diff --git a/src/App/App.css b/src/App/App.css
index 8953f8961c6b13a8861ca44fdf8e21e1d310a6a5..537319542c778a51a3ad5bbce32fd71153026cbd 100644
--- a/src/App/App.css
+++ b/src/App/App.css
@@ -62,6 +62,7 @@
--markmap-a-color: var(--text-primary) !important;
--node-size: 60px;
+ --simple-margin: 20px;
}
html{
scroll-behavior: smooth;
@@ -3967,17 +3968,6 @@ height: min-content !important;
}
-figure {
- align-self: center;
- display: table;
- text-align: center;
- font-style: italic;
- font-size: medium;
- text-indent: 0;
- margin: 0.5em;
-
-}
-
figcaption{
display: table-caption;
caption-side: bottom;
@@ -3991,11 +3981,7 @@ figure .row div{
justify-content: center;
}
-.figure-wrapper{
- display: flex;
- align-items: center;
- justify-content: center;
-}
+
figure img{
object-fit: cover !important;
@@ -4163,4 +4149,41 @@ figure img{
display: flex;
justify-content: center;
align-items: center;
+}
+
+
+
+.figure-wrapper {
+ border-color: var(--darkerbeige);
+ border-width: 5px;
+ background-color: white;
+ border-style: solid;
+ width: 100%;
+ display: flex;
+ justify-content: center;
+ margin-top: var(--simple-margin);
+ margin-bottom: var(--simple-margin);
+}
+figure {
+ align-self: center;
+ text-align: center;
+ font-style: italic;
+ font-size: medium;
+ text-indent: 0;
+ display: grid !important;
+ width: 100%;
+ text-align: center;
+ margin: 0 !important;
+}
+.responsive-image {
+ margin: auto;
+ padding-left: var(--simple-margin);
+ padding-right: var(--simple-margin);
+ margin-top: var(--simple-margin);
+ margin-bottom: var(--simple-margin);
+ align-self: center !important;
+ max-height: 40vh; /* Set the maximum height for tall images */
+ width: auto; /* Keeps the image's aspect ratio */
+ max-width: 100%; /* Limits the width to container's width */
+ object-fit: contain !important; /* Adds space around the image if it's narrower than the container */
}
\ No newline at end of file
diff --git a/src/components/Figures.tsx b/src/components/Figures.tsx
index 299ebd5fb7b2ec115ffa95324e5b8fdb07647cad..c1df2de82d3d5cfcc93ca2460ec01899778b37e4 100644
--- a/src/components/Figures.tsx
+++ b/src/components/Figures.tsx
@@ -5,81 +5,100 @@ interface FigureProps{
pic4?: string,
pic5?: string,
pic6?: string,
+ alt1: string,
description: React.ReactNode | string,
num: string |number;
}
-export function ThreeVertical({description, num, pic1, pic2, pic3}:FigureProps){
+export function ThreeVertical({description, num, pic1, pic2, pic3, alt1}:FigureProps){
return(
- <figure>
- <img src={pic1}/>
- <img src={pic2}/>
- <img src={pic3}/>
- <figcaption><b>Figure {num}.</b> <span>{description}</span></figcaption>
- </figure>
+ <div className="figure-wrapper">
+ <figure>
+ <img src={pic1} alt={alt1} className="responsive-image"/>
+ <img src={pic2} className="responsive-image"/>
+ <img src={pic3} className="responsive-image"/>
+ <figcaption><b>Figure {num}.</b> <span>{description}</span></figcaption>
+ </figure>
+
+ </div>
)
}
-export function TwoVertical({description, num, pic1, pic2}:FigureProps){
+export function TwoVertical({description, num, pic1, pic2, alt1}:FigureProps){
return(
+ <div className="figure-wrapper">
+
<figure>
- <img src={pic1}/>
- <img src={pic2}/>
+ <img src={pic1} alt={alt1} className="responsive-image"/>
+ <img src={pic2} className="responsive-image"/>
<figcaption><b>Figure {num}.</b> <span>{description}</span></figcaption>
</figure>
+
+ </div>
+
)
}
-export function TwoHorizontal({description, num, pic1, pic2}:FigureProps){
+export function TwoHorizontal({description, num, pic1, pic2, alt1}:FigureProps){
return(
+ <div className="figure-wrapper">
+
<figure>
<div className="row align-items-center">
<div className="col">
- <img src={pic1}/>
+ <img src={pic1} alt={alt1} className="responsive-image"/>
</div>
<div className="col">
- <img src={pic2}/>
+ <img src={pic2} className="responsive-image"/>
</div>
</div>
<figcaption><b>Figure {num}.</b> <span>{description}</span></figcaption>
</figure>
+
+ </div>
+
)
}
-export function ThreeHorizontal({description, num, pic1, pic2, pic3}:FigureProps){
+export function ThreeHorizontal({description, num, pic1, pic2, pic3, alt1}:FigureProps){
return(
+ <div className="figure-wrapper">
<figure>
<div className="row align-items-center">
<div className="col">
- <img src={pic1}/>
+ <img src={pic1} alt={alt1} className="responsive-image"/>
</div>
<div className="col">
- <img src={pic2}/>
+ <img src={pic2}className="responsive-image"/>
</div>
<div className="col">
- <img src={pic3}/>
+ <img src={pic3} className="responsive-image"/>
</div>
</div>
<figcaption><b>Figure {num}.</b> <span>{description}</span></figcaption>
