Difference between revisions of "Team:Fudan-TSI/Composite Part"

m
m
Line 56: Line 56:
 
         /** 重置表格元素 **/
 
         /** 重置表格元素 **/
 
         table { border-collapse: collapse; border-spacing: 0; }
 
         table { border-collapse: collapse; border-spacing: 0; }
−
#FudanBody .imgNoM img{
 
−
    width:100%;
 
−
}
 
−
#FudanBody .imgNoM{
 
−
    text-align:center;
 
−
}
 
 
     </style>
 
     </style>
−
     <title>2019 Team:Fudan-TSI Demonstrate</title>
+
     <title>2019 Team:Fudan-TSI Composite_Part</title>
 
</head>
 
</head>
  
Line 95: Line 89:
 
                     <div class="background">
 
                     <div class="background">
 
                     </div>
 
                     </div>
−
                     <p class="flow-text" style="width:100%;text-align:center"><span class="white-text">Demonstration</span></p>
+
                     <p class="flow-text" style="width:100%;text-align:center"><span class="white-text">Composite parts</span></p>
 
                 </div></li>
 
                 </div></li>
 
                 <li>
 
                 <li>
 
                     <ul class="collapsible expandable">
 
                     <ul class="collapsible expandable">
−
                        <li class="onThisPageNav"><span>On this page</span></li>
 
−
                        <li class="onThisPageNav"><a href="#section1">div with id section1</a></li>
 
−
                        <li class="onThisPageNav"><a href="#section2">div with id section2</a></li>
 
−
                        <li class="onThisPageNav"><a href="#section3">div with id section3</a></li>
 
−
                        <li class="onThisPageNav"><a href="#section4">div with id section4</a></li>
 
−
                        <li class="onThisPageNav"><a href="#section5">div with id section5</a></li>
 
 
                         <li class="onThisPageNav"><span>Team: Fudan-TSI</span></li>
 
                         <li class="onThisPageNav"><span>Team: Fudan-TSI</span></li>
 
<li><div class="collapsible-header"><span>Project</span></div>
 
<li><div class="collapsible-header"><span>Project</span></div>
Line 161: Line 149:
  
 
         <div id="pageContent" style="">
 
         <div id="pageContent" style="">
−
 
−
 
 
             <div id="contentBanner" class="figureBanner">
 
             <div id="contentBanner" class="figureBanner">
 
                 <div class="row">
 
                 <div class="row">
−
                     <div class="col s12 hide-on-med-and-up">
+
                     <div class="col s12 m6 valign-wrapper hide-on-med-and-up">
−
                         <h1>Demonstration</h1>
+
                         <h1>Composite parts</h1>
 
                     </div>
 
                     </div>
−
                     <div class="col s12 hide-on-med-and-up">
+
                     <div class="col s12 m6 valign-wrapper hide-on-med-and-up">
−
                         <span></span>
+
                         <span>This year, our BioBrick submission includes 7 versions of SynNotch receptors, with our favorite being &alpha;CD19-mN1c-tTAA.</span>
 
                     </div>
 
                     </div>
 
                 </div>
 
                 </div>
 
                 <div id="figureBannerTitle" class="hide-on-small-only">
 
                 <div id="figureBannerTitle" class="hide-on-small-only">
−
                     <h1>Demonstration</h1>
+
                     <h1>Composite parts</h1>
−
                     <p class="flow-text"><span></span></p>
+
                     <p class="flow-text"><span>This year, our BioBrick submission includes 7 versions of SynNotch receptors, with our favorite being &alpha;CD19-mN1c-tTAA.</span></p>
 
                 </div>
 
                 </div>
 
                 <div class="hide-on-small-only">
 
                 <div class="hide-on-small-only">
−
                     <img src="https://static.igem.org/mediawiki/2018/a/a5/T--Fudan--title_demonstration.jpg">
+
                     <img src="https://static.igem.org/mediawiki/2018/a/ad/T--Fudan--title_composite.jpg">
 
