Difference between revisions of "Team:Fudan/Parts Collection"
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table { border-collapse: collapse; border-spacing: 0; } | table { border-collapse: collapse; border-spacing: 0; } | ||
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| + | <title>2018 iGEM Team:Fudan - Parts collection</title> | ||
</head> | </head> | ||
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<div class="nav-wrapper"> | <div class="nav-wrapper"> | ||
<div id="teamLogo" class="brand-logo"> | <div id="teamLogo" class="brand-logo"> | ||
| − | <a href="https://2018.igem.org/Team:Fudan" target="_self"><img src="https://static.igem.org/mediawiki/2018/c/c4/T--Fudan--teamLogo.svg"></a> | + | <a href="https://2018.igem.org/Team:Fudan" target="_self"><img alt="2018 team Fudan logo" src="https://static.igem.org/mediawiki/2018/c/c4/T--Fudan--teamLogo.svg"></a> |
</div> | </div> | ||
<ul id="nav-mobile" class="right blueBorder"> | <ul id="nav-mobile" class="right blueBorder"> | ||
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<!-- Dropdown and List elements in navigation bar --> | <!-- Dropdown and List elements in navigation bar --> | ||
<ul id="dropdown1" class="dropdown-content"> | <ul id="dropdown1" class="dropdown-content"> | ||
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Demonstrate">Demonstration</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Antigen_Receptors">Antigen, Receptors</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Results">Transmembrane logic</a></li> |
</ul> | </ul> | ||
<ul id="dropdown2" class="dropdown-content"> | <ul id="dropdown2" class="dropdown-content"> | ||
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Addon#ribo">Addon: ribo</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Addon#TALE">Addon: TALE</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Addon#T2">Addon: T2</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Model#Transcriptional_Amplifer">Model: transcriptional amplifer</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Model#NotchLigandKinetics">Model: Notch-ligand kinetics</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Software">Software</a></li> |
</ul> | </ul> | ||
<ul id="dropdown3" class="dropdown-content"> | <ul id="dropdown3" class="dropdown-content"> | ||
| − | <li><a href=" | + | <li><a href="/Team:Fudan/InterLab">iGEM interLab</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Notebook">Our notebook</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Primers">Primers used</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Protocols">Protocols</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Safety">Safety</a></li> |
</ul> | </ul> | ||
<ul id="dropdown4" class="dropdown-content"> | <ul id="dropdown4" class="dropdown-content"> | ||
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Basic_Parts">Basic parts</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Composite_Parts">Composite parts</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Optimization">Optimization</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Parts_Collection">Parts collection</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Improve">Parts improvement</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Measurement">Quantification</a></li> |
</ul> | </ul> | ||
<ul id="dropdown5" class="dropdown-content"> | <ul id="dropdown5" class="dropdown-content"> | ||
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Bio-Art">Bio-Art display</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Collaborations">Collaborations</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Design_Intention">Design intention</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Human_Practices">Human practices</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Public_Engagement">Public engagement</a></li> |
</ul> | </ul> | ||
<ul id="dropdown6" class="dropdown-content"> | <ul id="dropdown6" class="dropdown-content"> | ||
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Acknowledgement">Acknowledgement</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Attributions">Attributions</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Heritage">Heritage</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Team">Members</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Sponsors">Sponsors</a></li> |
</ul> | </ul> | ||
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<div class="collapsible-body"> | <div class="collapsible-body"> | ||
<ul> | <ul> | ||
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Demonstrate">Demonstration</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Antigen_Receptors">Antigen, Receptors</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Results">Transmembrane logic</a></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="collapsible-body"> | <div class="collapsible-body"> | ||
<ul> | <ul> | ||
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Addon#ribo">Addon: ribo</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Addon#TALE">Addon: TALE</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Addon#T2">Addon: T2</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Model#Transcriptional_Amplifer">Model: transcriptional amplifer</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Model#NotchLigandKinetics">Model: Notch-ligand kinetics</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Software">Software</a></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="collapsible-body"> | <div class="collapsible-body"> | ||
<ul> | <ul> | ||
| − | <li><a href=" | + | <li><a href="/Team:Fudan/InterLab">iGEM interLab</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Notebook">Our notebook</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Primers">Primers used</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Protocols">Protocols</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Safety">Safety</a></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="collapsible-body"> | <div class="collapsible-body"> | ||
<ul> | <ul> | ||
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Basic_Parts">Basic parts</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Composite_Parts">Composite parts</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Optimization">Optimization</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Parts_Collection">Parts collection</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Improve">Parts improvement</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Measurement">Quantification</a></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="collapsible-body"> | <div class="collapsible-body"> | ||