</figure>
+
+ </div>
+
)
}
-export function OneFigure({description, num, pic1}:FigureProps){
+export function OneFigure({description, num, pic1, alt1}:FigureProps){
return(
- <figure>
- <div className="row align-items-center">
- <div className="col">
- <img src={pic1}/>
- </div>
- </div>
+ <div className="figure-wrapper">
+
+ <figure>
+ <img src={pic1} alt={alt1} className="responsive-image"/>
<figcaption><b>Figure {num}.</b> <span>{description}</span></figcaption>
</figure>
+
+ </div>
+
)
}
diff --git a/src/components/Table.tsx b/src/components/Table.tsx
index 35cc61634aa5eeeb5b245220231ab92efbbce395..773251db2818c7521d9571078c1c143370227eea 100644
--- a/src/components/Table.tsx
+++ b/src/components/Table.tsx
@@ -21,7 +21,7 @@ export function PartTable({data, cols}: {data: Array<Part>, cols: Array<string>}
return(
<div className="flex flex-col">
<div className="min-w-full overflow-x-auto">
- <div className="inline-block min-w-full py-4">
+ <div className="inline-block min-w-full">
<div className="overflow-hidden p-2">
<table className="text-center parttable">
<thead className="bg-d">
@@ -62,7 +62,7 @@ export function ResTable({data, cols}: {data: Array<Res>, cols: Array<string>}){
return(
<div className="flex flex-col">
<div className="min-w-full overflow-x-auto">
- <div className="inline-block min-w-full py-4">
+ <div className="inline-block min-w-full">
<div className="overflow-hidden p-2">
<table className="text-center restable">
<thead className="bg-d">
diff --git a/src/contents/results.tsx b/src/contents/results.tsx
index 96f7ec7de7a29ac82f040989ec7e86ae27cc8477..a9cb0f43110994e3cf8249a2b0f1c2a36b088a04 100644
--- a/src/contents/results.tsx
+++ b/src/contents/results.tsx
@@ -42,6 +42,7 @@ export function Results() {
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={1}
pic1="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/bild1-1.png"
+ alt1="pZMB"
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"
/>
@@ -53,6 +54,7 @@ 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}
+ 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"
/>
@@ -63,6 +65,7 @@ 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}
+ alt1="pZMB and pDAS12124"
pic1="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/bild3-1.png"
pic2="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/bild3-2.png"
pic3="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/bild3-3.png"
@@ -74,6 +77,7 @@ 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}
+ 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"
/>
@@ -81,15 +85,17 @@ export function Results() {
<p>HEK cells were thawed and another prelimary test was conducted. In this test two different transfection agents were used (Lipofectamine 3000 & CaCl2) to check which one is better suited for our experiments. The literature uses lipofectamine 3000 but CaCl2 transfection is much cheaper.</p>
<H5 text="Conclusion"/>
<p>Both transfections are working out well but the efficiency of the lipofectamine transfection was much higher.</p>
- <div className="figure-wrapper">
- <figure>
- <img src="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/bild5.png" style={{height: "10%", width: "auto"}}/>
- <figcaption> <b>Figure 5.</b>Microscopy of HEK293 72h post transfection with lipofectamine 3000 with 1000 ng or 1500 ng technical positive control pZMB938. Both transfections show fluorescence signals.</figcaption>
- </figure>
- </div>
+ <OneFigure
+ pic1="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/bild5.png"
+ num={5}
+ 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"
+ />
+
<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}
+ 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"
/>
@@ -98,6 +104,7 @@ export function Results() {
<H5 text="Conclusion"/>
<p>The transfection efficiency was much better. Our proof-of-concept was working correctly. The reporter system pDAS12189 only led to production of a fluorescent signal when co transfected with a prime editing complex as pCMV-PE2.</p>
<TwoVertical
+ alt1=""
description="Microscopy of HEK293 72h post transfection with lipofectamine 2000. Transfection with technical positive control pZMB938, internal positive control pDAS12124-preedited, co-transfection of pDAS12489 with pCMV-PE2, NTC, PE2 as control and pDAS12489 as control. All controls are negative and both positve controls as well as pDAS12489+pCMV-PE2 show fluorescence signals."
num={7}
pic1="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/bild7-1.png"
@@ -114,19 +121,24 @@ export function Results() {
description="Flow Cytometry analysis of pegRNAs with and without silent edits."