                     <svg width="10" height="10" xmlns="http://www.w3.org/2000/svg" style="position:absolute; left:0;top:0; width: 4%;height: 100%;">
 
                     <svg width="10" height="10" xmlns="http://www.w3.org/2000/svg" style="position:absolute; left:0;top:0; width: 4%;height: 100%;">
 
                         <defs>
 
                         <defs>
Line 206: Line 192:
 
             <div class="container">
 
             <div class="container">
 
                 <main style="margin: 0">
 
                 <main style="margin: 0">
−
                     <div class="section container scrolSpy" id="section1">
+
                     <div class="section container">
−
<h2>Video abstract</h2>
+
                        <h2>Part:BBa_K2549021 <a href="http://parts.igem.org/Part:BBa_K2549021" target=_blank>&alpha;CD19-mN1c-tTAA</a></h2>
−
<video class="responsive-video" style="margin-top:23px;" controls>
+
                         <h3>Introduction</h3>
−
    <source src="https://static.igem.org/mediawiki/2018/4/45/T--Fudan--demostrate-video.mp4" type="video/mp4">
+
                        <p class="flow-text">This year we have provided 7 versions of SynNotch receptors in our BioBrick submission, enabling others to wire their contact-dependent signal transduction in mammalian cells. Multiple combinations of extracellular domains, transmembrane core regions and intracellular domains make it even easier for others to readily assemble their own desirable genetic circuits. </p>
−
  </video>
+
                        <p class="flow-text"><b>Among the 7 SynNotch receptors, our favorite one is &alpha;CD19-mN1c-tTAA <a href="http://parts.igem.org/Part:BBa_K2549021" target="_blank">(BBa_K2549021)</a></b><br/>
−
                        <h2>Transmembrane Logic
+
<img alt="part BBa_K2549021" src="https://static.igem.org/mediawiki/2018/thumb/0/0c/T--Fudan--BBa_K2549021.png/1339px-T--Fudan--BBa_K2549021.png" />
−
                        </h2>
+
</p>
−
                         <h3>Sensing and integrating various transmembrane signals is a key aspect of cellular decision making.
+
                        <h3>How &alpha;CD19-mN1c-tTAA works
−
                        </h3><p class="flow-text">
+
                        </h3>
−
                            For example, activation of CD8+ cells requires co-activation of TCR and CD28 molecules, meanwhile, this activation can be inhibited by the PD-1 pathway  <a href="https://www.ncbi.nlm.nih.gov/pubmed/28280247" target=_blank>(Hui et al., 2017)</a>. By abstracting this biological process, we can get: the activation of CD8+ cell = activated TCR AND (activated CD28 NIMPLY activated PD-1). Programming cells with predictable complex transmembrane signal inputs – customized intracellular signal outputs logic relationships are significant for expanding the widespread applications of mammalian cells, such as cellular immunotherapy <a href="https://www.ncbi.nlm.nih.gov/pubmed/24337479" target=_blank>(Fedorov et al., 2013;</a> <a href="https://www.ncbi.nlm.nih.gov/pubmed/23242161" target=_blank>Kloss et al., 2013;</a> <a href="https://www.ncbi.nlm.nih.gov/pubmed/26830879" target=_blank>Roybal et al., 2016a;</a> <a href="https://www.ncbi.nlm.nih.gov/pubmed/27693353" target=_blank>Roybal et al., 2016b), tissue patterning </a><a href="https://www.ncbi.nlm.nih.gov/pubmed/26830878" target=_blank>(Morsut et al., 2016;</a><a href="https://www.ncbi.nlm.nih.gov/pubmed/27693353" target=_blank>Roybal et al., 2016;</a> <a href="https://www.ncbi.nlm.nih.gov/pubmed/29853554" target=_blank>Toda et al., 2018)</a>.
+
                        <p class="flow-text">It receives ligand-dependent signal via the CD19 scFv and undergoes a cleavage process in which the tTA advance is released, then entering into the nucleus to activate the expression of TRE3GV promotor. Thus it can be served as a signal input module.
 
