diff --git a/static/style.css b/static/style.css
index e86c4a5b18f5cfaadba3fb76a70d42146c381cf2..1c3ad5f4a22e98a2cbf7691a311168087872cacb 100644
--- a/static/style.css
+++ b/static/style.css
@@ -92,7 +92,7 @@ h2 {
}
:root {
- cursor: url('https://static.igem.wiki/teams/4764/wiki/miscellaneous/cursors/carp-cursor.png'), auto;
+ cursor: url('https://static.igem.wiki/teams/4764/wiki/miscellaneous/cursors/megayumcursor.png'), auto;
}
diff --git a/wiki/pages/contribution.html b/wiki/pages/contribution.html
index 00d43e30cce1bd96bf413903d756ecff93236914..07d1fdc2401f12daf746d2b566522a10a6a08418 100644
--- a/wiki/pages/contribution.html
+++ b/wiki/pages/contribution.html
@@ -6,10 +6,10 @@
<div class="bd-callout bd-callout-info">
<h1>A toolkit to facilitate chimeric antigen receptor assembly</h1>
</div>
-<p>While designing our chimeric antigen receptors (CARs), we noticed that CARs contain an inherently
-modular structure: every CAR requires an extracellular domain to recognize a target,
-a transmembrane domain to transduce signal into the cell, and an intracellular domain to
-induce some reaction by the cell. This realization inspired us to design CAR-TK, a
+<p>While designing our chimeric antigen receptors (CARs), we noticed that CARs are inherently
+modular in structure: every CAR requires an extracellular domain to recognize a target,
+a transmembrane domain to transduce the signal into the cell, and an intracellular domain to
+induce some response by the cell. This realization inspired us to design CAR-TK, a
chimeric antigen receptor toolkit that facilitates modular assembly of CARs.
Learn more on our <a href="{{ url_for('pages', page='engineering') }}" target="_blank">engineering page</a>
</p>
@@ -23,7 +23,7 @@ Learn more on our <a href="{{ url_for('pages', page='engineering') }}" target="_
and hardware for future teams and researchers to construct their own end-over-end spinner.
<br>
<br>
- <p>Below is a video demonstrating how our rotator functions in practice. </p>
+ <p>Below is a video demonstrating how our rotator functions in practice. <strong>Warning that the audio may be loud!</strong></p>
<iframe style="margin-left:auto; margin-right:auto" title="MIT: End-over-end microcentrifuge tube rotating hardware (2023) [English]" width="800" height="387" src="https://video.igem.org/videos/embed/6b0f67ed-c568-4b42-8138-bd4c99896661" frameborder="0" allowfullscreen="" sandbox="allow-same-origin allow-scripts allow-popups"></iframe>
<br>
<br>
diff --git a/wiki/pages/description.html b/wiki/pages/description.html
index ec82caf9f4cc9da86bc4ab9ef8c02abae18d6cac..4225960c1a995c46a96fd7660a5d2bc796cc2c40 100644
--- a/wiki/pages/description.html
+++ b/wiki/pages/description.html
@@ -118,7 +118,7 @@
<ul>
<li><strong>Extracellular receptor:</strong></li> Anti-IL-6 antibody single chain variable fragment, to bind IL-6<sup><a href="#cite4">4</a></sup>
<li><strong>Transmembrane signal transduction and stability domain:</strong></li> CD8, known to transduce signal and increase CAR stability<sup><a href="#cite5">5</a></sup>
- <li><strong>Intracellular signaling domain:</strong></li> Either Fcgamma or Megf10, known to induce phagocytosis<sup><a href="#cite3">3,</a></sup><sup><a href="#cite6">6</a></sup>
+ <li><strong>Intracellular signaling domain:</strong></li> Either Fcγ or Megf10, known to induce phagocytosis<sup><a href="#cite3">3,</a></sup><sup><a href="#cite6">6</a></sup>
<li><strong>Fluorescent tagging:</strong></li> eGFP, for visualization and for easy folding of the fusion protein
</ul>
</div>
diff --git a/wiki/pages/education.html b/wiki/pages/education.html
index 0a0f8738faad23a7d984860d6b7adcd60a27e73c..effcca78e732880374740ccc59476bd7ae99ee3f 100644
--- a/wiki/pages/education.html
+++ b/wiki/pages/education.html
@@ -20,12 +20,7 @@ Our team has explored multiple avenues of educating the global community, includ
<div class="bd-callout bd-callout-info">
<h1>Summer camp collaboration with Nazarbayev University</h1>
</div>
-<p>In July, the iGEM team at Nazarbayev University was kind enough to reach out to us seeking collaboration for their
- wonderful Summer Camp. NU organized a set of online synthetic biology lectures with the goal
- of educating high school students around the world on the applications of synthetic biology, in order to inspire young, aspiring scientists
- to pursue their goals! They
- provided us with an opportunity to record a lecture for their attendees in which we explained the background and importance of cancer cachexia, along with how our
- therapy uses synthetic biology to tackle the condition. You can view our lecture <a href="https://www.youtube.com/watch?v=WxaT0Im_T1A" target="_blank">here</a>.