<ul> | <ul> | ||
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Bio-Art">Bio-Art display</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Collaborations">Collaborations</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Design_Intention">Design intention</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Human_Practices">Human practices</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Public_Engagement">Public engagement</a></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="collapsible-body"> | <div class="collapsible-body"> | ||
<ul> | <ul> | ||
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Acknowledgement">Acknowledgement</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Attributions">Attributions</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Heritage">Heritage</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Team">Members</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Sponsors">Sponsors</a></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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</div> | </div> | ||
<div class="col s12 m6 valign-wrapper hide-on-med-and-up"> | <div class="col s12 m6 valign-wrapper hide-on-med-and-up"> | ||
| − | <span>Our parts collection | + | <span>Our parts collection provides an extensive toolbox for researchers to construct their own transmembrane logic gates in mammalian cells.</span> |
</div> | </div> | ||
</div> | </div> | ||
<div id="figureBannerTitle" class="hide-on-small-only"> | <div id="figureBannerTitle" class="hide-on-small-only"> | ||
<h1>Parts collection</h1> | <h1>Parts collection</h1> | ||
| − | <p><span>Our parts collection | + | <p><span>Our parts collection provides an extensive toolbox for researchers to construct their own transmembrane logic gates in mammalian cells.</span></p> |
</div> | </div> | ||
<div class="hide-on-small-only"> | <div class="hide-on-small-only"> | ||
| Line 271: | Line 273: | ||
<!-- side navigator of page content --> | <!-- side navigator of page content --> | ||
<ul id="pageContentNav" class="hide-on-med-and-down z-depth-0"> | <ul id="pageContentNav" class="hide-on-med-and-down z-depth-0"> | ||
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Basic_Parts">Basic parts</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Composite_Parts">Composite parts</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Optimization">Optimization</a></li> |
<li>Parts collection</li> | <li>Parts collection</li> | ||
<li class="onThisPageNav"><a href="#section1">SynNotch receptors set</a></li> | <li class="onThisPageNav"><a href="#section1">SynNotch receptors set</a></li> | ||
| Line 279: | Line 281: | ||
<li class="onThisPageNav"><a href="#section3">Amplifiers & combiners</a></li> | <li class="onThisPageNav"><a href="#section3">Amplifiers & combiners</a></li> | ||
<li class="onThisPageNav"><a href="#section4">ENABLE</a></li> | <li class="onThisPageNav"><a href="#section4">ENABLE</a></li> | ||
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Improve">Parts improvement</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Measurement">Quantification</a></li> |
</ul> | </ul> | ||
<main> | <main> | ||
<div class="section container"> | <div class="section container"> | ||
| − | <p>Our parts collection | + | <p>Our parts collection provides an extensive toolbox for researchers to construct their own transmembrane logic gates in mammalian cells. These parts were made as BioBricks, and are NOT ready for transfecting mammalian cells. Plasmids with these parts in eukaryotic expression backbone are available upon request. |
</p> | </p> | ||
| − | <p>Our parts collection contains all essential components of our ENABLE toolbox, that could also be divided into several sets, enabling others to readily utilize them to improve their existing genetic circuits. | + | <p>Our parts collection contains <b>all essential components</b> of our ENABLE toolbox, that could also be divided into several sets, enabling others to readily utilize them to improve their existing genetic circuits. |
</p> | </p> | ||
| − | <p> | + | <p>Following our 3-layer design principle, all ENABLE components have been well characterized, including our SynNotch receptors, Amplifiers and Combiners. In the collection, we have provided 7 versions of SynNotch receptors with (1) different combinations of extracellular domains which can bind to different antigens with different affinities, (2) transmembrane domains with varying cleavage efficiency and (3) intracellular domains executing diverse transcriptional amplifications. |
</p> | </p> | ||
| − | <p>We have provided a set of transcriptional amplifiers including zinc finger-based, TEV protease-based and split intein-based, allowing others to build their own genetic circuits, preferably through transcription (we have | + | <p>We have provided a set of transcriptional amplifiers including zinc finger-based, TEV protease-based and split intein-based ones, allowing others to <b>build their own genetic circuits</b>, preferably through transcription (we have tested), but not limited to (we have not tested). Besides, a set of combiners with different copies of response elements are provided, which makes it <b>easy for others to tune the activation or repression threshold</b>. This feature was experimental confirmed previously and mathematically <a href="/Team:Fudan/Model#Transcriptional_Amplifier" target="_blank">modeled</a> in our 3-layer design again this year. Our modeling process could be used by others to predict their own genetic circuits before any wet-lab experiments. |
</p> | </p> | ||
<p> | <p> | ||
| Line 296: | Line 298: | ||
</p> | </p> | ||
</div> | </div> | ||
| − | <div id="section1" class="section container | + | <div id="section1" class="section container scrolSpy"> |
<h2>SynNotch receptors set</h2> | <h2>SynNotch receptors set</h2> | ||