num={8}
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"
- />
+ pic2="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/bild8.png" alt1={""} />
<H4 text="Screening of pegRNA variances"/>
<H5 text="Workflow"/>
<p>Cotransfection of pPEAR_CFTR and PE2 and also 1 of the 14 pegRNAs to compare the transfection efficiency of all of our designed pegRNAs.</p>
<H5 text="Conclusion"/>
- <p>The pegRNAs lead to differing amounts of cells showing fluorescence, which, assuming comparable transfection efficiencies, indicates varying prime editing efficiency. The pegRNA7 showed the highest transfection efficiency (see Figure 9).</p>
- <ResTable cols={headercols} data={resultdata}/>
- <div className="figure-wrapper">
- <figure>
- <img src="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/bild9.png" style={{height: "10%", width: "auto"}}/>
- <figcaption> <b>Figure 9.</b>Flow Cytometry results of all screened pegRNAs</figcaption>
- </figure>
+ <div className="row">
+ <div className="col">
+ <ResTable cols={headercols} data={resultdata}/>
+ </div>
+ <div className="col">
+ <p>The pegRNAs lead to differing amounts of cells showing fluorescence, which, assuming comparable transfection efficiencies, indicates varying prime editing efficiency. The pegRNA7 showed the highest transfection efficiency (see Figure 9).</p>
+ <OneFigure
+ pic1="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/bild9.png"
+ num={9}
+ alt1="Flow Cytometry results of all screened pegRNAs"
+ description="Flow Cytometry results of all screened pegRNAs"
+ />
+ </div>
</div>
<H4 text="Application lung epithelial cell lines"/>
<H5 text="Workflow"/>
@@ -134,6 +146,7 @@ export function Results() {
<H5 text="Conclusion"/>
<p>Transfection of CFBE41o- with pDAS12124-preedited was successful (see Figure 10). After 24 hours a successful co transfection of pPEAR_CFTR with PE6c and pegRNA4 was visible, although the transfection efficiency was really bad (see Figure 10).</p>
<TwoVertical
+ alt1=""
description="Microscopy results after 24h or 48h. Transfection of pDAS12124-preedited with lipofectamine 3000 was successfully done in CFBE41o- cell line and visible after 48h. CFBE41o- cell line was transfected with pDAS-IDT with Lipofectamine 3000 and afterwards with LNPs including PE6c and pegRNA4 and was after 24h fluorescence visible."
num={10}
pic1="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/bild10-1.png"
@@ -141,6 +154,7 @@ export function Results() {
/>
<p>Moreover transfection was conducted in human nasal epithelial cells (hNECs) in Air-liquid interface cultures as well as apical-out airway organoids (see Figure 11). No fluorescence was visible. </p>
<TwoVertical
+ alt1=""
description="Microscopy of HEK 72h post transfection with lipofectamine 3000. Co-transfection of pPEAR_CFTR with PE6c and pegRNA4. Both show no fluorescence signals."
num={11}
pic1="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/ali-tr.png"
@@ -152,12 +166,15 @@ export function Results() {
<H5 text="Conclusion"/>
<p>The Flow Cytometry results show that transfection with pCMV-PE2 as the prime editing complex had editing efficiency of 52.90% when normalized on pDAS12124-preedited. When pCMV-PE_CO-Mini was used as a prime editing complex it had a transfection efficiency of 2.54% (see Figure 11, 12).</p>
<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}
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"
/>
<OneFigure
+ alt1=""
pic1="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/pe2-pe-co.png"
num={12}
description="Flow Cytometry analysis to compare prime editing complexes PE2 and PE_CO-Mini"
@@ -166,14 +183,15 @@ export function Results() {
<p>We compared the 3 different Prime Editing complexes (pCMV-PE2, pCMV-PE2_CO-Mini & pCMV-PE6c) to check which one has the best transfection efficiency.</p>
<H5 text="Conclusion"/>
<p>The Flow Cytometry measurement shows the fluorescence rate cells co-transfected with pDAS12489 and pCMV-PE6c as a prime editing complex. The editing efficiency off PE6c was by far the highest (81.88%) (see Figure 13, 14). The efficiency was 1.55 higher than the efficiency when pCMV-PE2 was used as prime editing complex (see Figure 13).</p>
-
- <div className="figure-wrapper">
- <figure>
- <img src="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/bild13.png" style={{height: "10%", width: "auto"}}/>
- <figcaption> <b>Figure 13</b>Microscopy of HEK293 72h post transfection with lipofectamine 3000 and co transfection with pCMV-PE6c and pDAS12489.</figcaption>
- </figure>
- </div>
+ <OneFigure
+ pic1="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/bild13.png"
+ num={13}
+ 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."
+ />
+
<TwoHorizontal
+ alt1=""
description="Flow Cytometry results for evaluation of PE6c performance."