+<p>In July, the iGEM team at Nazarbayev University was kind enough to reach out to us seeking collaboration for their wonderful Summer Camp. NU organized a hybrid camp in three languages with the goal of educating local grade school students on the applications of synthetic biology, especially in the oncology realm, in order to inspire young, aspiring scientists to pursue their goals! They provided us with an opportunity to record a lecture for their attendees in which we explained the background and importance of cancer cachexia, along with how our therapy uses synthetic biology to tackle the condition. You can view our lecture <a href="https://www.youtube.com/watch?v=WxaT0Im_T1A" target="_blank">here</a>.
</p>
<div class="bd-callout bd-callout-info">
@@ -49,7 +44,7 @@ Our team has explored multiple avenues of educating the global community, includ
<h2>Cell passaging</h2>
<p class="learn-more">Allison walks us through the passaging protocol in <a href="https://www.youtube.com/watch?v=yEPGskCDgF4" target="_blank">this tutorial</a>. </p>
-<h2>Miniprepping (STILL EDITING but we will remember to update this, if you see this mentors :D )</h2>
-<p class="learn-more">Katie and Allison demonstrate how to extract plasmid DNA from bacteria via miniprep in <a href="https://www.youtube.com/watch?v=yEPGskCDgF4" target="_blank">this tutorial</a>. </p>
+<h2>Extraction of plasmids from bacteria (miniprep) </h2>
+<p class="learn-more">Katie and Allison demonstrate how to extract plasmid DNA from bacteria via miniprep in <a href="https://www.youtube.com/watch?v=r0EhrlEW7QE" target="_blank">this tutorial</a>. </p>
{% endblock %}
diff --git a/wiki/pages/engineering.html b/wiki/pages/engineering.html
index 5c48d1c5934cf78f392ed7d6f9f256124bb69c81..6284c80b5d99dae96dc098ee1ff51f8a0a1c5e91 100644
--- a/wiki/pages/engineering.html
+++ b/wiki/pages/engineering.html
@@ -29,7 +29,7 @@
</p>
<h3>Second iteration</h3>
Having ruled out photoconversion, we moved on to the second round of this cycle by researching optimal CREs for transfection of HEK293 cells.
- Literature review led us to select
+ Literature review led us to select FINISH THISSS
</p>
diff --git a/wiki/pages/home.html b/wiki/pages/home.html
index 30594af754a67c77eb0e59d57c56e62caf870580..dbcdb46b6d27990a14ea6a6bbd7aa70d7cce09c8 100644
--- a/wiki/pages/home.html
+++ b/wiki/pages/home.html
@@ -82,7 +82,9 @@
health, fight cancer, and seize the day!
</h2>
</div>
-
+<br>
+<p style="margin:0 auto 10px auto">To quickly learn more about our project, view our project promotion video below.</p>
+<iframe style="margin:auto" title="MIT: CAR-P Diem: Seize the day! (2023) - Project Promotion [English]" width="800" height="450" src="https://video.igem.org/videos/embed/6fef497f-dd98-4717-bee8-710a642ddcb8" frameborder="0" allowfullscreen="" sandbox="allow-same-origin allow-scripts allow-popups"></iframe>
<br>
diff --git a/wiki/pages/results.html b/wiki/pages/results.html
index d3279dd9129f3cd3b0a22a9953e19b0bc68f0206..998e9f11b6613ec7d84505b60947233335493917 100644
--- a/wiki/pages/results.html
+++ b/wiki/pages/results.html
@@ -29,7 +29,7 @@ Our CAR plasmids have the following components linked together with glycine/seri
<ul>
<li>extracellular: anti-Il-6 receptor</li>
<li>transmembrane: CD8</li>
- <li>intracellular: Fcgamma vs. Megf10</li>
+ <li>intracellular: Fcγ vs. Megf10</li>
<li>reporter: eGFP</li>
</ul>
The extracellular component was taken directly from sirukumab, a monoclonal antibody for IL-6.