| − | <p>We have provided 7 versions of SynNotch receptors. Each of them can transduce signal from out of the cell via a contact-dependent antigen-stimulated cleavage process. When | + | <p>We have provided 7 versions of SynNotch receptors. Each of them can transduce signal from out of the cell via a contact-dependent antigen-stimulated cleavage process. When expressing two SynNotch receptors with two different extracellular domains recognizing two types of antigens, the cell is able to accept dual inputs. The chosen nanobodies are highly specific against their antigens, and the chosen intracellular domains are transcriptionally orthogonal. For more specific details, please refer to our parts listed below.</p> |
<div class="tableHolder"> | <div class="tableHolder"> | ||
<table class="striped"> | <table class="striped"> | ||
| Line 351: | Line 353: | ||
<tr> | <tr> | ||
<td><a href="http://parts.igem.org/Part:BBa_K2549021" target="_blank">BBa_K2549021</a></td> | <td><a href="http://parts.igem.org/Part:BBa_K2549021" target="_blank">BBa_K2549021</a></td> | ||
| − | <td> | + | <td> αCD19-mN1c-tTAA</td> |
<td>CD19</td> | <td>CD19</td> | ||
<td>mouse Notch1 core</td> | <td>mouse Notch1 core</td> | ||
| Line 359: | Line 361: | ||
<tr> | <tr> | ||
<td><a href="http://parts.igem.org/Part:BBa_K2549022" target="_blank">BBa_K2549022</a></td> | <td><a href="http://parts.igem.org/Part:BBa_K2549022" target="_blank">BBa_K2549022</a></td> | ||
| − | <td> | + | <td> αCD19-mN1c-GV2</td> |
<td>CD19</td> | <td>CD19</td> | ||
<td>mouse Notch1 core</td> | <td>mouse Notch1 core</td> | ||
| Line 368: | Line 370: | ||
</div> | </div> | ||
</div> | </div> | ||
| − | <div id="section2" class="section container | + | <div id="section2" class="section container scrolSpy"> |
<h2>Transcriptional amplifiers set</h2> | <h2>Transcriptional amplifiers set</h2> | ||
| − | <p>Last year, we have constructed <a href="https://2017.igem.org/Team:Fudan/Part_Collection" target="_blank">3 zinc finger-based transcription repressors</a>. This year, we have expanded our zinc finger-based transcription factors library by | + | <p>Last year, we have constructed <a href="https://2017.igem.org/Team:Fudan/Part_Collection" target="_blank">3 zinc finger-based transcription repressors</a>. This year, we have expanded our zinc finger-based transcription factors library by adding 3 more zinc finger-based transcription activators.</p> |
<p> | <p> | ||
| − | Although we prefer to use eight copies of response elements (RE) for a balance between not-too-hard molecular cloning and sufficient signal-to-noise ratio, we have provided 4 promotors with different repeats of response elements, allowing others to explore and tune their own transcriptional amplifiers. <a href="" target="_blank">Latest modeling | + | Although we prefer to use eight copies of response elements (RE) for a balance between not-too-hard molecular cloning and sufficient signal-to-noise ratio, we have provided 4 promotors with different repeats of response elements, <b>allowing others to explore and tune their own transcriptional amplifiers</b>. <a href="/Team:Fudan/Model#Transcriptional_Amplifer" target="_blank">Latest modeling</a> strongly supports our experimental preference, showing that eight copies of response elements can render the best result in line with our needs.</p> |
<div class="tableHolder"> | <div class="tableHolder"> | ||
<table class="striped"> | <table class="striped"> | ||
| Line 462: | Line 464: | ||
<p>As is stated above, we have provided three types of transcriptional-based Amplifiers, including zinc finger-based, TEV protease-based and split intein-based. These Amplifiers must be used with our Combiners to execute designed binary logic function.</p> | <p>As is stated above, we have provided three types of transcriptional-based Amplifiers, including zinc finger-based, TEV protease-based and split intein-based. These Amplifiers must be used with our Combiners to execute designed binary logic function.</p> | ||
</div> | </div> | ||
| − | <div id="section3" class="section container | + | <div id="section3" class="section container scrolSpy"> |
<h2>Amplifiers and Combiners work together to execute binary computation</h2> | <h2>Amplifiers and Combiners work together to execute binary computation</h2> | ||
<h3>Zinc finger-based</h3> | <h3>Zinc finger-based</h3> | ||
| − | <p>For simple binary logic function, such as A gate, NOT A gate, OR gate, NOR gate, and NIMPLY gate, placing response elements upstream and downstream of the promoter is sufficient. For more details, please visit our <a href=" | + | <p>For simple binary logic function, such as A gate, NOT A gate, OR gate, NOR gate, and NIMPLY gate, placing response elements upstream and downstream of the promoter is sufficient. For more details, please visit our <a href="/Team:Fudan/Results" target=”_blank”>results page</a>. |
</p> | </p> | ||
<div class="tableHolder"> | <div class="tableHolder"> | ||
| Line 514: | Line 516: | ||
</div> | </div> | ||
<h3> TEV protease-based</h3> | <h3> TEV protease-based</h3> | ||
| − | <p>For IMPLY gate, we placed TEV protease-controlled destroyable nuclear localization sequence between zinc finger DNA binding domain (DBD) and transcription factor (TF). In the presence of intracellular TEV protease, | + | <p>For IMPLY gate, we placed TEV protease-controlled destroyable nuclear localization sequence between zinc finger DNA binding domain (DBD) and transcription factor (TF). In the presence of intracellular TEV protease, DBD-TF would be cleaved, thus destroying its transcriptional regulation function and efficiently blocking its signal. For more details, please visit our <a href="/Team:Fudan/Results" target=”_blank”>results page</a>.</p> |
<div class="tableHolder"> | <div class="tableHolder"> | ||
<table class="striped"> | <table class="striped"> | ||
| Line 540: | Line 542: | ||
</div> | </div> | ||
<h3>Split intein-based</h3> | <h3>Split intein-based</h3> | ||
| − | <p>We built complex binary logic gates, | + | <p>We built the more complex binary logic gates, such as XOR and XNOR gate, using split intein-based Amplifiers. Utilizing intein's ability to fuse the split ends of a transcription factor together, we're able to present these logic gates consistent with our 3-layer design principle. For more details, please visit our <a href="/Team:Fudan/Results" target=”_blank”>results page</a>.</p> |
<div class="tableHolder"> | <div class="tableHolder"> | ||
<table class="striped"> | <table class="striped"> | ||
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</div> | </div> | ||
</div> | </div> | ||
| − | <div id="section4" class="section container | + | <div id="section4" class="section container scrolSpy"> |
<h2>ENABLE - 16 logic gates for transmembrane signaling</h2> | <h2>ENABLE - 16 logic gates for transmembrane signaling</h2> | ||