num={14}
pic1="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/pe2-pe-co-pe6c.png"
@@ -184,13 +202,12 @@ export function Results() {
<H4 text="RNA Synthesis"/>
<div className="row align-items-center">
<div className="col">
- <figure>
- <img src="https://static.igem.wiki/teams/5247/delivery/results/rna-gel-final.png"/>
- <figcaption>
- <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>
+ <OneFigure
+ num={16}
+ pic1="https://static.igem.wiki/teams/5247/delivery/results/rna-gel-final.png"
+ description="Gel of Denaturing RNA Gel Electrophoresis for mRNA synthesized from pcDNA 3.1 eYFP indicating successful RNA synthesis. Lane 1: Low Range Ribo Ruler, Lane 2: FLuc Control Template, Lane 3: Negative Control, Lane 4-9 mRNA from pcDNA 3.1 eYFP"
+ alt1="Gel of Denaturing RNA Gel Electrophoresis for mRNA synthesized from pcDNA 3.1 eYFP indicating successful RNA synthesis. Lane 1: Low Range Ribo Ruler, Lane 2: FLuc Control Template, Lane 3: Negative Control, Lane 4-9 mRNA from pcDNA 3.1 eYFP"
+ />
</div>
<div className="col">
<p>We began by synthesizing mRNA <i>in vitro</i> using a plasmid with a eYFP reporter from Addgene (pcDNA 3.1 eYFP) before proceeding with the synthesis of our approximately 6000 bp prime editing RNA. This was done to test the transfection efficiency and compatibility of our LNPs. The synthesis was successful, yielding an average of 1400 ng/µl of purified mRNA from 1 µg of plasmid DNA determined by Nanodrop measurement (data not shown). The size and integrity of the synthesized RNA were confirmed using a Denaturing RNA Gel, where we expected to see a product of 900 nucleotides. As anticipated, a strong and prominent band corresponding to this size was observed (Figure 16). This mRNA was subsequently used in further LNP formulations with RNA.</p>
@@ -202,50 +219,49 @@ export function Results() {
<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 reporter fluorescence. After consulting with expert <a onClick={() => goToPagesAndOpenTab('radukic', '/human-practices')}>Dr. Marco Radukic</a> and adjusting our LNP formulation and transfection protocols, specifically by pre-acidifying the OptiMEM medium, we were able to successfully assemble and transfect the LNPs. We also got from him Minicircle DNA from <a href="https://www.plasmidfactory.com/custom-dna/minicircle-dna/" title="PlasmidFactory" >PlasmidFactory</a> as a small plasmid carrying an eYFP gene and easy to transform, by that serving as a positive control in our experiments. Upon pipetting the components together, the solution immediately turned cloudy and bluish, indicating successful LNP formation (Figure 17).</p>
</div>
<div className="col">
- <figure>
- <img src="https://static.igem.wiki/teams/5247/delivery/results/caymanlnpblue.webp"/>
- <figcaption>
- <b>Figure 17. </b>
- Cayman LNP Formation indicated by blue color and turbidity. Mini DNA = Minicircle DNA from PlasmidFactory.
- </figcaption>
- </figure>
+ <OneFigure
+ description="Cayman LNP Formation indicated by blue color and turbidity. Mini DNA = Minicircle DNA from PlasmidFactory"
+ num={17}
+ pic1="https://static.igem.wiki/teams/5247/delivery/results/caymanlnpblue.webp"
+ alt1="Cayman LNP Formation indicated by blue color and turbidity. Mini DNA = Minicircle DNA from PlasmidFactory"
+ />
+
</div>
</div>
<H5 text="Transfection"/>
<p>To evaluate the efficiency of transfection, we performed fluorescence microscopy (Leica DMI6000 B at 20x magnification) on HEK293 cells transfected with LNP-formulated DNA and mRNA of pcDNA 3.1 eYFP, Minicircle DNA as the positive control and LNP without cargo as the negative control. Moreover, transfection via lipofectamine was tested for delivery method comparison.</p>
<p>Until 72h post-transfection, we observed in the conditions with lipofectamine alone or combined with RNA, no fluorescence, indicating unsuccessful transfection with RNA. Similarly, no fluorescence was seen in cells treated with LNPs alone or in combination with DNA or RNA. When LNPs were combined with Minicircle DNA or the cells were transfected with lipofectamine and Minicircle DNA, clear fluorescence was observed, indicating successful transfection and expression of our eYFP reporter under this condition (Figure 18). However, a strong background fluorescence from the OptiMEM medium was observed, complicating the analysis.</p>
<p>Overall, among all the tested conditions, the LNP formulation with Minicircle DNA was the only combination that resulted in noticeable fluorescence, suggesting it to be the most effective transfection method for HEK293 cells in this experiment.</p>
- <figure>
- <img src="https://static.igem.wiki/teams/5247/delivery/results/precyse/cayman.png"/>
- <figcaption>
- <b>Figure 18. </b>
- 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.