@@ -41,7 +41,7 @@ The transmembrane and intracellular components were inspired by Morrissey et al.
<div id="moclo-info">
After receiving our plasmids from IDT, we cloned them into a plasmid backbone (YTK001) provided by the Yeast Toolkit (YTK). The other components of the plasmid were directly taken from the YTK. Our designs are noted on our lab notebook.
<br><br>
-After verification of the plasmids via diagnostic digests, we cloned them again to include mammalian cis-regulatory elements and to form a complete level 1 transcriptional unit plasmid. We initially started with the SV40 promoter and SV40 3’ UTR, but saw nearly 0 transfection efficiency, meaning that extremely few cells per well in our 12 well plate successfully transfected and fluoresced. However, we also ordered and used the CD22-Megf10 plasmid from Morrissey et al. as a positive control; we saw that the CD22-Megf10 plasmid had extremely high transfection efficiency, so we thought to compare our existing promoter and 3’UTR to those used by Morrissey et al. Through many iterations of cloning, detailed on our engineering page, we finally ended on using the pEF1alpha promoter and SV40 3’UTR.
+After verification of the plasmids via diagnostic digests, we cloned them again to include mammalian cis-regulatory elements and to form a complete level 1 transcriptional unit plasmid. We initially started with the SV40 promoter and SV40 3’ UTR, but saw nearly 0 transfection efficiency, meaning that extremely few cells per well in our 12 well plate successfully transfected and fluoresced. However, we also ordered and used the CD22-Megf10 plasmid from Morrissey et al. as a positive control; we saw that the CD22-Megf10 plasmid had extremely high transfection efficiency, so we thought to compare our existing promoter and 3’UTR to those used by Morrissey et al. Through many iterations of cloning, detailed on our engineering page, we finally ended on using the pEF1α promoter and SV40 3’UTR.
</div>
<div class="bd-callout bd-callout-info">
@@ -64,9 +64,7 @@ Our initial plan at the start of the summer was to use HEK293 cells to produce o
<p style='display: inline'>
As detailed in our engineering page and the modular cloning section of this page,
we initially struggled to achieve any transfection efficiency. Representative
- photos of our first attempts are to the right. <br>
- <em>Left: 3xFLAG-IL6-mCherry <br>
- Right: anti-IL6 Megf10 CAR</em>
+ photos of our first attempts are to the right.
</p>
</div>
<div class="col-6">
@@ -80,19 +78,24 @@ Our initial plan at the start of the summer was to use HEK293 cells to produce o
<p style='display: inline'>
After our many iterations of plasmid construction, we ended up seeing substantial
fluorescence from all three of our constructs! Representative
- photos from transfection of our finalized designs are to the right. <br>
- <em>Left: 3xFLAG-IL6-mCherry <br>
- Right: anti-IL6 Megf10 CAR</em>
+ photos from transfection of our finalized designs are to the right.
</p>
</div>
<div class="col-6">
<img src="https://static.igem.wiki/teams/4764/wiki/flow/red-good.png" width="49%">
<img src="https://static.igem.wiki/teams/4764/wiki/flow/green-good.png" width="49%">
+ <div>
+ <p style="margin:auto; text-shadow: 1px 3px 3px #fd0db04a;"><em><strong>Figure 1: Transfection failures and successes.<br></strong></em> </p>
+ <em><strong>Top row: first attempt at transfecting 3xFLAG-IL6-mCherry (left) and anti-IL6 Megf10 CAR (right).</strong></em>
+ <br><em><strong>Bottom row: finalized transfection of 3xFLAG-IL6-mCherry (left) and anti-IL6 Megf10 CAR (right).</strong></em>
+ <br><br>
+ </div>
+
</div>
</div>
</div>
-<h4 id="flow-button" class="learn-more dropdown-toggle">Analysis of flow cytometry data</h4>
+<h4 id="flow-button" class="learn-more dropdown-toggle">Flow cytometry data</h4>
<div id="flow-info">
<p>
Ultimately, we have successfully achieved a proof-of-concept for the IL-6 binding component of our therapeutic
@@ -101,7 +104,7 @@ Our initial plan at the start of the summer was to use HEK293 cells to produce o
It took a few attempts to run everything smoothly, but our final protocol followed a precise timeline that allowed us to see encouraging results!