<div class="tableHolder"> | <div class="tableHolder"> | ||
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<tr> | <tr> | ||
<th> Gate</th> | <th> Gate</th> | ||
| − | <th> | + | <th>Intracellular Domain of the Receptor</th> |
| − | <th>Amplifier</th> | + | <th>the Amplifier</th> |
| − | <th>Combiner</th> | + | <th>the Combiner</th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 611: | Line 613: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td> NOT A</td> | + | <td> NOT A</td> |
<td>tTAA + Gal4-VP64</td> | <td>tTAA + Gal4-VP64</td> | ||
<td>TRE3GV-<a href="http://parts.igem.org/Part:BBa_K2446039" target="_blank">ZF21.16-KRAB</a></td> | <td>TRE3GV-<a href="http://parts.igem.org/Part:BBa_K2446039" target="_blank">ZF21.16-KRAB</a></td> | ||
| Line 617: | Line 619: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td> NOT B</td> | + | <td> NOT B</td> |
<td>tTAA + Gal4-VP64</td> | <td>tTAA + Gal4-VP64</td> | ||
<td>4*UAS-<a href="http://parts.igem.org/Part:BBa_K2446039" target="_blank">ZF21.16-KRAB</a></td> | <td>4*UAS-<a href="http://parts.igem.org/Part:BBa_K2446039" target="_blank">ZF21.16-KRAB</a></td> | ||
| Line 625: | Line 627: | ||
<td> OR</td> | <td> OR</td> | ||
<td>tTAA + Gal4-VP64</td> | <td>tTAA + Gal4-VP64</td> | ||
| − | <td>TRE3GV-<a href="http://parts.igem.org/Part:BBa_K2549023" target="_blank">ZF21.16-VP64</a>< | + | <td>TRE3GV-<a href="http://parts.igem.org/Part:BBa_K2549023" target="_blank">ZF21.16-VP64</a><div class="divider"></div> |
4*UAS-minCMV-<a href="http://parts.igem.org/Part:BBa_K2549023" target="_blank">ZF21.16-VP64</a></td> | 4*UAS-minCMV-<a href="http://parts.igem.org/Part:BBa_K2549023" target="_blank">ZF21.16-VP64</a></td> | ||
<td><a href="http://parts.igem.org/Part:BBa_K2549026" target="_blank">8*ZF21.16-minCMV</a>-d2EGFP</td> | <td><a href="http://parts.igem.org/Part:BBa_K2549026" target="_blank">8*ZF21.16-minCMV</a>-d2EGFP</td> | ||
| Line 632: | Line 634: | ||
<td> NOR</td> | <td> NOR</td> | ||
<td>tTAA + Gal4-VP64</td> | <td>tTAA + Gal4-VP64</td> | ||
| − | <td>TRE3GV-<a href="http://parts.igem.org/Part:BBa_K2446039" target="_blank">ZF21.16-KRAB</a>< | + | <td>TRE3GV-<a href="http://parts.igem.org/Part:BBa_K2446039" target="_blank">ZF21.16-KRAB</a><div class="divider"></div> |
4*UAS-minCMV-<a href="http://parts.igem.org/Part:BBa_K2446039" target="_blank">ZF21.16-KRAB</a></td> | 4*UAS-minCMV-<a href="http://parts.igem.org/Part:BBa_K2446039" target="_blank">ZF21.16-KRAB</a></td> | ||
<td>8*ZF21.16-CMV<a href="http://parts.igem.org/Part:BBa_K2549029" target="_blank"></a>-d2EGFP</td> | <td>8*ZF21.16-CMV<a href="http://parts.igem.org/Part:BBa_K2549029" target="_blank"></a>-d2EGFP</td> | ||
| Line 639: | Line 641: | ||
<td> XOR</td> | <td> XOR</td> | ||
<td>tTAA + Gal4-VP64</td> | <td>tTAA + Gal4-VP64</td> | ||
| − | <td>TRE3GV-<a href="http://parts.igem.org/Part:BBa_K2549039" target="_blank">VP64-dNLS-ZF21.16</a>-T2A-<a href="http://parts.igem.org/Part:BBa_K2549042" target="_blank">NLS-TEVpN-CfaN</a>< | + | <td>TRE3GV-<a href="http://parts.igem.org/Part:BBa_K2549039" target="_blank">VP64-dNLS-ZF21.16</a>-T2A-<a href="http://parts.igem.org/Part:BBa_K2549042" target="_blank">NLS-TEVpN-CfaN</a><div class="divider"></div> |
4*UAS-minCMV-<a href="http://parts.igem.org/Part:BBa_K2549043" target="_blank">CfaC-TEVpC</a>-T2A-<a href="http://parts.igem.org/Part:BBa_K2549039" target="_blank">VP64-dNLS-ZF21.16</a></td> | 4*UAS-minCMV-<a href="http://parts.igem.org/Part:BBa_K2549043" target="_blank">CfaC-TEVpC</a>-T2A-<a href="http://parts.igem.org/Part:BBa_K2549039" target="_blank">VP64-dNLS-ZF21.16</a></td> | ||
<td><a href="http://parts.igem.org/Part:BBa_K2549026" target="_blank">8*ZF21.16-minCMV</a>-d2EGFP</td> | <td><a href="http://parts.igem.org/Part:BBa_K2549026" target="_blank">8*ZF21.16-minCMV</a>-d2EGFP</td> | ||
| Line 646: | Line 648: | ||
<td> AND</td> | <td> AND</td> | ||
<td>tTAA + Gal4-VP64</td> | <td>tTAA + Gal4-VP64</td> | ||
| − | <td>TRE3GV-<a href="http://parts.igem.org/Part:BBa_K2549036" target="_blank">VP64-ZF21.16N-CfaN</a>< | + | <td>TRE3GV-<a href="http://parts.igem.org/Part:BBa_K2549036" target="_blank">VP64-ZF21.16N-CfaN</a><div class="divider"></div> |
4*UAS-minCMV-<a href="http://parts.igem.org/Part:BBa_K2549038" target="_blank">CfaC-ZF21.16C-NLS</a></td> | 4*UAS-minCMV-<a href="http://parts.igem.org/Part:BBa_K2549038" target="_blank">CfaC-ZF21.16C-NLS</a></td> | ||
<td><a href="http://parts.igem.org/Part:BBa_K2549026" target="_blank">8*ZF21.16-minCMV</a>-d2EGFP </td> | <td><a href="http://parts.igem.org/Part:BBa_K2549026" target="_blank">8*ZF21.16-minCMV</a>-d2EGFP </td> | ||
| Line 653: | Line 655: | ||
<td> NAND</td> | <td> NAND</td> | ||
<td>tTAA + Gal4-VP64</td> | <td>tTAA + Gal4-VP64</td> | ||
| − | <td>TRE3GV-<a href="http://parts.igem.org/Part:BBa_K2549037" target="_blank">KRAB-ZF21.16N-CfaN</a>< | + | <td>TRE3GV-<a href="http://parts.igem.org/Part:BBa_K2549037" target="_blank">KRAB-ZF21.16N-CfaN</a><div class="divider"></div> |
4*UAS-minCMV-<a href="http://parts.igem.org/Part:BBa_K2549038" target="_blank">CfaC-ZF21.16C-NLS</a></td> | 4*UAS-minCMV-<a href="http://parts.igem.org/Part:BBa_K2549038" target="_blank">CfaC-ZF21.16C-NLS</a></td> | ||
<td><a href="http://parts.igem.org/Part:BBa_K2549029" target="_blank">8*ZF21.16-CMV</a>-d2EGFP</td> | <td><a href="http://parts.igem.org/Part:BBa_K2549029" target="_blank">8*ZF21.16-CMV</a>-d2EGFP</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td> A IMPLY B</td> | + | <td> A IMPLY B</td> |
<td>tTAA + Gal4-VP64</td> | <td>tTAA + Gal4-VP64</td> | ||
| − | <td>TRE3GV-<a href="http://parts.igem.org/Part:BBa_K2549040" target="_blank">KRAB-dNLS-ZF21.16</a>< | + | <td>TRE3GV-<a href="http://parts.igem.org/Part:BBa_K2549040" target="_blank">KRAB-dNLS-ZF21.16</a><div class="divider"></div> |
4*UAS-minCMV-<a href="http://parts.igem.org/Part:BBa_K2549041" target="_blank">NLS-TEVp</a></td> | 4*UAS-minCMV-<a href="http://parts.igem.org/Part:BBa_K2549041" target="_blank">NLS-TEVp</a></td> | ||
<td><a href="http://parts.igem.org/Part:BBa_K2549029" target="_blank">8*ZF21.16-CMV</a>-d2EGFP</td> | <td><a href="http://parts.igem.org/Part:BBa_K2549029" target="_blank">8*ZF21.16-CMV</a>-d2EGFP</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td> B IMPLY A</td> | + | <td> B IMPLY A</td> |
<td>tTAA + Gal4-VP64</td> | <td>tTAA + Gal4-VP64</td> | ||
