Difference between revisions of "Team:Fudan/Protocols"
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| + | <title>2018 iGEM Team:Fudan - Protocols</title> | ||
</head> | </head> | ||
<body> | <body> | ||
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<div id="FudanWrapper" class="white"> | <div id="FudanWrapper" class="white"> | ||
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<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 | + | <ul id="nav-mobile" class="right blueBorder"> |
<li class="hide-on-med-and-down"><a class="dropdown-trigger" data-target="dropdown1">Project</a></li> | <li class="hide-on-med-and-down"><a class="dropdown-trigger" data-target="dropdown1">Project</a></li> | ||
<li class="hide-on-med-and-down"><a class="dropdown-trigger" data-target="dropdown2">Dry lab</a></li> | <li class="hide-on-med-and-down"><a class="dropdown-trigger" data-target="dropdown2">Dry lab</a></li> | ||
| − | <li class="hide-on-med-and-down"><a class="dropdown-trigger" data-target="dropdown3">Wet lab</a></li> | + | <li class="hide-on-med-and-down"><a class="dropdown-trigger thisPageLink" data-target="dropdown3">Wet lab</a></li> |
<li class="hide-on-med-and-down"><a class="dropdown-trigger" data-target="dropdown4">Toolbox</a></li> | <li class="hide-on-med-and-down"><a class="dropdown-trigger" data-target="dropdown4">Toolbox</a></li> | ||
<li class="hide-on-med-and-down"><a class="dropdown-trigger" data-target="dropdown5">Outreach</a></li> | <li class="hide-on-med-and-down"><a class="dropdown-trigger" data-target="dropdown5">Outreach</a></li> | ||
| − | <li class="hide-on-med-and-down"><a class="dropdown-trigger | + | <li class="hide-on-med-and-down"><a class="dropdown-trigger" data-target="dropdown6">Team</a></li> |
<li> | <li> | ||
<a id="navList" data-target="slide-out" class="waves-effect waves-light sidenav-trigger right"> | <a id="navList" data-target="slide-out" class="waves-effect waves-light sidenav-trigger right"> | ||
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<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=" | + | <li><a href="/Team:Fudan/Antigen_Receptors">Antigen, Receptors</a></li> |
| + | <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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<ul id="slide-out" class="sidenav"> | <ul id="slide-out" class="sidenav"> | ||
<li style="padding: 0"><div class="sidenavBanner"> | <li style="padding: 0"><div class="sidenavBanner"> | ||
| − | <div class="background | + | <div class="background"> |
</div> | </div> | ||
| − | <p style="width: 100%;text-align: center;font-size: 24px"><span class="white-text"> | + | <p style="width: 100%;text-align: center;font-size: 24px"><span class="white-text">Our notebook</span></p> |
</div></li> | </div></li> | ||
<li> | <li> | ||
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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="/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="/Team:Fudan/Addon#ribo">Addon: ribo</a></li> | |
| − | + | <li><a href="/Team:Fudan/Addon#TALE">Addon: TALE</a></li> | |
| − | + | <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 id="pageContent" style=""> | <div id="pageContent" style=""> | ||
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<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> | + | <h1>Protocols</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>The full-length protocols are provided as individual PDF files for download, with brief English descriptions on this page.</span> |
</div> | </div> | ||
</div> | </div> | ||
<div id="figureBannerTitle" class="hide-on-small-only"> | <div id="figureBannerTitle" class="hide-on-small-only"> | ||
| − | <h1> | + | <h1>Protocols</h1> |
| − | <p><span> | + | <p><span>The full-length protocols are provided as individual PDF files for download, with brief English descriptions on this page.</span></p> |
</div> | </div> | ||
<div class="hide-on-small-only"> | <div class="hide-on-small-only"> | ||
| − | <img src="https://static.igem.org/mediawiki/2018/ | + | <img src="https://static.igem.org/mediawiki/2018/4/47/T--Fudan--title_protocols.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> | ||
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<!-- @@@@ main content of the page --> | <!-- @@@@ main content of the page --> | ||
<div class="container"> | <div class="container"> | ||