<br><br>
<em>Day 0:</em> We transfected a 10cm dish of HEK293 cells with our 3xFLAG-IL6-mCherry fusion protein. mCherry control wells were transfected too.<br>
- <em>Day 1:</em> We transfected a 12-well plate of HEK293 cells, half of the wells with our anti-IL6 Fcgamma CAR and half with our anti-IL6 Megf10 CAR. GFP control wells were transfected too.<br>
+ <em>Day 1:</em> We transfected a 12-well plate of HEK293 cells, half of the wells with our anti-IL6 Fcγ CAR and half with our anti-IL6 Megf10 CAR. GFP control wells were transfected too.<br>
<em>Day 2:</em> We extracted the cells of the IL6-transfected dish using a cell scraper and processed the cells in 2 different ways.
<ol>
<li>Scraped cells were lysed according to a freeze-thaw protocol (detailed on our experiments page) then co-cultured with wells containing CAR-expressing cells (detailed in our lab notebook).</li>
@@ -113,35 +116,36 @@ Our initial plan at the start of the summer was to use HEK293 cells to produce o
our CAR plus IL-6 experimental cells.
<br><br>
Due to our team's inexperience running flow cytometry, we struggled with establishing appropriate voltages and determining settings for parameters such as forward scattering and back scattering.
- Our post-flow analysis using the software FlowJo was challenging because of this, and we had to plot data with unconcentional methods such as switching from
- linear to logarithmic axes to ensure all of our data points fit on graphs. However, with the help of mentors with much more flow experience than us—Noor Radde, in particular—we were able to
+ Our post-flow analysis using the software FlowJo was challenging because of this, and we had to plot data with unconventional methods such as switching from
+ linear to logarithmic axes to ensure all of our data points fit on graphs. However, with the help of mentors with much more flow experience than us—Hayden Sandt and Noor Radde, in particular—we were able to
effectively and objectively re-format our data to better match academic standards.
<br><br>
- We then went about gating our data to determine cell populations, based on whether they fluoresced green (i.e., expressed CARs) and/or whether they fluoresced red (i.e., bound to IL6-mCherry). For full
- transparency, our backgating data and analysis is presented below.
+ We then gated our data to determine cell populations based on whether they fluoresced green (i.e., expressed CARs) and/or whether they fluoresced red (i.e., bound to IL6-mCherry). For full
+ transparency, our backgating data and analysis is presented below. In each figure below,
+ the left graph illustrates gating for HEK293 cells; the middle graph illustrates
+ the subset of cells that are GFP positive (CAR+); and the right graph
+ illustrates the subset of CAR+ cells that are mCherry positive (CAR+IL6).
</p>
<div class="row">
- <div class="col-8">
- <img src="https://static.igem.wiki/teams/4764/wiki/flow/backgating-negative-control.png" width="100%">
+ <div class="col-lg-8 col-sm-4">
+ <img src="https://static.igem.wiki/teams/4764/wiki/flow/backgating-negative-control1.png" width="100%">
+ <p style="margin:auto; text-shadow: 1px 3px 3px #fd0db04a;"><em><strong>Figure 2: Negative control backgating.</strong></em></p> <br><br>
</div>
- <div class="col-4">
+ <div class="col-lg-4 col-sm-8">
<p class="class="col-6"">
- First, we used negative control cells to separate cell debris from actual HEK293 cells. This was performed under the assumption that debris is significantly smaller in size than actual HEK293 cells.
- Retroactively, we calculated green and red fluorescence of these populations to verify correct gating; as expected, no green or red fluorescence was observed in negative control cells.
- </p>
+ First, we used negative control cells to separate cell debris from actual HEK293 cells in our data. This was performed under the assumption that debris is significantly smaller in size than actual HEK293 cells. Thus, we strictly gated for the area around the hotspot and above, but discarded the area that tailed underneath. Retroactively, we applied our eGFP+ and mCherry+ gating to the negative control population to verify our gating stringency; as expected, neither eGFP+ nor mCherry+ cell populations were observed in the negative control HEK293 cells.</p>
</div>
</div>
<div class="row">
- <div class="col-8">
- <img src="https://static.igem.wiki/teams/4764/wiki/flow/backgating-egfp.png" width="100%">
+ <div class="col-lg-8 col-sm-4">
+ <img src="https://static.igem.wiki/teams/4764/wiki/flow/backgating-egfp1.png" width="100%">
+ <p style="margin:auto; text-shadow: 1px 3px 3px #fd0db04a;"><em><strong>Figure 3: eGFP control backgating.</strong></em></p> <br><br>
</div>
- <div class="col-4">
+ <div class="col-lg-4 col-sm-8">
<p class="class="col-6"">
- Next, we used eGFP-expressing control cells to separate green fluorescing cells from non-green cells.