| − | <td>4*UAS-minCMV-<a href="http://parts.igem.org/Part::BBa_K254904" target="_blank">KRAB-dNLS-ZF21.16</a>< | + | <td>4*UAS-minCMV-<a href="http://parts.igem.org/Part::BBa_K254904" target="_blank">KRAB-dNLS-ZF21.16</a><div class="divider"></div> |
TRE3GV-<a href="http://parts.igem.org/Part:BBa_K2549041" target="_blank">NLS-TEVp</a></td> | TRE3GV-<a href="http://parts.igem.org/Part:BBa_K2549041" target="_blank">NLS-TEVp</a></td> | ||
<td><a href="http://parts.igem.org/Part:BBa_K2549029" target="_blank">8*ZF21.16-CMV</a>-d2EGFP</td> | <td><a href="http://parts.igem.org/Part:BBa_K2549029" target="_blank">8*ZF21.16-CMV</a>-d2EGFP</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td> A NIMPLY B</td> | + | <td> A NIMPLY B</td> |
<td>tTAA + Gal4-VP64</td> | <td>tTAA + Gal4-VP64</td> | ||
| − | <td>TRE3GV-<a href="http://parts.igem.org/Part:BBa_K2549023" target="_blank">ZF21.16-VP64</a>< | + | <td>TRE3GV-<a href="http://parts.igem.org/Part:BBa_K2549023" target="_blank">ZF21.16-VP64</a><div class="divider"></div> |
4*UAS-minCMV-<a href="http://parts.igem.org/Part:BBa_K2446041" target="_blank">ZF43.8-KRAB</a></td> | 4*UAS-minCMV-<a href="http://parts.igem.org/Part:BBa_K2446041" target="_blank">ZF43.8-KRAB</a></td> | ||
| − | <td><a href="http://parts.igem.org/Part:BBa_K2549035" target="_blank">8*ZF21.16-minCMV- | + | <td><a href="http://parts.igem.org/Part:BBa_K2549035" target="_blank">8*ZF21.16-minCMV-2*ZF43.8</a>-d2EGFP</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td> B NIMPLY A</td> | + | <td> B NIMPLY A</td> |
<td>tTAA + Gal4-VP64</td> | <td>tTAA + Gal4-VP64</td> | ||
| − | <td>4*UAS-minCMV-<a href="http://parts.igem.org/Part:BBa_K2549023" target="_blank">ZF21.16-VP64</a>< | + | <td>4*UAS-minCMV-<a href="http://parts.igem.org/Part:BBa_K2549023" target="_blank">ZF21.16-VP64</a><div class="divider"></div> |
TRE3GV-<a href="http://parts.igem.org/Part:BBa_K2446041" target="_blank">ZF43.8-KRAB</a></td> | TRE3GV-<a href="http://parts.igem.org/Part:BBa_K2446041" target="_blank">ZF43.8-KRAB</a></td> | ||
| − | <td><a href="http://parts.igem.org/Part:BBa_K2549035" target="_blank">8*ZF21.16-minCMV- | + | <td><a href="http://parts.igem.org/Part:BBa_K2549035" target="_blank">8*ZF21.16-minCMV-2*ZF43.8</a>-d2EGFP</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td> XNOR</td> | <td> XNOR</td> | ||
<td>tTAA + Gal4-VP64</td> | <td>tTAA + Gal4-VP64</td> | ||
| − | <td>TRE3GV-<a href="http://parts.igem.org/Part:BBa_K2549040" target="_blank">KRAB-dNLS-ZF21.16</a>-T2A-<a href="http://parts.igem.org/Part:BBa_K2549042" target="_blank">NLS-TEVpN-CfaN</a>< | + | <td>TRE3GV-<a href="http://parts.igem.org/Part:BBa_K2549040" target="_blank">KRAB-dNLS-ZF21.16</a>-T2A-<a href="http://parts.igem.org/Part:BBa_K2549042" target="_blank">NLS-TEVpN-CfaN</a><div class="divider"></div> |
4*UAS-minCMV-<a href="http://parts.igem.org/Part:BBa_K2549043" target="_blank">CfaC-TEVpC</a>-T2A-<a href="http://parts.igem.org/Part:BBa_K2549040" target="_blank">KRAB-dNLS-ZF21.16</a></td> | 4*UAS-minCMV-<a href="http://parts.igem.org/Part:BBa_K2549043" target="_blank">CfaC-TEVpC</a>-T2A-<a href="http://parts.igem.org/Part:BBa_K2549040" target="_blank">KRAB-dNLS-ZF21.16</a></td> | ||
<td><a href="http://parts.igem.org/Part:BBa_K2549029" target="_blank">8*ZF21.16-CMV</a>-d2EGFP</td> | <td><a href="http://parts.igem.org/Part:BBa_K2549029" target="_blank">8*ZF21.16-CMV</a>-d2EGFP</td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
| + | </div> | ||
| + | <p>Here is our 3-layer design of transmembrane binary logic gates. On the left lying a truth table which indicates the inputs and outputs of the according logic computation. What is set on the right is how it works. The double crossline on the top represents the plasma membrane. A and B signify the signal input, which are surface-expressed EGFP and surface-expressed CD19 in our system. What are embed on the membrane are our SynNotch receptors, the intracellular domain of which are transcriptional activators, which are tTAA and GV2 in our assay. The large rectangle represents the nucleus. The elongated rectangle with an array before represents the genetic circuit that are under control of the transcriptional factors. Squares in groups of three represent transcriptional factors that are amplified by the amplifier layer. You can swipe the bar to detect other gates by yourself.</p> | ||
| + | |||
| + | |||
| + | <div class="FudanGallery" style="width: 100%;"> | ||
| + | <div class="galleryActive"> | ||
| + | <img src="https://static.igem.org/mediawiki/2018/8/8d/T--Fudan--16-1.png"> | ||
| + | </div> | ||
| + | <div class="galleryThumbnail"> | ||
| + | <img style="opacity: 1;" src="https://static.igem.org/mediawiki/2018/8/8d/T--Fudan--16-1.png"> | ||
| + | |||
| + | <img src="https://static.igem.org/mediawiki/2018/1/14/T--Fudan--16-2.png"> | ||
| + | <img src="https://static.igem.org/mediawiki/2018/1/1f/T--Fudan--16-3.png"> | ||
| + | <img src="https://static.igem.org/mediawiki/2018/5/54/T--Fudan--16-4.png"> | ||
| + | <img src="https://static.igem.org/mediawiki/2018/6/60/T--Fudan--16-5.png"> | ||
| + | <img src="https://static.igem.org/mediawiki/2018/0/0c/T--Fudan--16-6.png"> | ||
| + | <img src="https://static.igem.org/mediawiki/2018/a/a2/T--Fudan--16-7.png"> | ||
| + | <img src="https://static.igem.org/mediawiki/2018/8/83/T--Fudan--16-8.png"> | ||
| + | <img src="https://static.igem.org/mediawiki/2018/1/13/T--Fudan--16-9.png"> | ||
| + | <img src="https://static.igem.org/mediawiki/2018/6/67/T--Fudan--16-10.png"> | ||
| + | <img src="https://static.igem.org/mediawiki/2018/3/30/T--Fudan--16-11.png"> | ||
| + | <img src="https://static.igem.org/mediawiki/2018/2/2e/T--Fudan--16-12.png"> | ||
| + | <img src="https://static.igem.org/mediawiki/2018/5/5e/T--Fudan--16-13.png"> | ||
| + | <img src="https://static.igem.org/mediawiki/2018/d/db/T--Fudan--16-14.png"> | ||
| + | <img src="https://static.igem.org/mediawiki/2018/b/bc/T--Fudan--16-15.png"> | ||
| + | <img src="https://static.igem.org/mediawiki/2018/9/92/T--Fudan--16-16.png"> | ||
| + | </div> | ||
| + | <div class="galleryBtnLeft hide-on-small-only"><i class="fa fa-angle-left"></i></div> | ||
| + | <div class="galleryBtnRight hide-on-small-only"><i class="fa fa-angle-right"></i></div> | ||
</div> | </div> | ||
</div> | </div> | ||
| Line 700: | Line 731: | ||
<!--Abstract on content page--> | <!--Abstract on content page--> | ||
<div id="abstractContent" class="z-depth-2"> | <div id="abstractContent" class="z-depth-2"> | ||