| − | < | + | <main style="margin:0"> |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
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| − | + | ||
<div class="section container"> | <div class="section container"> | ||
<div class="figureHolder"> | <div class="figureHolder"> | ||
| − | <p>PCR and subcloning were performed using standard methods. Detailed primer sequences are <a href=" | + | <p>PCR and subcloning were performed using standard methods. Detailed primer sequences are <a href="/Team:Fudan/Primers">provided</a>. All constructs were verified by Sanger sequencing. |
</p> | </p> | ||
<p>Cells were cultured in DMEM supplemented with 10% FBS (HyClone), 100 U/ml penicillin, 100 μg/ml streptomycin and 1x GlutaMax (Gibco). Transient transfections were performed using Lipofectamine 2000 (Invitrogen) and Opti-MEM (Gibco). Viral packaging, infection and fluorescence-activated cell sorting were performed using standard methods. | <p>Cells were cultured in DMEM supplemented with 10% FBS (HyClone), 100 U/ml penicillin, 100 μg/ml streptomycin and 1x GlutaMax (Gibco). Transient transfections were performed using Lipofectamine 2000 (Invitrogen) and Opti-MEM (Gibco). Viral packaging, infection and fluorescence-activated cell sorting were performed using standard methods. | ||
</p> | </p> | ||
| − | <p>Images, unless otherwise indicated, were captured using | + | <p>Images, unless otherwise indicated, were captured using an inverted epifluorescence microscope (IX-81, Olympus) and a sCMOS camera (pixel size = 0.3222 μm; Zyla 5.5, Andor; 20x objective N.A. 0.75) and were controlled by <a href="http://www.micro-manager.org/" target=_blank>Micro-Manager software</a>. |
</p> | </p> | ||
<p>All statistical analysis was performed using Prism (Graphpad) and <a href="http://rsbweb.nih.gov/ij/developer/macro/macros.html" target=_blank>ImageJ</a>. All experiments were independently performed in triplicates; unless otherwise indicated. Images were combined and annotated in Powerpoint for presentation. Representative images are shown. | <p>All statistical analysis was performed using Prism (Graphpad) and <a href="http://rsbweb.nih.gov/ij/developer/macro/macros.html" target=_blank>ImageJ</a>. All experiments were independently performed in triplicates; unless otherwise indicated. Images were combined and annotated in Powerpoint for presentation. Representative images are shown. | ||
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</div> | </div> | ||
| − | <div class="tableHolder"> | + | <div class="tableHolder"> |
| − | <table> | + | <table> |
| − | <tr> | + | <tr> |
| − | <th>Our protocols as PDF files</th><th> </th> | + | <th>Our protocols as PDF files</th><th> </th> |
| − | <th>Download</th> | + | <th>Download</th> |
| − | </tr> | + | </tr> |
| − | <tr> | + | <tr> |
| − | <td>Be a Good Lab Member</td><td><i>GoodLabPractices.pdf</i></td> | + | <td>Be a Good Lab Member</td><td><i>GoodLabPractices.pdf</i></td> |
| − | <td><a href="https://2018.igem.org/File:T--Fudan--GoodLabPractices.pdf" target="_blank"><i class="fa fa-download"></i></a></td> | + | <td><a href="https://2018.igem.org/File:T--Fudan--GoodLabPractices.pdf" target="_blank"><i class="fa fa-download"></i></a></td> |
| − | </tr> | + | </tr> |
| − | <tr> | + | <tr> |
| − | <td>Molecular Cloning</td><td><i>MolecularCloning.pdf</i></td> | + | <td>Molecular Cloning</td><td><i>MolecularCloning.pdf</i></td> |
| − | <td><a href="https://2018.igem.org/File:T--Fudan--MolecularCloning.pdf" target="_blank"><i class="fa fa-download"></i></a></td> | + | <td><a href="https://2018.igem.org/File:T--Fudan--MolecularCloning.pdf" target="_blank"><i class="fa fa-download"></i></a></td> |
| − | </tr> | + | </tr> |
| − | <tr> | + | <tr> |
| − | <td>Tissue Culture</td><td><i>CellCulture.pdf</i></td> | + | <td>Tissue Culture</td><td><i>CellCulture.pdf</i></td> |
| − | <td><a href="https://2018.igem.org/File:T--Fudan--CellCulture.pdf" target="_blank"><i class="fa fa-download"></i></a></td> | + | <td><a href="https://2018.igem.org/File:T--Fudan--CellCulture.pdf" target="_blank"><i class="fa fa-download"></i></a></td> |
| − | </tr> | + | </tr> |
| − | <tr> | + | <tr> |
| − | <td>Make a Stable Cell Line</td><td><i>MakeStableCellLine.pdf</i></td> | + | <td>Make a Stable Cell Line</td><td><i>MakeStableCellLine.pdf</i></td> |