- Retroactively, we plotted green and red fluorescence of these populations to verify correct gating; as expected, green fluorescence was observed in GFP-expressing control cells, but no red fluorescence was observed.
- In a similar manner, we used mCherry-expressing control cells to separate red fluorescing cells from non-red cells and retroactively confirmed correct gating.
+ In Figure 2, we next used eGFP-expressing cells as a positive control. We intended to gate for green fluorescing cells (eGFP+) in this population, but due to the realization that we did not have a clear second peak representing the eGFP+ population, we decided to use this population to gate for mCherry+ instead. We knew these eGFP+ cells had no mCherry expression; therefore, our mCherry gate was decided where there was no fluorescent signal on the histogram. In the same manner, we determined our eGFP+ gate by using the mCherry-expressing population and selecting the portion with low to no mCherry signal. Retroactively, we verified that the majority of the eGFP-expressing population was correctly gated for eGFP+, and similarly for mCherry+.
</p>
</div>
</div>
@@ -149,55 +153,52 @@ Our initial plan at the start of the summer was to use HEK293 cells to produce o
Having gated for green expression, red expression, and no color, we then analyzed our CAR plus IL-6 experimental cell populations.
<br><br>
<div class="row">
- <div class="col-8">
- <img src="https://static.igem.wiki/teams/4764/wiki/flow/backgating-megf10-igg.png" width="100%">
+ <div class="col-lg-8 col-sm-4">
+ <img src="https://static.igem.wiki/teams/4764/wiki/flow/backgating-megf10-igg1.png" width="100%">
+ <p style="margin:auto; text-shadow: 1px 3px 3px #fd0db04a;"><em><strong>Figure 4: Megf10 CAR with IgG-purified IL-6 backgating.</strong></em></p> <br><br>
</div>
- <div class="col-4">
+ <div class="col-lg-4 col-sm-8">
<p class="class="col-6"">
- This figure corresponds to cells transfected with our Megf10 CAR and incubated with IgG-purified IL-6.
- We first subsetted the entire population to cells fluorescing green, implying CAR expression.
- We then further subsetted this green population to cells also fluorescing red, implying binding to IL6-mCherry.
- As desired, a significant portion of the entire population fluoresces both green and red.
- </p>
+ Figure 3 corresponds to cells transfected with our Megf10 CAR and incubated with IgG-purified IL-6.
+ We first subsetted the entire population to only HEK293 cells, removing debris. Next, we gated for cells which were eGFP+, implying CAR expression. We then further subsetted this green population to cells which also fluoresced red (doubly positive), implying that the CAR bound to our fusion IL6-mCherry protein. As desired, a significant portion of the entire population fluoresces both green and red, indicating successful binding.
+</p>
</div>
</div>
<div class="row">
- <div class="col-8">
- <img src="https://static.igem.wiki/teams/4764/wiki/flow/backgating-megf10-ft.png" width="100%">
+ <div class="col-lg-8 col-sm-4">
+ <img src="https://static.igem.wiki/teams/4764/wiki/flow/backgating-megf10-ft1.png" width="100%">
+ <p style="margin:auto; text-shadow: 1px 3px 3px #fd0db04a;"><em><strong>Figure 5: Megf10 CAR with IL-6 freeze-thaw lysate backgating.</strong></em></p> <br><br>
</div>
- <div class="col-4">
+ <div class="col-lg-4 col-sm-8">
<p class="class="col-6"">
- This figure corresponds to cells transfected with our Megf10 CAR and co-cultured with IL-6 freeze-thaw lysate.
- We first subsetted the entire population to cells fluorescing green, implying CAR expression.
- We then further subsetted this green population to cells also fluorescing red, implying binding to IL6-mCherry.
- As desired, a significant portion of the entire population fluoresces both green and red.
- </p>
+ Figure 4 corresponds to cells transfected with our Megf10 CAR and co-cultured with IL-6 freeze-thaw lysate.
+ We first subsetted the entire population to only HEK293 cells, removing debris. Next, we gated for cells which were eGFP+, implying CAR expression. We then further subsetted this green population to cells which also fluoresced red (doubly positive), implying that the CAR bound to our fusion IL6-mCherry protein. As desired, a significant portion of the entire population fluoresces both green and red, indicating successful binding.