| − | <a href="#!"><img src="https://static.igem.org/mediawiki/2018/9/96/T--Fudan--X.svg"></a> | + | <a href="#!"><img alt="2018 team Fudan abstract" src="https://static.igem.org/mediawiki/2018/9/96/T--Fudan--X.svg"></a> |
<div class="container"> | <div class="container"> | ||
<h2 style="margin: 0;padding: 10px 0;">Abstract</h2> | <h2 style="margin: 0;padding: 10px 0;">Abstract</h2> | ||
| Line 737: | Line 768: | ||
<div class="row"> | <div class="row"> | ||
<div id="sponsor" class="col m3 s12 row"> | <div id="sponsor" class="col m3 s12 row"> | ||
| − | <a href="https://2018.igem.org/Team:Fudan" target="_blank"><img class="col s3 m6 l3" style="position:relative; padding: 0 0.3rem; margin:-0.15rem 0; left: -0.45rem;" src="https://static.igem.org/mediawiki/2018/7/73/T--Fudan--teamLogoWhite.png"> | + | <a href="https://2018.igem.org/Team:Fudan" target="_blank"><img alt="2018 Team:Fudan logo white" class="col s3 m6 l3" style="position:relative; padding: 0 0.3rem; margin:-0.15rem 0; left: -0.45rem;" src="https://static.igem.org/mediawiki/2018/7/73/T--Fudan--teamLogoWhite.png"> |
</a><a href="http://www.fudan.edu.cn/en/" target="_blank"><img class="col s3 m6 l3" alt="Fudan University" src="https://static.igem.org/mediawiki/2018/f/f7/T--Fudan--schoolLogo.png"> | </a><a href="http://www.fudan.edu.cn/en/" target="_blank"><img class="col s3 m6 l3" alt="Fudan University" src="https://static.igem.org/mediawiki/2018/f/f7/T--Fudan--schoolLogo.png"> | ||
</a><a href="http://life.fudan.edu.cn/" target="_blank"><img class="col s3 m6 l3" style="margin-bottom: 4%;/* 该图比其他小一点，排版需要 */" alt="School of Life Sciences, Fudan University" src="https://static.igem.org/mediawiki/2018/1/1d/T--Fudan--schoolOfLifeSciencesIcon.png"> | </a><a href="http://life.fudan.edu.cn/" target="_blank"><img class="col s3 m6 l3" style="margin-bottom: 4%;/* 该图比其他小一点，排版需要 */" alt="School of Life Sciences, Fudan University" src="https://static.igem.org/mediawiki/2018/1/1d/T--Fudan--schoolOfLifeSciencesIcon.png"> | ||
| Line 749: | Line 780: | ||
<span>Project</span> | <span>Project</span> | ||
<ul> | <ul> | ||
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Demonstrate">Demonstration</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Antigen_Receptors">Antigen, Receptors</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Results">Transmembrane logic</a></li> |
<li><a href="https://2017.igem.org/Team:Fudan">2017.iGEM</a></li> | <li><a href="https://2017.igem.org/Team:Fudan">2017.iGEM</a></li> | ||
</ul> | </ul> | ||
| Line 759: | Line 790: | ||
<span>Dry lab</span> | <span>Dry lab</span> | ||
<ul> | <ul> | ||
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Addon#ribo">Addon: ribo</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Addon#TALE">Addon: TALE</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Addon#T2">Addon: T2</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Model#Transcriptional_Amplifer">Model: transcriptional amplifer</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Model#NotchLigandKinetics">Model: Notch-ligand kinetics</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Software">Software</a></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 770: | Line 801: | ||
<span>Wet lab</span> | <span>Wet lab</span> | ||
<ul> | <ul> | ||
| − | <li><a href=" | + | <li><a href="/Team:Fudan/InterLab">iGEM interLab</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Notebook">Our notebook</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Primers">Primers used</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Protocols">Protocols</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Safety">Safety</a></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 782: | Line 813: | ||
<span>Toolbox</span> | <span>Toolbox</span> | ||
<ul> | <ul> | ||
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Basic_Parts">Basic parts</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Composite_Parts">Composite parts</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Optimization">Optimization</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Parts_Collection">Parts collection</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Improve">Parts improvement</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Measurement">Quantification</a></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 793: | Line 824: | ||
<span>Outreach</span> | <span>Outreach</span> | ||
<ul> | <ul> | ||
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Bio-Art">Bio-Art display</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Collaborations">Collaborations</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Design_Intention">Design intention</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Human_Practices">Human practices</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Public_Engagement">Public engagement</a></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 803: | Line 834: | ||
<span>Team</span> | <span>Team</span> | ||
<ul> | <ul> | ||
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Acknowledgement">Acknowledgement</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Attributions">Attributions</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Heritage">Heritage</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Team">Members</a></li> |
| − | <li><a href=" | + | <li><a href="/Team:Fudan/Sponsors">Sponsors</a></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 816: | Line 847: | ||
<div class="footer-copyright"> | <div class="footer-copyright"> | ||
<div class="container"> | <div class="container"> | ||
| − | + | <div class="contactUS row"> | |
| − | + | <div class="col s12 m6 l4"><i class="fa fa-location-arrow"></i> Life Sci Bldg E301, 2005 Songhu Rd, Shanghai | |
| − | + | </div><div class="col s12 m6 l2"><i class="fa fa-fax"></i> +86-21-31246727 | |
| − | + | </div><div class="col s12 m6 l2"><i class="fa fa-envelope-o"></i> igem@fudan.edu.cn | |
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Latest revision as of 15:20, 7 November 2018
- Addon: ribo
- Addon: TALE
- Addon: T2
- Model: transcriptional amplifer
- Model: Notch-ligand kinetics
- Software
Parts collection
Our parts collection provides an extensive toolbox for researchers to construct their own transmembrane logic gates in mammalian cells. These parts were made as BioBricks, and are NOT ready for transfecting mammalian cells. Plasmids with these parts in eukaryotic expression backbone are available upon request.
