatem.webp"
+ alt1="PC1"
+ description="Sample preparation for SEM: Sputtering in Argon plasma"
+ num={8}
+ />
</div>
</div>
</Subesction>
diff --git a/src/contents/parts.tsx b/src/contents/parts.tsx
index ae8be736..04a84a4b 100644
--- a/src/contents/parts.tsx
+++ b/src/contents/parts.tsx
@@ -70,6 +70,7 @@ export function Parts() {
<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"
diff --git a/src/contents/results.tsx b/src/contents/results.tsx
index b72bcd91..09b9c242 100644
--- a/src/contents/results.tsx
+++ b/src/contents/results.tsx
@@ -43,6 +43,7 @@ export function Results() {
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"
/>
@@ -54,6 +55,7 @@ export function Results() {
<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."
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"
@@ -65,6 +67,7 @@ export function Results() {
<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."
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"
@@ -77,6 +80,7 @@ export function Results() {
<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."
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"
@@ -88,6 +92,7 @@ export function Results() {
<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"
/>
@@ -95,6 +100,7 @@ export function Results() {
<TwoVertical
description="Microscopy of HEK293 72h post transfection with CaCl2 with 500 ng, 1000 ng or 1500 ng pZMB938. All transfections show fluorescence signals."
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"
@@ -107,6 +113,7 @@ export function Results() {
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."
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"
/>
@@ -120,6 +127,7 @@ export function Results() {
<TwoHorizontal
description="Flow Cytometry analysis of pegRNAs with and without silent edits."
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"/>
@@ -149,6 +157,7 @@ export function Results() {
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."
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"
/>
@@ -157,6 +166,7 @@ export function Results() {
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."
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"
/>
@@ -170,7 +180,8 @@ export function Results() {
<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."
- num={11}
+ 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"
@@ -180,7 +191,8 @@ export function Results() {
<OneFigure
alt1=""
pic1="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/pe2-pe-co.png"
- num={12}
+ num={13}
+ bg="white"
description="Flow Cytometry analysis to compare prime editing complexes PE2 and PE_CO-Mini"
/>
</div>
@@ -193,7 +205,8 @@ export function Results() {
<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={13}
+ num={14}
+ bg="white"
description="Microscopy of HEK293 72h post transfection with lipofectamine 3000 and co transfection with pCMV-PE6c and pDAS12489."
alt1="Microscopy of HEK293 72h post transfection with lipofectamine 3000 and co transfection with pCMV-PE6c and pDAS12489."
/>
@@ -201,7 +214,8 @@ export function Results() {
<TwoHorizontal
alt1=""
description="Flow Cytometry results for evaluation of PE6c performance."
- num={14}
+ 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"
/>
@@ -243,6 +257,7 @@ export function Results() {
<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"
/>
@@ -281,6 +296,7 @@ export function Results() {
<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"
/>
@@ -306,6 +322,7 @@ export function Results() {
<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"
/>
@@ -317,6 +334,7 @@ export function Results() {
<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"
/>
@@ -334,6 +352,7 @@ export function Results() {
<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"
/>
@@ -344,6 +363,7 @@ export function Results() {
<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"
/>
@@ -375,6 +395,7 @@ export function Results() {
<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"
/>
@@ -390,6 +411,7 @@ export function Results() {
<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"
/>
@@ -485,6 +507,7 @@ export function Results() {
<OneFigure
pic1="https://static.igem.wiki/teams/5247/photos/results/patchclamp/pc.webp"
num={35}
+ 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"
/>
@@ -503,6 +526,7 @@ export function Results() {
alt1=""
pic1="https://static.igem.wiki/teams/5247/photos/results/patchclamp/pc2.webp"
num={36}
+ 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"
/>
--
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