- </figcaption>
- </figure>
+ <OneFigure
+ pic1="https://static.igem.wiki/teams/5247/delivery/results/precyse/cayman.png"
+ num={18}
+ description="Overlay of phase contrast and fluorescence microscopic images of transfected HEK293 cells at 20x magnification 72h post-transfection with different Cayman LNP formulations recorded with Leica DMI6000 B. Lipo= lipofectamine, Mini DNA = Minicircle DNA"
+ alt1="Overlay of phase contrast and fluorescence microscopic images of transfected HEK293 cells at 20x magnification 72h post-transfection with different Cayman LNP formulations recorded with Leica DMI6000 B. Lipo= lipofectamine, Mini DNA = Minicircle DNA"
+ />
+
<H5 text="SEM"/>
<div className="row align-items-center">
<div className="col">
<p>Scanning Electron Microscopy (SEM) (Phenom ProX, Thermo Fisher) was employed by us to examine the morphology and surface characteristics of Cayman LNPs. The SEM images revealed that the LNPs displayed a generally spherical morphology with a relatively smooth surface (Figure 19). The average particle size was approximately 200 nm. However, a heterogeneous distribution of particle sizes was observed, with some larger, round structures present. These larger structures could potentially indicate aggregated LNPs.</p>
</div>
<div className="col">
- <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 19. </b>
- SEM image of Cayman LNPs (10,000x magnification) with Topography mode. </figcaption>
- </figure>
+ <OneFigure
+ pic1="https://static.igem.wiki/teams/5247/delivery/results/screenshot-2024-10-01-200629.png"
+ alt1="CayREM"
+ description="SEM image of Cayman LNPs (10,000x magnification) with Topography mode"
+ num={19}/>
</div>
</div>
<p>While many particles retained their structural integrity, the presence of these aggregates suggests that, under certain conditions, the LNPs may tend to cluster. It is important to note that for SEM analysis, the samples were dried and observed under vacuum, which probably have affected the structure and shape of the LNPs. This preparation process can introduce artifacts that would not typically be present in solution and should be considered when interpreting the results. Additionally, the contrast under vacuum conditions was too low to reliably distinguish the LNPs with sufficient detail. It provided a useful initial glimpse into the world of nanoparticles. Further complementary techniques will be needed for a more accurate and detailed characterization.</p>
<H4 text="Corden LNP"/>
<div className="row align-items-center">
<div className="col">
- <figure>
- <img src="https://static.igem.wiki/teams/5247/delivery/results/whatsapp-image-2024-09-24-at-12-57-59.jpeg"/>
- <figcaption>
- <b>Figure 20. </b>
- Turbidity after components of the Corden LNP have been pipetted together indicates particle formation. </figcaption>
- </figure>
+ <OneFigure
+ pic1="https://static.igem.wiki/teams/5247/delivery/results/whatsapp-image-2024-09-24-at-12-57-59.jpeg"
+ num={20}
+ description="Turbidity after components of the Corden LNP have been pipetted together indicates particle formation"
+ alt1="Turbidity after components of the Corden LNP have been pipetted together indicates particle formation"
+ />
</div>
<div className="col">
<p>During the preparation of the LNPs, the solution became turbid and bluish, indicating successful nanoparticle formation (Figure 20). This was further confirmed by cryo-EM (cryogenic electron microscopy) analysis, which revealed the presence of well-formed LNPs. Despite the successful formation of LNPs, no detectable fluorescence was observed in the cells treated with LNPs containing pcDNA 3.1 eYFP DNA or mRNA at any of the measured time points, indicating that transfection did not occur under these conditions.</p>
@@ -254,24 +270,22 @@ export function Results() {
<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 24h, 48h, and 72h post-transfection.</p>
<p>Retrospectively, after 72h none of the LNP-treated samples showed significant fluorescence, indicating a failure in transfection (Figure 21). The lack of fluorescence in all experimental groups, except lipofectamine and Minicircle DNA, suggests either insufficient uptake of the LNPs by the cells or a failure in expression of the YFP reporter, indicating that the Corden LNP may not suited as our delivery system. Also the deformed morphology and decreased growth are indicators for negative effects of the Cayman LNP on HEK293, probably reasoned in the employment of more cytotoxic mPEG-DSPE compared to DMG-PEG in the Cayman and SORT LNP.</p>
- <figure>
- <img src="https://static.igem.wiki/teams/5247/delivery/results/precyse/corden.png"/>
- <figcaption>
- <b>Figure 21. </b>
- 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.
- </figcaption>
- </figure>
+ <OneFigure
+ pic1="https://static.igem.wiki/teams/5247/delivery/results/precyse/corden.png"
+ num={21}
+ description="Overlay of phase contrast and fluorescence microscopic images of transfected HEK293 cells at 20x magnification 72h post-transfection with different Corden LNP formulations recorded with Leica DMI6000 B. For lipofectamine (lipo) + Minicircle DNA (Mini DNA) only the fluorescence image is shown."
+ alt1="Overlay of phase contrast and fluorescence microscopic images of transfected HEK293 cells"
+ />
<H5 text="Cryo-EM"/>
<p>Cryo-EM as a form of transmission electron microscopy (TEM) was performed by us using a JEOL JEM-2200FS electron microscope (JEOL, Freising, Germany) operating at 200kV, equipped with a cold field emission electron gun. The sample preparation and imaging were carried out at cryogenic temperatures, which allowed for the visualization of LNPs in their native hydrated state.</p>
<div className="row align-items-center">
<div className="col">
- <figure>
- <img src="https://static.igem.wiki/teams/5247/delivery/results/corden-lnp.jpg"/>
- <figcaption>
- <b>Figure 22. </b>
- Cryo-EM image of Corden LNPs.
- </figcaption>
- </figure>
+ <OneFigure
+ pic1="https://static.igem.wiki/teams/5247/delivery/results/corden-lnp.jpg"
+ num={22}
+ description="Cryo-EM image of Corden LNPs."