Our parts collection contains all essential components of our ENABLE toolbox, that could also be divided into several sets, enabling others to readily utilize them to improve their existing genetic circuits.
Following our 3-layer design principle, all ENABLE components have been well characterized, including our SynNotch receptors, Amplifiers and Combiners. In the collection, we have provided 7 versions of SynNotch receptors with (1) different combinations of extracellular domains which can bind to different antigens with different affinities, (2) transmembrane domains with varying cleavage efficiency and (3) intracellular domains executing diverse transcriptional amplifications.
We have provided a set of transcriptional amplifiers including zinc finger-based, TEV protease-based and split intein-based ones, allowing others to build their own genetic circuits, preferably through transcription (we have tested), but not limited to (we have not tested). Besides, a set of combiners with different copies of response elements are provided, which makes it easy for others to tune the activation or repression threshold. This feature was experimental confirmed previously and mathematically modeled in our 3-layer design again this year. Our modeling process could be used by others to predict their own genetic circuits before any wet-lab experiments.
Each part (sometimes two combined) constitutes one of our ENABLE module, which has its unique function. Yet they consolidate together to create all the 16 transmembrane binary logic gates for mammalian cells.
SynNotch receptors set
We have provided 7 versions of SynNotch receptors. Each of them can transduce signal from out of the cell via a contact-dependent antigen-stimulated cleavage process. When expressing two SynNotch receptors with two different extracellular domains recognizing two types of antigens, the cell is able to accept dual inputs. The chosen nanobodies are highly specific against their antigens, and the chosen intracellular domains are transcriptionally orthogonal. For more specific details, please refer to our parts listed below.
| BioBrick ID | Protein Name | Recognized Antigen (affinity) | Transmembrane Domain | Intracellular Domain | Sequence Verification |
|---|---|---|---|---|---|
| BBa_K2549016 | LaG17-mN1c-tTAA | GFP (low) | mouse Notch1 core | tTA advance | 016.ab1 |
| BBa_K2549017 | LaG17-mN1ce-tTAA | GFP (low) | mouse Notch1 extended core | tTA advance | 017.ab1 |
| BBa_K2549018 | LaG16-mN1ce-tTAA | GFP (high) | mouse Notch1 extended core | tTA advance | 018.ab1 |
| BBa_K2549019 | LaG16-2-mN1c-tTAA | GFP (ultrahigh) | mouse Notch1 core | tTA advance | 019.ab1 |
| BBa_K2549020 | LaG16-2-mN1c-GV2 | GFP (ultrahigh) | mouse Notch1 core | Gal4-VP64 | 020.ab1 |
| BBa_K2549021 | αCD19-mN1c-tTAA | CD19 | mouse Notch1 core | Gal4-VP64 | 021.ab1 |
| BBa_K2549022 | αCD19-mN1c-GV2 | CD19 | mouse Notch1 core | Gal4-VP64 | 022.ab1 |
Transcriptional amplifiers set
Last year, we have constructed 3 zinc finger-based transcription repressors. This year, we have expanded our zinc finger-based transcription factors library by adding 3 more zinc finger-based transcription activators.
Although we prefer to use eight copies of response elements (RE) for a balance between not-too-hard molecular cloning and sufficient signal-to-noise ratio, we have provided 4 promotors with different repeats of response elements, allowing others to explore and tune their own transcriptional amplifiers. Latest modeling strongly supports our experimental preference, showing that eight copies of response elements can render the best result in line with our needs.
| BioBrick ID | Protein Name | DNA Binding Domain | Transcriptional | Sequence Verification |
|---|---|---|---|---|
| BBa_K2549023 | ZF21.16-VP64 | ZF21.16 | activation | 023.ab1 |
| BBa_K2549024 | ZF42.10-VP64 | ZF42.10 | activation | 024.ab1 |
| BBa_K2549025 | ZF43.8-VP64 | ZF43.8 | activation | 025.ab1 |
| BBa_K2446039 | ZF21.16-KRAB | ZF21.16 | repression | 039.abl |
| BBa_K2446040 | ZF42.10-KRAB | ZF42.10 | repression | 040.abl |
| BBa_K2446041 | ZF43.8-KRAB | ZF43.8 | repression | 041.abl |
| BioBrick ID | DNA Name | Usage | Sequence Verification |
|---|---|---|---|
| BBa_K2549032 | 4*ZF21.16-minCMV-2*ZF43.8 | 4 copies of RE for ZF21.16 transcriptional activator to bind, and 2 copies of RE for ZF43.8 transcriptional repressor to bind | 032.ab1 |
| BBa_K2549033 | 6*ZF21.16-minCMV-2*ZF43.8 | 6 copies of RE for ZF21.16 transcriptional activator to bind, and 2 copies of RE for ZF43.8 transcriptional repressor to bind | 033.ab1 |
| BBa_K2549034 | 8*ZF21.16-minCMV-2*ZF43.8 | 8 copies of RE for ZF21.16 transcriptional activator to bind, and 2 copies of RE for ZF43.8 transcriptional repressor to bind | 034.ab1 |
| BBa_K2549035 | 8*ZF21.16-minCMV-8*ZF43.8 | 8 copies of RE for ZF21.16 transcriptional activator to bind, and 8 copies of RE for ZF43.8 transcriptional repressor to bind | 035.ab1 |
As is stated above, we have provided three types of transcriptional-based Amplifiers, including zinc finger-based, TEV protease-based and split intein-based. These Amplifiers must be used with our Combiners to execute designed binary logic function.
Amplifiers and Combiners work together to execute binary computation
Zinc finger-based
For simple binary logic function, such as A gate, NOT A gate, OR gate, NOR gate, and NIMPLY gate, placing response elements upstream and downstream of the promoter is sufficient. For more details, please visit our results page.
| BioBrick ID | Combiner Name | Usage | Sequence Verification |
|---|---|---|---|
| BBa_K2549026 | 8*ZF21.16-minCMV | 8 copies of RE for ZF21.16 transcriptional activator to bind | 026.ab1 |
| BBa_K2549027 | 8*ZF42.10-minCMV | 8 copies of RE for ZF42.10 transcriptional activator to bind | 027.ab1 |
| BBa_K2549028 | 8*ZF43.8-minCMV | 8 copies of RE for ZF43.8 transcriptional activator to bind | 028.ab1 |
| BBa_K2549029 | 8*ZF21.16-CMV | 8 copies of RE for ZF21.16 transcriptional activator to bind | 029.ab1 |
| BBa_K2549030 | 8*ZF42.10-CMV | 8 copies of RE for ZF42.10 transcriptional activator to bind | 030.ab1 |
| BBa_K2549031 | 8*ZF43.8-CMV | 8 copies of RE for ZF43.8 transcriptional activator to bind | 031.ab1 |
TEV protease-based
For IMPLY gate, we placed TEV protease-controlled destroyable nuclear localization sequence between zinc finger DNA binding domain (DBD) and transcription factor (TF). In the presence of intracellular TEV protease, DBD-TF would be cleaved, thus destroying its transcriptional regulation function and efficiently blocking its signal. For more details, please visit our results page.
