Difference between revisions of "Team:Fudan/Protocols"

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                         <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 src="https://static.igem.org/mediawiki/2018/c/c4/T--Fudan--teamLogo.svg"></a>
 
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                         <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>
 
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                         <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>
 
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                 <li><a href="https://2018.igem.org/Team:Fudan/Demonstrate">Demonstration</a></li>
 
                 <li><a href="https://2018.igem.org/Team:Fudan/Demonstrate">Demonstration</a></li>
 +
 
                 <li><a href="https://2018.igem.org/Team:Fudan/Antigen_Receptors">Antigen, Receptors</a></li>
 
                 <li><a href="https://2018.igem.org/Team:Fudan/Antigen_Receptors">Antigen, Receptors</a></li>
 
                 <li><a href="https://2018.igem.org/Team:Fudan/Results">Transmembrane logic</a></li>
 
                 <li><a href="https://2018.igem.org/Team:Fudan/Results">Transmembrane logic</a></li>
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                     <p style="width: 100%;text-align: center;font-size: 24px"><span class="white-text">Sponsors</span></p>
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                     <p style="width: 100%;text-align: center;font-size: 24px"><span class="white-text">Our notebook</span></p>
 
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                 <li>
 
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                                 <ul>
 
                                 <ul>
 
                                     <li><a href="https://2018.igem.org/Team:Fudan/Demonstrate">Demonstration</a></li>
 
                                     <li><a href="https://2018.igem.org/Team:Fudan/Demonstrate">Demonstration</a></li>
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                <li><a href="https://2018.igem.org/Team:Fudan/Antigen_Receptors">Antigen, Receptors</a></li>
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 +
                                    <li><a href="https://2018.igem.org/Team:Fudan/Antigen_Receptors">Antigen, Receptors</a></li>
 
                                     <li><a href="https://2018.igem.org/Team:Fudan/Results">Transmembrane logic</a></li>
 
                                     <li><a href="https://2018.igem.org/Team:Fudan/Results">Transmembrane logic</a></li>
 
                                 </ul>
 
                                 </ul>
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                                 <ul>
 
                                 <ul>
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                                  <li><a href="https://2018.igem.org/Team:Fudan/Addon#ribo">Addon: ribo</a></li>
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                                    <li><a href="https://2018.igem.org/Team:Fudan/Addon#ribo">Addon: ribo</a></li>
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                <li><a href="https://2018.igem.org/Team:Fudan/Addon#TALE">Addon: TALE</a></li>
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                                    <li><a href="https://2018.igem.org/Team:Fudan/Addon#TALE">Addon: TALE</a></li>
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                <li><a href="https://2018.igem.org/Team:Fudan/Addon#T2">Addon: T2</a></li>
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                                    <li><a href="https://2018.igem.org/Team:Fudan/Addon#T2">Addon: T2</a></li>
 
                                     <li><a href="https://2018.igem.org/Team:Fudan/Model#Transcriptional_Amplifer">Model: transcriptional amplifer</a></li>
 
                                     <li><a href="https://2018.igem.org/Team:Fudan/Model#Transcriptional_Amplifer">Model: transcriptional amplifer</a></li>
 
                                     <li><a href="https://2018.igem.org/Team:Fudan/Model#War_Predictor">Model: war&nbsp;predictor</a></li>
 
                                     <li><a href="https://2018.igem.org/Team:Fudan/Model#War_Predictor">Model: war&nbsp;predictor</a></li>
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                         <h1>Our protocols</h1>
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                         <h1>Protocols</h1>
 
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                         <span>We provide the full-length protocols as individual PDF files for download, with brief English descriptions on this page.</span>
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                         <span>...</span>
 
                     </div>
 
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                     <h1>Our protocols</h1>
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                     <h1>Protocols</h1>
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                     <p><span>We provide the full-length protocols as individual PDF files for download, with brief English descriptions on this page. </span></p>
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                     <p><span>...</span></p>
 