+ alt1="Cryo-EM image of Corden LNPs"
+/>
</div>
<div className="col">
<p>The images reveal the presence of spherical LNP structures with an approximate size of 100 nm (Figure 22). The LNPs appear well-formed, with uniform morphology, indicating successful nanoparticle formation. In addition to individual particles, some larger, round structures were also observed, which could represent aggregated LNPs. These aggregations are a common phenomenon in LNP systems and could be attributed to interactions between particles under certain conditions.</p>
@@ -281,25 +295,23 @@ export function Results() {
<H4 text="SORT LNP"/>
<H5 text="Transfection"/>
<p>Already at 48h of 72 monitored hours post-transfection of HEK293 with the SORT LNP, clear and strong fluorescence was already observed when using LNPs combined with Minicircle DNA, as well as in the lipofectamine and Minicircle DNA condition, confirming successful transfection and robust expression of the eYFP reporter (Figure 23). This early detection of fluorescence highlights the efficiency of SORT LNPs as delivery system. In contrast, no fluorescence was detected in any conditions involving lipofectamine alone, lipofectamine with RNA, or LNPs combined with DNA or RNA, further emphasizing the superior performance of the Minicircle DNA formulations and highlighting the need for improvement of cargo stabilization and our cargo choice.</p>
- <figure>
- <img src="https://static.igem.wiki/teams/5247/delivery/results/precyse/sort.png"/>
- <figcaption>
- <b>Figure 23. </b>
- 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.
- </figcaption>
- </figure>
+ <OneFigure
+ pic1="https://static.igem.wiki/teams/5247/delivery/results/precyse/sort.png"
+ num={23}
+ description="Overlay of phase contrast and fluorescence microscopic images of transfected HEK293 cells at 20x magnification 48h post-transfection with different SORT LNP formulations recorded with Leica DMI6000 B. Lipo = lipofectamine, Mini DNA = Minicircle DNA."
+ alt1="Overlay of phase contrast and fluorescence microscopic images of transfected HEK293 cells"
+/>
<p>Notably, the SORT LNP formulation with Minicircle DNA showed significant transfection efficiency within 48 hours, making it the most effective delivery method tested among all three LNPs. This early and robust expression demonstrates the clear advantage of this approach for HEK293 cells.</p>
<H5 text="Flow Cytometry"/>
<div className="row align-items-center">
<p>We performed flow cytometry analysis 72h post-transfection to evaluate the transfection efficiency of the SORT LNP in HEK293. The relative percentage of fluorescent cells was determined by measuring the percentage of FITC-A+ cells, followed by normalization to the negative control and fold change calculation.</p>
<div className="col">
- <figure>
- <img src="https://static.igem.wiki/teams/5247/delivery/results/sortlnp-facs.png" alt="SORTFACS" style={{maxHeight: "200pt"}}/>
- <figcaption>
- <b>Figure 24. </b>
- 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.
- </figcaption>
- </figure>
+ <OneFigure
+ pic1="https://static.igem.wiki/teams/5247/delivery/results/sortlnp-facs.png"
+ num={24}
+ description="Percentage of fluorescent cells (FITC-A+) performed 72h post-transfection of SORT LNP in HEK293. Mean +/- SEM for n=3. For statistics one-way ANOVA was performed."
+ alt1="Percentage of fluorescent cells post-transfection of SORT LNP in HEK293"
+/>
</div>
<div className="col">
<p>The SORT LNP-transfected sample carrying Minicircle DNA exhibited a significant increase in fluorescence compared to the lipofectamine transfection of Minicircle DNA, with approximately 14 times more fluorescent cells compared to the lipofectamine-transfected sample (Figure 24). This substantial difference indicates that the transfection efficiency with LNPs is markedly higher than with lipofectamine, demonstrating the superior performance of our LNP formulation in delivering nucleic acids to HEK cells.</p>
@@ -311,23 +323,22 @@ export function Results() {
<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 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>
+ <OneFigure
+ pic1="https://static.igem.wiki/teams/5247/delivery/results/sort-zeta.webp"
+ num={25}
+ description="Zeta potential of SORT LNP with different cargos measured with Nanotrack Wave II indicating varying degrees of stability but most important good stability for the SORT LNP loaded with pcDNA 3.1 eYFP (LNP DNA). Mean +/- SEM for n=5. For statistics one-way ANOVA was performed."
+ alt1="Zeta potential of SORT LNP with different cargos"
+/>
</div>
</div>
<div className="row align-items-center">
<div className="col">
- <figure>
- <img src="https://static.igem.wiki/teams/5247/delivery/results/screenshot-2024-10-01-204938.png"/>
- <figcaption>
- <b>Figure 26. </b>
- Size distribution for the SORT LNP with different cargos weighted by scattering intensity measured with Nanotrack Wave II.
- </figcaption>
- </figure>
+ <OneFigure
+ pic1="https://static.igem.wiki/teams/5247/delivery/results/screenshot-2024-10-01-204938.png"
+ num={26}
+ description="Size distribution for the SORT LNP with different cargos weighted by scattering intensity measured with Nanotrack Wave II."