+</p>
</div>
</div>
<div class="row">
- <div class="col-8">
- <img src="https://static.igem.wiki/teams/4764/wiki/flow/backgating-fcgamma-igg.png" width="100%">
+ <div class="col-lg-8 col-sm-4">
+ <img src="https://static.igem.wiki/teams/4764/wiki/flow/backgating-fcgamma-igg1.png" width="100%">
+ <p style="margin:auto; text-shadow: 1px 3px 3px #fd0db04a;"><em><strong>Figure 6: Fcγ CAR with IgG-purified IL-6 backgating.</strong></em></p> <br><br>
+
</div>
- <div class="col-4">
+ <div class="col-lg-4 col-sm-8">
<p class="class="col-6"">
- This figure corresponds to cells transfected with our Fcgamma CAR and incubated with IgG-purified IL-6.
- We first subsetted the entire population to cells fluorescing green, implying CAR expression.
- We then further subsetted this green population to cells also fluorescing red, implying binding to IL6-mCherry.
- As desired, a significant portion of the entire population fluoresces both green and red.
- </p>
+ Figure 5 corresponds to cells transfected with our Fcγ CAR and incubated with IgG-purified IL-6.
+ We first subsetted the entire population to only HEK293 cells, removing debris. Next, we gated for cells which were eGFP+, implying CAR expression. We then further subsetted this green population to cells which also fluoresced red (doubly positive), implying that the CAR bound to our fusion IL6-mCherry protein. As desired, a significant portion of the entire population fluoresces both green and red, indicating successful binding.
+</p>
</div>
</div>
<div class="row">
- <div class="col-8">
- <img src="https://static.igem.wiki/teams/4764/wiki/flow/backgating-fcgamma-ft.png" width="100%">
+ <div class="col-lg-8 col-sm-4">
+ <img src="https://static.igem.wiki/teams/4764/wiki/flow/backgating-fcgamma-ft1.png" width="100%">
+ <p style="margin:auto; text-shadow: 1px 3px 3px #fd0db04a;"><em><strong>Figure 7: Fcγ CAR with IL-6 freeze-thaw lysate backgating.</strong></em></p> <br><br>
</div>
- <div class="col-4">
+ <div class="col-lg-4 col-sm-8">
<p class="class="col-6"">
- This figure corresponds to cells transfected with our Fcgamma CAR and co-cultured with IL-6 freeze-thaw lysate.
- We first subsetted the entire population to cells fluorescing green, implying CAR expression.
- We then further subsetted this green population to cells also fluorescing red, implying binding to IL6-mCherry.
- As desired, a significant portion of the entire population fluoresces both green and red.
- </p>
+ Figure 6 corresponds to cells transfected with our Fcγ CAR and co-cultured with IL-6 freeze-thaw lysate.
+ We first subsetted the entire population to only HEK293 cells, removing debris. Next, we gated for cells which were eGFP+, implying CAR expression. We then further subsetted this green population to cells which also fluoresced red (doubly positive), implying that the CAR bound to our fusion IL6-mCherry protein. As desired, a significant portion of the entire population fluoresces both green and red, indicating successful binding.
+</p>
</div>
</div>
@@ -205,8 +206,11 @@ Our initial plan at the start of the summer was to use HEK293 cells to produce o
<p>Finally, we aggregated the above data and concluded with exciting results: <strong>a significant percentage of cells expressed CARs <em>and</em> bound IL6-mCherry!!</strong></p>
<div class="row">
<div class="col-6">
- This figure depicts 6 cell populations and the percentage of each population that is positive for both green and red fluorescence. Being positive for both implies successful CAR expression <em>and</em> ability to bind IL-6.</p>
- <img src="https://static.igem.wiki/teams/4764/wiki/flow/layout-with-legend.png" width="100%">
+ <img src="https://static.igem.wiki/teams/4764/wiki/flow/finalhist.png" width="100%">
+ <img src="https://static.igem.wiki/teams/4764/wiki/flow/finalfreqs.png" width="100%">
+ <p style="margin:auto; text-shadow: 1px 3px 3px #fd0db04a;"><em><strong>Figure 8: Control and experimental cell populations positive for both green and red fluorescence. Being positive for both implies successful CAR expression <em>and</em> ability to bind IL-6.</p>
+ </strong></em></p> <br><br>
+
</div>
<div class="col-6">
<p class="class="col-6"">
@@ -215,8 +219,8 @@ Our initial plan at the start of the summer was to use HEK293 cells to produce o
<li>Negative control: as expected, cells not transfected with CARs bound no IL-6.</li>