| − | <td><a href="https://2018.igem.org/File:T--Fudan--MakeStableCellLine.pdf" target="_blank"><i class="fa fa-download"></i></a></td> | + | <td><a href="https://2018.igem.org/File:T--Fudan--MakeStableCellLine.pdf" target="_blank"><i class="fa fa-download"></i></a></td> |
| − | </tr> | + | </tr> |
| − | <tr> | + | <tr> |
| − | <td>Cell Sorting</td><td><i>FACS.pdf</i></td> | + | <td>Cell Sorting</td><td><i>FACS.pdf</i></td> |
| − | <td><a href="https://2018.igem.org/File:T--Fudan--FACS.pdf" target="_blank"><i class="fa fa-download"></i></a></td> | + | <td><a href="https://2018.igem.org/File:T--Fudan--FACS.pdf" target="_blank"><i class="fa fa-download"></i></a></td> |
| − | </tr> | + | </tr> |
| − | <tr> | + | <tr> |
| − | <td>Cell Staining</td><td><i>FixStain.pdf</i></td> | + | <td>Cell Staining</td><td><i>FixStain.pdf</i></td> |
| − | <td><a href="https://2018.igem.org/File:T--Fudan--FixStain.pdf" target="_blank"><i class="fa fa-download"></i></a></td> | + | <td><a href="https://2018.igem.org/File:T--Fudan--FixStain.pdf" target="_blank"><i class="fa fa-download"></i></a></td> |
| − | </tr> | + | </tr> |
| − | <tr> | + | <tr> |
| − | <td>Time-lapse Live-cell Imaging</td><td><i>TimeLapseImaging.pdf</i></td> | + | <td>Time-lapse Live-cell Imaging</td><td><i>TimeLapseImaging.pdf</i></td> |
| − | <td><a href="https://2018.igem.org/File:T--Fudan--TimeLapseImaging.pdf" target="_blank"><i class="fa fa-download"></i></a></td> | + | <td><a href="https://2018.igem.org/File:T--Fudan--TimeLapseImaging.pdf" target="_blank"><i class="fa fa-download"></i></a></td> |
| − | </tr> | + | </tr> |
| − | </table> | + | </table> |
| − | </div> | + | </div> |
| − | <p> | + | <p style="color:grey"> |
For practical reasons, all full-length protocols are in Chinese. | For practical reasons, all full-length protocols are in Chinese. | ||
</p> | </p> | ||
</div> | </div> | ||
| − | |||
</main> | </main> | ||
</div> | </div> | ||
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<!--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> | ||
| − | <p style="margin:0 | + | <p style="margin: 0">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. | |
</p> | </p> | ||
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| − | <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> | ||
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| − | <li><a href=" | + | <li><a href="/Team:Fudan/Addon#ribo">Addon: ribo</a></li> |
| − | + | <li><a href="/Team:Fudan/Addon#TALE">Addon: TALE</a></li> | |
| − | + | <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> |
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| − | <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> |
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| − | <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> |
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| − | <li><a href=" | + | <li><a href="/Team:Fudan/Acknowledgement">Acknowledgement</a></li> |
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| − | <li><a href=" | + | <li><a href="/Team:Fudan/Team">Members</a></li> |
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Latest revision as of 15:21, 7 November 2018
- Addon: ribo
- Addon: TALE
- Addon: T2
- Model: transcriptional amplifer
- Model: Notch-ligand kinetics
- Software
Our notebook
PCR and subcloning were performed using standard methods. Detailed primer sequences are provided. All constructs were verified by Sanger sequencing.
Cells were cultured in DMEM supplemented with 10% FBS (HyClone), 100 U/ml penicillin, 100 μg/ml streptomycin and 1x GlutaMax (Gibco). Transient transfections were performed using Lipofectamine 2000 (Invitrogen) and Opti-MEM (Gibco). Viral packaging, infection and fluorescence-activated cell sorting were performed using standard methods.
Images, unless otherwise indicated, were captured using an inverted epifluorescence microscope (IX-81, Olympus) and a sCMOS camera (pixel size = 0.3222 μm; Zyla 5.5, Andor; 20x objective N.A. 0.75) and were controlled by Micro-Manager software.
All statistical analysis was performed using Prism (Graphpad) and ImageJ. All experiments were independently performed in triplicates; unless otherwise indicated. Images were combined and annotated in Powerpoint for presentation. Representative images are shown.
For practical reasons, all full-length protocols are in Chinese.
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.