+ alt1="Size distribution for the SORT LNP with different cargos"
+/>
</div>
<div className="col">
<p>The size distribution for all three samples shows a predominantly monomodal, yet broad, distribution with diameters ranging between 50 nm and 700 nm, with the peak of the distribution lying between 150 nm and 200 nm (Figure 26). SORT LNPs without DNA exhibited larger radii, with a peak around 300 nm. The SORT LNP containing Minicircle DNA suggests the presence of larger aggregates with diameters exceeding 1 µm. The likely reason for this variable particle size distribution, despite loading with different types of DNA, could be attributed to the manufacturing method. Since the LNPs were not produced using an extruder but rather via dialysis, this is highly plausible.</p>
@@ -339,13 +350,12 @@ export function Results() {
<p>Cryo-EM analysis was also performed of SORT LNPs by us with the same JEOL JEM-2200FS microscope at 200kV, allowing visualization of LNPs in their native hydrated state. The images show spherical LNP structures around 100 nm, with some larger aggregates also present. These aggregates likely result from interactions between particles due to the non-extrusion-based preparation method, which may explain the variability in particle size. Additionally, particles potentially representing different LNP populations or overlapped structures with low contrast were observed (Figure 27). The low sample concentration likely contributed to the limited number of visible particles.</p>
</div>
<div className="col">
- <figure>
- <img src="https://static.igem.wiki/teams/5247/delivery/results/sortcryoem.png"/>
- <figcaption>
- <b>Figure 27. </b>
- Cryo-EM image of SORT LNPs. The different colored outlines indicate different size populations of LNPs.
- </figcaption>
- </figure>
+ <OneFigure
+ pic1="https://static.igem.wiki/teams/5247/delivery/results/sortcryoem.png"
+ num={27}
+ description="Cryo-EM image of SORT LNPs. The different colored outlines indicate different size populations of LNPs."
+ alt1="Cryo-EM image of SORT LNPs with different size populations"
+/>
</div>
</div>
<p>Overall, while the Cryo-EM data confirm the presence and general morphology of LNPs that also fall within the diameter range specified by Wang et al. for SORT LNPs at smaller than 200 nm [1]. The variability in size and the presence of aggregates highlight potential areas for optimization, such as refining sample concentration and preparation methods to achieve more consistent particle formation.</p>
@@ -353,13 +363,14 @@ export function Results() {
<p>We used Dynamic Light Scattering (DLS) to assess the size distribution of our SORT LNPs by measuring the fluctuations in scattered light due to particle motion. The hydrodynamic diameter was calculated using the Stokes-Einstein equation, considering the diffusion coefficient, temperature, and viscosity of the medium.</p>
<div className="row align-items-center">
<div className="col">
- <figure>
- <img src="https://static.igem.wiki/teams/5247/delivery/results/sort-dls.webp"/>
- <figcaption>
- <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>
+
+<OneFigure
+ pic1="https://static.igem.wiki/teams/5247/delivery/results/sort-dls.webp"
+ num={28}
+ description="Results for hydrodynamic radius determination by DLS Measurements for our SORT LNP, indicating a radius of approximately 100 nm."
+ alt1="Hydrodynamic radius of SORT LNP measured by DLS"
+/>
+
</div>
<div className="col">
<p>The results showed a hydrodynamic diameter of SORT LNPs yielding an average radius of approximately 100 nm (Figure 28). These findings are consistent with our previous applied size determination methods, such as Zeta potential and Cryo-EM, which also indicated similar particle dimensions in appropriate range for our research and medical applications.</p>
@@ -368,12 +379,12 @@ export function Results() {
<H5 text="MTT Assay"/>
<div className="row align-items-center">
<div className="col">
- <figure>
- <img src="https://static.igem.wiki/teams/5247/fanzor/sort-mtt.webp"/>
- <figcaption>
- <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>
+ <OneFigure
+ pic1="https://static.igem.wiki/teams/5247/fanzor/sort-mtt.webp"
+ num={29}
+ description="MTT Assay of LNPs from all iterations performed on HEK293 including Triton as negative control and untreated cells as positive control. Mean +/- SEM for n=6. For statistics one-way ANOVA was performed."
+ alt1="MTT Assay results of LNPs on HEK293 cells"
+/>
</div>
<div className="col">
{/* <p>In order to evaluate the <a onClick={() => goToPageAndScroll ('Biosafety2', '/safety')}>biosafety</a> of our lung-specific LNPs, particularly concerning the choice of <a onClick={() => goToPagesAndOpenTab({collapseId: 'Col1', path: '/engineering', tabId: 'delivery' })}>PEG</a> - known to cause cytotoxicity issues - we performed MTT assays using HEK293 cells with various LNP formulations. The results demonstrated that the Cayman LNP achieved 74.90% viability and SORT LNP showed 75.01% viability, exhibiting lower cytotoxicity due to the inclusion of DMG-PEG, a less cytotoxic PEG variant compared to mPEG-2000-DSPE, which resulted in 66.69% viability in the Corden LNP (Figure t). These findings prove we made the best decision by choosing the SORT LNP as the least cytotoxic LNPs.</p> */}
@@ -389,13 +400,12 @@ export function Results() {
<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 24h post-transfection recorded with Leica DMI6000 B.
- </figcaption>
- </figure>
+ <OneFigure
+ pic1="https://static.igem.wiki/teams/5247/delivery/results/precyse/lnp-chito-rna500-t24.webp"
+ num={30}
+ description="Overlay of phase contrast and fluorescence microscopic images of with SORT + chitosan 500 transfected CFBE41o- cells at 20x magnification 24h post-transfection recorded with Leica DMI6000 B."