                         </p>
 
                         </p>
−
                         <div class="expFigureHolder" style="width:100%">
+
                         <h3>Advantages of &alpha;CD19-mN1c-tTAA
−
                            <img class="responsive-img" src="https://static.igem.org/mediawiki/2018/2/28/T--Fudan--results-1.svg">
+
−
 
+
−
                        </div>
+
−
                        <h3>a sequence-specific toolbox for continuous mutagenesis
+
 
                         </h3>
 
                         </h3>
−
                         <p class="flow-text"><b>
+
                         <p class="flow-text">We have conducted flow cytometry experiments to test our SynNotch receptors and after testing, &alpha;CD19-mN1c-tTAA have stood out for showing the highest signal-to-noise ratio. We have also discovered that it has the highest activation ratio when activated by surface-expressed CD19 antigen. Moreover, it also shows only a few amount of false activation which can be tolerated. As it performs great modularity and has a great potential to be utilized by others to assemble their own CD19-dependent signal transduction module, this especially enables the possibility of the clinical application of SynNotch receptors.
−
                            The ingenious design of nature makes us amazing, and inspires us to explore new possibilities. By designing the ENABLE (Engineered, Across-membrane, Binary Logic in Eukaryotes) system and achieving the first complete transmembrane binary Boolean logic in mammals, we could make the pupil outdo the master, make cells even smarter.
+
−
                        </b></p>
+
−
                    </div>
+
−
                    <div id="section2" class="section container">
+
−
                        <h2>Optimizing the Receptor, aims for reduced background activation
+
−
                        </h2>
+
−
                        <p class="flow-text">In order to be able to implement a custom multiplexed transmembrane signal input/output relationship, the first condition is that engineering modular receptor to enable it to recognize extracellular signals and transduce them into customized intracellular signals. <a href="https://www.nature.com/articles/nrc.2016.17" target=_blank>SynNotch</a> is a transmembrane receptor with high programmability. Therefore, we can use this tool to receive extracellular signals and output customized intracellular signals.
+
−
                        </p><p class="flow-text">
+
−
                            Good tools are prerequisite to the successful execution of a job. In order to make SynNotch more suitable as a tool for receiving complex extracellular signals, we have optimized it. Please visit <a href="/Team:Fudan-TSI/Optimization">Optimization page</a> for more details.
+
 
                         </p>
 
                         </p>
−
                    </div>
+
                         <div class="figureHolder width40" style="margin: 23px auto 0 auto;">
−
                    <div id="section3" class="section container">
+
                             <img class="responsive-img" src="https://static.igem.org/mediawiki/2018/b/bc/T--Fudan--composite-1.png">
−
                        <h2>Three-layer design for dual transmembrane signals
+
−
 
+
−
                        </h2>
+
−
                        <p class="flow-text">The expression of membrane proteins on the cell membrane is limited. Our modeling tells us how to make cells sensitive to the limited signal molecules, and perform function efficiently is the key to make the transmembrane logic gate system work.
+
−
 
+
−
                        </p><p class="flow-text"> Based on this, we developed the ENABLE system with a 3-layers design: Receptor, Amplifier, and Combiner. By adding an intermediate layer, the “Amplifier” layer, we are able to effectively amplify the transmembrane signal, thus enabling our intracellular components to effectively travel logic functions.
+
−
 
+
−
                    </p><p class="flow-text">    The 3-layer design principle underlying ENABLE empowers any future development of transmembrane logic circuits, thus contributes a foundational advance to synthetic biology.
+
−
 
+
−
                    </p>
+
−
                         <div class="expFigureHolder" style="width:50%;margin: 23px auto 0 auto">
+
−
                             <img class="responsive-img" src="https://static.igem.org/mediawiki/2018/a/a3/T--Fudan--results-3.svg">
+
  