<li>anti-IL6 Megf10 CAR (IgG method): 10.4% of cells transfected with our Megf10 CAR and incubated with IgG-purified IL-6 were double positive.</li>
<li>anti-IL6 Megf10 CAR (freeze-thaw method): 12.0% of cells transfected with our Megf10 CAR and co-cultured with IL-6 freeze-thaw lysate were double positive.</li>
- <li>anti-IL6 Fcgamma CAR (IgG method): 11.0% of cells transfected with our Fcgamma CAR and incubated with IgG-purified IL-6 were double positive.</li>
- <li>anti-IL6 Fcgamma CAR (freeze-thaw method): 16.2% of cells transfected with our Fcgamma CAR and co-cultured with IL-6 freeze-thaw lysate were double positive.</li>
+ <li>anti-IL6 Fcγ CAR (IgG method): 11.0% of cells transfected with our Fcγ CAR and incubated with IgG-purified IL-6 were double positive.</li>
+ <li>anti-IL6 Fcγ CAR (freeze-thaw method): 16.2% of cells transfected with our Fcγ CAR and co-cultured with IL-6 freeze-thaw lysate were double positive.</li>
<li>eGFP control: as expected, cells transfected only with an eGFP plasmid bound effectively no IL-6.</li>
</ol>
</p>
@@ -225,13 +229,54 @@ Our initial plan at the start of the summer was to use HEK293 cells to produce o
</div>
+<!-- hey-->
+
+<h4 id="analysis-button" class="learn-more dropdown-toggle">Analysis of results</h4>
+<div id="analysis-info">
+<p>
+Before running this final experiment, we were struggling to achieve a significant, nonzero concentration of IL-6 protein with which we could incubate the CAR-expressing macrophages. We had attempted to optimize the protein purification protocol a number of times using the Pierce anti-FLAG Magnetic Agarose Beads. Namely, we attempted using three elution buffers: our SDS-page sample loading buffer (1x Laemmli buffer), IgG elution buffer, and 0.1 M glycine. We were repeatedly unable to detect significant differences between the elution buffers after trying to quantify our protein concentration using the Bradford assay and NanoDrop spectrophotometry.
+<br><br>
+We had not considered forgoing the step of protein purification for the IL-6 fusion protein. However, after encountering the reality that we had extremely low final concentration of purified protein regardless of the elution buffer we used, we backed up a few steps to consider what could be changed. This was when a mentor, Noor, brought up the idea of incubating the CAR-expressing HEK293 cells directly with the lysate of IL6-expressing HEK293 cells.
+<br><br>
+We examined the lysis and purification steps that we had been applying – so far, we had been lysing the IL6-expressing HEK293 cells with ThermoFisher M-PER reagent. Since M-PER was a detergent that would unintentionally lyse our CAR-expressing cells and could not be extracted from the final lysate solution, we turned to another form of mammalian cell lysis to accomplish our goal: freeze-thaw lysis. The final lysate was not purified; we directly added it to wells of CAR-expressing HEK293 cells to observe the resulting behavior. This way, we knew IL-6 was in the lysate and would no longer act as a limiting factor.
+<br><br>
+From the final chart comparing our different variables against each other, we can conclude that the Fcgamma CAR incubated with IL-6 freeze-thaw lysate had the greatest double positive frequency of the parent population, but the other three samples were significant as well and not too different from each other. We are pleased to find that the incubations with freeze-thaw lysate were both higher than the incubations with IgG-eluted IL-6. This supports our suspicion that the protein purification protocol had annihilated the concentration of IL-6 in those solutions. We suspect that the manyfold greater concentration of IL-6 in the freeze-thaw lysate improved the probability of CAR binding events.
+<br><br>
+Additionally, we were eager to compare the behavior of the Fcgamma vs. Megf10 intracellular component. Although it is important to note that we were not able to compare its impact on the phagocytotic behavior of a macrophage, we theorize that the intracellular component had an impact on the folding of the protein or even the binding affinity of the protein. Due to the limited scope of the experiments we ran, we cannot conclude which is more optimal than the other; we determine that they are both great candidates to move forward with and perform macrophage experiments with. Based on phagocytic activity, it may become more apparent which CAR is preferable.