+ alt1="Images of SORT + chitosan 500 transfected CFBE41o- cells"
+/>
</div>
<div className="col">
<p>The sample containing 500 ng/µl of chitosan and pcDNA 3.1 eYFP mRNA, encapsulated within SORT LNPs, exhibited fluorescence 24 hours post-transfection (Figure 30). This result indicates that the RNA was successfully delivered into the CFBE41o- cells, where it was transcribed and translated into the YFP protein. The presence of YFP fluorescence confirms not only successful transfection but also robust expression of the reporter gene.</p>
@@ -406,45 +416,42 @@ export function Results() {
<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 24h post-transfection recorded with Leica DMI6000 B.
- </figcaption>
- </figure>
+ <OneFigure
+ pic1="https://static.igem.wiki/teams/5247/delivery/results/precyse/lnpchito-rna50-t24.webp"
+ num={31}
+ description="Overlay of phase contrast and fluorescence microscopic images of with SORT + chitosan 50 transfected CFBE41o- cells at 20x magnification 24h post-transfection recorded with Leica DMI6000 B."
+ alt1="Images of SORT + chitosan 50 transfected CFBE41o- cells"
+/>
</div>
</div>
<p>The samples containing only chitosan (Figure 32) and only SORT LNP (Figure 33) without any mRNA, did not exhibit any detectable fluorescence. This result is consistent with expectations, as chitosan alone and SORT LNP alone are not fluorescent. These samples served as important negative controls to confirm that the chitosan itself and the SORT LNP itself don't interfere with the fluorescence signal.</p>
<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 24h post-transfection recorded with Leica DMI6000 B.
- </figcaption>
- </figure>
+ <OneFigure
+ pic1="https://static.igem.wiki/teams/5247/delivery/results/precyse/chito-t24-1.jpeg"
+ num={32}
+ description="Overlay of phase contrast and fluorescence microscopic images of with SORT transfected CFBE41o- cells at 20x magnification 24h post-transfection recorded with Leica DMI6000 B."
+ alt1="Images of SORT transfected CFBE41o- cells"
+/>
</div>
<div className="col">
- <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 24h post-transfection recorded with Leica DMI6000 B.
- </figcaption>
- </figure>
+ <OneFigure
+ pic1="https://static.igem.wiki/teams/5247/delivery/results/precyse/lnp-leer-t24.webp"
+ num={33}
+ description="Overlay of phase contrast and fluorescence microscopic images of with chitosan transfected CFBE41o- cells at 20x magnification 24h post-transfection recorded with Leica DMI6000 B."
+ alt1="Images of chitosan transfected CFBE41o- cells"
+/>
</div>
</div>
<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 24h post-transfection recorded with Leica DMI6000 B.
- </figcaption>
- </figure>
+
+<OneFigure
+ pic1="https://static.igem.wiki/teams/5247/delivery/results/precyse/ntc-t24.webp"
+ num={34}
+ description="Overlay of phase contrast and fluorescence microscopic images of untransfected CFBE41o- cells at 20x magnification 24h post-transfection recorded with Leica DMI6000 B."
+ alt1="Images of untransfected CFBE41o- cells"
+/>
</div>
<div className="col">
<p>The untreated control, which did not receive any chitosan, RNA, or LNPs, did not show any fluorescence (Figure 34). This negative control confirms that the cells themselves do not express YFP under normal conditions, and that any observed fluorescence in the experimental groups is directly attributable to the transfection and expression of the delivered RNA.</p>
@@ -467,13 +474,12 @@ export function Results() {
<H4 text="Initial Measurements"/>
<div className='row align-items-center'>
<div className='col'>
- <figure>
- <img src="https://static.igem.wiki/teams/5247/photos/results/patchclamp/pc.webp" alt="PC" style={{maxHeight: "300pt"}}/>
- <figcaption>
- <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>
+ <OneFigure
+ pic1="https://static.igem.wiki/teams/5247/photos/results/patchclamp/pc.webp"
+ num={35}
+ description="Current density of HEK293, HEK293T CFTR WT and HEK293T CFTR F508del showing significant differences of both HEK293T cell lines compared to HEK293 but no significant differences between them. For statistics one-way ANOVA was performed."
+ alt1="Current density differences between HEK293 and HEK293T CFTR cell lines"
+/>
</div>
<div className='col'>
<p>In our first set of experiments, we measured current density in <a onClick={() => goToPageAndScroll ('Cell Culture', '/materials-methods')}>HEK293T CFTR wild type (WT) and HEK293T F508del</a> cell lines, comparing them with regular HEK293. The results demonstrated significant differences in chloride ion conductance, with the CFTR-expressing cell lines showing enhanced conductivity compared to HEK293 (Figure 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[2], impairing chloride ion transport through the CFTR channel.</p>
@@ -485,13 +491,19 @@ export function Results() {
<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 36. </b>
- 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.
- </figcaption>
- </figure>
+ <OneFigure
+ alt1=""
+ pic1="https://static.igem.wiki/teams/5247/photos/results/patchclamp/pc2.webp"
+ num={36}
+ 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"
+ />
+ <OneFigure
+ pic1=""
+ alt1="PC1"
+ num={37}
+ description=""
+ />
</div>
</div>
<H4 text="Next Steps"/>