 
                         </div>
 
                         </div>
−
                    </div>
+
                        <p style="margin-top:0;text-indent: 0;"><b>Figure 1. Flow cytometry characterization of SynNotch receptors.</b> TRE3GV-EGFP circuit was set to indicate the activation level of SynNotch receptors. It is shown that &alpha;CD19-mN1c-tTAA has the highest signal-to-noise ratio.</p>
−
                    <div id="section4" class="section container">
+
                         <p class="flow-text">For more details, please check <a href="/Team:Fudan-TSI/Part_Collection">our parts collection page</a>.
−
                        <h2>Unified intracellular logic gates layout
+
−
 
+
−
                        </h2>
+
−
                         <p class="flow-text">In order to be able to receive dual transmembrane signals and produce complete binary Boolean logic, we designed a set of interactive grammar for the interaction between the "Amplifier" layer and the "Combiner" layer within the cell membrane. This grammar consists mainly of the following elements, a transcription system based on the activating-form, silencing-from or NIMPLY-form promoters (the three are collectively referred to as a synthetic transcription factor-promoter pairs based transcription system); intein-based protein in vivo fusion systems, proteolytic enzyme-based protein in vivo destruction systems (the two are collectively referred to as a protein fusion/destruction-based transcription factor logic). We have test them and report in <a href="/Team:Fudan-TSI/Results">Results page</a>, as well as on <b>parts.igem</b> pages.
+
 
                         </p>
 
                         </p>
−
<div class="expFigureHolder" style="width:60%;margin: 23px auto 0 auto">
 
−
                            <img class="responsive-img" src="https://static.igem.org/mediawiki/2018/e/ec/T--Fudan--stack.gif">
 
−
 
−
                        </div>
 
 
                     </div>
 
                     </div>
−
                    <div id="section5" class="section container">
 
−
                        <h2>ENABLE logic gates
 
−
                        </h2>
 
−
                        <p class="flow-text">Below we show experimental verifications of six (OR, NOR, AND, NAND, IMPLY, NIMPLY) of the eight truly binary logics.
 
−
                        </p>
 
−
                        <div class="expFigureHolder" style="width:100%">
 
−
                            <img class="responsive-img" src="https://static.igem.org/mediawiki/2018/5/56/T--Fudan--LG.svg">
 
−
 
−
                        </div>
 
−
                        <p class="flow-text">Here, we use the OR gate as a proof of concept for a three-layer transmembrane binary Boolean logic during tissue patterning.
 
−
                        </p>
 
−
                        <p class="flow-text"> Through dynamic and static observations, we can see that the Receiver cell with transmembrane OR gate can be activated by two Sender cells expressing different membrane antigens.
 
−
                        </p>
 
−
                        <div class="row">
 
−
                            <img class="col s12 m4" src="https://static.igem.org/mediawiki/2018/9/98/T--Fudan--demon-1.png">
 
−
                            <img class="col s12 m4" src="https://static.igem.org/mediawiki/2018/d/d1/T--Fudan--results-rgb2.gif">
 
−
                        </div>
 
−
 
−
                        <h2>Public engagement and education
 
−
                        </h2>
 
−
                        <p class="flow-text">Our <a href="/Team:Fudan-TSI/Human_Practices">human practice</a> activities include <a href="/Team:Fudan-TSI/Public_Engagement">dialogues (debate and interviews), presentations</a>, <a href="/Team:Fudan-TSI/Bio-Art">Bio-Art display</a> and <a href="/Team:Fudan-TSI/Public_Engagement">hands-on practices</a>, all of which centered around farseeing. We want to unlock the creativity and intelligence in as many people as possible. Fully engaged with the public, we motivated by their openness and willingness, which reminds us why we initiated the research and why we work hard in the lab - for the good of the people, including ourselves.
 