| BioBrick ID | Protein Name | Sequence Verification |
|---|---|---|
| BBa_K2549039 | VP64-dNLS-ZF21.16 | 039.ab1 |
| BBa_K2549040 | KRAB-dNLS-ZF21.16 | 040.ab1 |
| BBa_K2549041 | NLS-TEVp | 041.ab1 |
Split intein-based
We built the more complex binary logic gates, such as XOR and XNOR gate, using split intein-based Amplifiers. Utilizing intein's ability to fuse the split ends of a transcription factor together, we're able to present these logic gates consistent with our 3-layer design principle. For more details, please visit our results page.
| BioBrick ID | Protein Name | Sequence Verification |
|---|---|---|
| BBa_K2549036 | VP64-ZF21.16N-CfaN | 036.ab1 |
| BBa_K2549037 | KRAB-ZF21.16N-CfaN | 037.ab1 |
| BBa_K2549038 | CfaC-ZF21.16C-NLS | 038.ab1 |
| BBa_K2549042 | NLS-TEVpN-CfaN | 042.ab1 |
| BBa_K2549043 | CfaC-TEVpC | 043.ab1 |
ENABLE - 16 logic gates for transmembrane signaling
| Gate | Intracellular Domain of the Receptor | the Amplifier | the Combiner |
|---|---|---|---|
| TRUE | tTAA + Gal4-VP64 | / | CMV-d2EGFP |
| FALSE | tTAA + Gal4-VP64 | / | / |
| A | tTAA + Gal4-VP64 | TRE3GV-ZF21.16-VP64 | 8*ZF21.16-minCMV-d2EGFP |
| B | tTAA + Gal4-VP64 | 4*UAS-ZF21.16-VP64 | 8*ZF21.16-minCMV-d2EGFP |
| NOT A | tTAA + Gal4-VP64 | TRE3GV-ZF21.16-KRAB | 8*ZF21.16-CMV-d2EGFP |
| NOT B | tTAA + Gal4-VP64 | 4*UAS-ZF21.16-KRAB | 8*ZF21.16-CMV-d2EGFP |
| OR | tTAA + Gal4-VP64 | TRE3GV-ZF21.16-VP64 4*UAS-minCMV-ZF21.16-VP64 | 8*ZF21.16-minCMV-d2EGFP |
| NOR | tTAA + Gal4-VP64 | TRE3GV-ZF21.16-KRAB 4*UAS-minCMV-ZF21.16-KRAB | 8*ZF21.16-CMV-d2EGFP |
| XOR | tTAA + Gal4-VP64 | TRE3GV-VP64-dNLS-ZF21.16-T2A-NLS-TEVpN-CfaN 4*UAS-minCMV-CfaC-TEVpC-T2A-VP64-dNLS-ZF21.16 | 8*ZF21.16-minCMV-d2EGFP |
| AND | tTAA + Gal4-VP64 | TRE3GV-VP64-ZF21.16N-CfaN 4*UAS-minCMV-CfaC-ZF21.16C-NLS | 8*ZF21.16-minCMV-d2EGFP |
| NAND | tTAA + Gal4-VP64 | TRE3GV-KRAB-ZF21.16N-CfaN 4*UAS-minCMV-CfaC-ZF21.16C-NLS | 8*ZF21.16-CMV-d2EGFP |
| A IMPLY B | tTAA + Gal4-VP64 | TRE3GV-KRAB-dNLS-ZF21.16 4*UAS-minCMV-NLS-TEVp | 8*ZF21.16-CMV-d2EGFP |
| B IMPLY A | tTAA + Gal4-VP64 | 4*UAS-minCMV-KRAB-dNLS-ZF21.16 TRE3GV-NLS-TEVp | 8*ZF21.16-CMV-d2EGFP |
| A NIMPLY B | tTAA + Gal4-VP64 | TRE3GV-ZF21.16-VP64 4*UAS-minCMV-ZF43.8-KRAB | 8*ZF21.16-minCMV-2*ZF43.8-d2EGFP |
| B NIMPLY A | tTAA + Gal4-VP64 | 4*UAS-minCMV-ZF21.16-VP64 TRE3GV-ZF43.8-KRAB | 8*ZF21.16-minCMV-2*ZF43.8-d2EGFP |
| XNOR | tTAA + Gal4-VP64 | TRE3GV-KRAB-dNLS-ZF21.16-T2A-NLS-TEVpN-CfaN 4*UAS-minCMV-CfaC-TEVpC-T2A-KRAB-dNLS-ZF21.16 | 8*ZF21.16-CMV-d2EGFP |
Here is our 3-layer design of transmembrane binary logic gates. On the left lying a truth table which indicates the inputs and outputs of the according logic computation. What is set on the right is how it works. The double crossline on the top represents the plasma membrane. A and B signify the signal input, which are surface-expressed EGFP and surface-expressed CD19 in our system. What are embed on the membrane are our SynNotch receptors, the intracellular domain of which are transcriptional activators, which are tTAA and GV2 in our assay. The large rectangle represents the nucleus. The elongated rectangle with an array before represents the genetic circuit that are under control of the transcriptional factors. Squares in groups of three represent transcriptional factors that are amplified by the amplifier layer. You can swipe the bar to detect other gates by yourself.
Abstract
Contact-dependent signaling is critical for multicellular biological events, yet customizing contact-dependent signal transduction between cells remains challenging. Here we have developed the ENABLE toolbox, a complete set of transmembrane binary logic gates. Each gate consists of 3 layers: Receptor, Amplifier, and Combiner. We first optimized synthetic Notch receptors to enable cells to respond to different signals across the membrane reliably. These signals, individually amplified intracellularly by transcription, are further combined for computing. Our engineered zinc finger-based transcription factors perform binary computation and output designed products. In summary, we have combined spatially different signals in mammalian cells, and revealed new potentials for biological oscillators, tissue engineering, cancer treatments, bio-computing, etc. ENABLE is a toolbox for constructing contact-dependent signaling networks in mammals. The 3-layer design principle underlying ENABLE empowers any future development of transmembrane logic circuits, thus contributes a foundational advance to Synthetic Biology.