+</p>
+</div>
+<!-- yoyoyo end-->
+
<div class="bd-callout bd-callout-info">
- <h1>Replicating our experiments; WE ARE EDITING THIS TODAY @mentors :)</h1>
+ <h1>Replicating our experiments</h1>
</div>
+<p>The precise protocols that we followed are detailed on this page and on
+ our experiments page and lab notebook. We have made every effort to thoroughly document our experiments
+ and ensure other researchers can reproduce our results, and we welcome questions or clarifications
+ addressed to igem-2023-students@mit.edu
+</p>
<div class="bd-callout bd-callout-info">
- <h1>Future plans; WE ARE EDITING THIS TODAY @mentors :)</h1>
+ <h1>Future plans</h1>
</div>
+<p>We are extremely proud of having achieved a proof-of-concept that HEK293 cells expressing our CARs
+ bind IL-6 significantly more strongly than cells lacking our CARs. Due to constraints on our time and resources, we were unable to
+ progress further than this level of achievement. However, we have planned potential next steps that
+ can be taken to achieve the loftier goal of developing a real, practical cachexia therapy.
+ <ol>
+ <li><strong style="color:#ee2b76">Transfecting macrophages:</strong></li>
+ <li><strong style="color:#ee2b76">Inducing IL-6 aggregation (if needed):</strong></li>
+ <li><strong style="color:#ee2b76">Designing a delivery system:</strong></li>
+ <li><strong style="color:#ee2b76">Testing in animal models of cachexia:</strong></li>
+ <li><strong style="color:#ee2b76">Quantifying pharmacokinetic/pharmacodynamic parameters:</strong></li>
+ <li><strong style="color:#ee2b76">Clinical testing:</strong></li>
+
+ </ol>
+</p>
+
+<br>
+
+<p>Our team </p>
{% endblock %}
diff --git a/wiki/pages/software.html b/wiki/pages/software.html
index 1754c4aeeb4ce29447834f359c1efd2ecaeca1c8..5c0d336034641b48d58d7fa9d6b7869fd7a53e29 100644
--- a/wiki/pages/software.html
+++ b/wiki/pages/software.html
@@ -1,7 +1,7 @@
{% extends "layout.html" %}
{% block title %}Software{% endblock %}
-{% block lead %}MIT's software enables easier codon optimization, pairwise sequence alignment, and primer design for CAR-TK parts.{% endblock %}
+{% block lead %}MIT's software enables easier codon optimization, pairwise sequence alignment, diagnostic digests, and primer design for CAR-TK parts.{% endblock %}
{% block page_content %}
@@ -42,6 +42,24 @@ We experienced these difficulties firsthand when we received sequencing results
</section>
<!-- end alignment tool -->
+<!-- start diagnostic digest aid -->
+<section>
+ <div class="bd-callout bd-callout-info">
+ <h1>Diagnostic digest aid</h1>
+ </div>
+ <p>Modern cloning methods are excellent and typically result in
+ correctly constructed plasmids, but errors are always possible, and it is
+ crucial to verify constructs before using them for experiments. Sequencing is a foolproof
+ method of verifying constructs, but costs can rapidly accumulate. Thus, for
+ daily lab activity, our team often turns to enzymatic digestion of plasmids followed by
+ agarose gel electrophoresis to roughly determine whether plasmids appear correct. </p>
+ <p>Planning these crucial diagnostic digests can be tedious and prone to human error. To mitigate these issues,
+ our team developed a user-friendly tool that makes planning digests quick and effortless.
+ </p>
+ <p>Click <a href="https://digests.k3s.solving.group/" target="_blank">here</a> to use our diagnostic digest aid!
+ </p>
+<!-- end diagnostic digest aid -->
+
<!-- start CAR-TK software -->
<section>
<div class="bd-callout bd-callout-info">
@@ -60,23 +78,4 @@ We experienced these difficulties firsthand when we received sequencing results
</section>
<!-- end CAR-TK software-->
-<!-- start diagnostic digest aid -->
-<!-- <section>
- <div class="bd-callout bd-callout-info">
- <h1>Diagnostic digest aid</h1>
- </div>
- <p>Modern cloning methods are excellent and typically result in
- correctly constructed plasmids, but errors are always possible, and it is
- crucial to verify constructs before using them for experiments. Sequencing is a foolproof
- method of verifying constructs, but costs can rapidly accumulate. Thus, for
- daily lab activity, our team often turns to enzymatic digestion of plasmids followed by
- agarose gel electrophoresis to roughly determine whether plasmids appear correct. </p>
- <p>Planning these crucial diagnostic digests can be tedious and prone to human error. To mitigate these issues,
- our team developed a user-friendly tool that makes planning digests quick and effortless.
- </p>
- <p>Click <a href="https://igem.science" target="_blank">here</a> to use our diagnostic digest aid!
-
- Note that you may encounter a safety warning; this is solely due to the site domain and not due to any genuine
-safety concerns, so we recommend pushing past this warning to use the tool. </p> -->
-<!-- end diagnostic digest aid -->
{% endblock %}