−
                        </p>
 
−
                    </div>
 
−
 
 
                 </main>
 
                 </main>
 
             </div>
 
             </div>
−
            <!-- end of main content of the page -->
 
  
 
<!--Abstract on content page-->
 
<!--Abstract on content page-->
Line 324: Line 252:
 
                         <div id="usefulLinks" class="col m9 s12 row">
 
                         <div id="usefulLinks" class="col m9 s12 row">
 
                             <div class="col s12 l6 row">
 
                             <div class="col s12 l6 row">
−
                                 <div class="col s12 m4 active">
+
                                 <div class="col s12 m4">
 
                                   <span><a href="/Team:Fudan-TSI/Description">Project</a></span>
 
                                   <span><a href="/Team:Fudan-TSI/Description">Project</a></span>
 
                                     <ul>
 
                                     <ul>
Line 354: Line 282:
 
                             </div>
 
                             </div>
 
                             <div class="col s12 l6 row">
 
                             <div class="col s12 l6 row">
−
                                 <div class="col s12 m4">
+
                                 <div class="col s12 m4 active">
 
                                   <span><a href="/Team:Fudan-TSI/Parts">Parts</a></span>
 
                                   <span><a href="/Team:Fudan-TSI/Parts">Parts</a></span>
 
                                     <ul>
 
                                     <ul>

Revision as of 07:06, 17 September 2019

<script src="https://code.jquery.com/jquery-1.11.3.min.js"></script> 2019 Team:Fudan-TSI Composite_Part

Composite parts

This year, our BioBrick submission includes 7 versions of SynNotch receptors, with our favorite being αCD19-mN1c-tTAA.

Composite parts

This year, our BioBrick submission includes 7 versions of SynNotch receptors, with our favorite being αCD19-mN1c-tTAA.

Part:BBa_K2549021 αCD19-mN1c-tTAA

Introduction

This year we have provided 7 versions of SynNotch receptors in our BioBrick submission, enabling others to wire their contact-dependent signal transduction in mammalian cells. Multiple combinations of extracellular domains, transmembrane core regions and intracellular domains make it even easier for others to readily assemble their own desirable genetic circuits.

Among the 7 SynNotch receptors, our favorite one is αCD19-mN1c-tTAA (BBa_K2549021)
part BBa_K2549021

How αCD19-mN1c-tTAA works

It receives ligand-dependent signal via the CD19 scFv and undergoes a cleavage process in which the tTA advance is released, then entering into the nucleus to activate the expression of TRE3GV promotor. Thus it can be served as a signal input module.

Advantages of αCD19-mN1c-tTAA

We have conducted flow cytometry experiments to test our SynNotch receptors and after testing, αCD19-mN1c-tTAA have stood out for showing the highest signal-to-noise ratio. We have also discovered that it has the highest activation ratio when activated by surface-expressed CD19 antigen. Moreover, it also shows only a few amount of false activation which can be tolerated. As it performs great modularity and has a great potential to be utilized by others to assemble their own CD19-dependent signal transduction module, this especially enables the possibility of the clinical application of SynNotch receptors.

Figure 1. Flow cytometry characterization of SynNotch receptors. TRE3GV-EGFP circuit was set to indicate the activation level of SynNotch receptors. It is shown that αCD19-mN1c-tTAA has the highest signal-to-noise ratio.

For more details, please check our parts collection page.

project summary

Project Summary

Mutation library generation is critical for biological and medical research, but current methods cannot mutate a specific sequence continuously without manual intervention. Here we present a toolbox for in vivo continuous mutation library construction. First, the target DNA is transcribed into RNA. Next, our reverse transcriptase reverts RNA into cDNA, during which the target is randomly mutated by enhanced error-prone reverse transcription. Finally, the mutated version replaces the original sequence through recombination. These steps will be carried out iteratively, generating a random mutation library of the target with high efficiency as mutations accumulate along with bacterial growth. Our toolbox is orthogonal and provides a wide range of applications among various species. R-Evolution could mutate coding sequences and regulatory sequences, which enables the in vivo evolution of individual proteins or multiple targets at a time, promotes high-throughput research, and serves as a foundational advance to synthetic biology.