                 </div>
 
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                         <defs>
 
                         <defs>
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                     <li><a href="./InterLab">iGEM interLab</a></li>
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                     <li><a href="https://2018.igem.org/Team:Fudan/InterLab">iGEM interLab</a></li>
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                     <li><a href="./Notebook">Our notebook</a></li>
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                     <li><a href="https://2018.igem.org/Team:Fudan/Notebook">Our notebook</a></li>
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                     <li><a href="./Primers">Primers used</a></li>
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                     <li><a href="https://2018.igem.org/Team:Fudan/Primers">Primers used</a></li>
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                     <li>Protocols</li>
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                     <li><a href="https://2018.igem.org/Team:Fudan/Protocols">Protocols</a></li>
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                     <li style="margin-bottom: 10px;"><a href="./Safety">Safety</a></li>
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                     <li><a href="https://2018.igem.org/Team:Fudan/Safety">Safety</a></li>
 +
                    <!--
 +
                    <li class="onThisPageNav"><a href="#section1">General safety concerns</a></li>
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                    <li class="onThisPageNav"><a href="#section2">Laboratory practice</a></li>
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                    <li class="onThisPageNav"><a href="#section3">Specific safety concerns</a></li>-->
 
                 </ul>
 
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                         </div>
 
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                            <table>
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<tr>
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                                <tr>
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<th>Our protocols as PDF files</th><th>&nbsp;</th>
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                                    <th>Our protocols as PDF files</th><th>&nbsp;</th>
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<th>Download</th>
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                                    <th>Download</th>
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</tr>
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                                </tr>
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<tr>
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                                <tr>
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<td>Be a Good Lab Member</td><td><i>GoodLabPractices.pdf</i></td>
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                                    <td>Be a Good Lab Member</td><td><i>GoodLabPractices.pdf</i></td>
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<td><a href="https://2018.igem.org/File:T--Fudan--GoodLabPractices.pdf" target="_blank"><i class="fa fa-download"></i></a></td>
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                                    <td><a href="https://2018.igem.org/File:T--Fudan--GoodLabPractices.pdf" target="_blank"><i class="fa fa-download"></i></a></td>
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<tr>
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                                <tr>
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<td>Molecular Cloning</td><td><i>MolecularCloning.pdf</i></td>
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                                    <td>Molecular Cloning</td><td><i>MolecularCloning.pdf</i></td>
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<td><a href="https://2018.igem.org/File:T--Fudan--MolecularCloning.pdf" target="_blank"><i class="fa fa-download"></i></a></td>
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                                    <td><a href="https://2018.igem.org/File:T--Fudan--MolecularCloning.pdf" target="_blank"><i class="fa fa-download"></i></a></td>
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                                </tr>
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<tr>
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<td>Tissue Culture</td><td><i>CellCulture.pdf</i></td>
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                                    <td>Tissue Culture</td><td><i>CellCulture.pdf</i></td>
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<td><a href="https://2018.igem.org/File:T--Fudan--CellCulture.pdf" target="_blank"><i class="fa fa-download"></i></a></td>
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                                    <td><a href="https://2018.igem.org/File:T--Fudan--CellCulture.pdf" target="_blank"><i class="fa fa-download"></i></a></td>
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<tr>
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                                <tr>
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<td>Make a Stable Cell Line</td><td><i>MakeStableCellLine.pdf</i></td>
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                                    <td>Make a Stable Cell Line</td><td><i>MakeStableCellLine.pdf</i></td>
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<td><a href="https://2018.igem.org/File:T--Fudan--MakeStableCellLine.pdf" target="_blank"><i class="fa fa-download"></i></a></td>
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                                    <td><a href="https://2018.igem.org/File:T--Fudan--MakeStableCellLine.pdf" target="_blank"><i class="fa fa-download"></i></a></td>
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<td>Cell Sorting</td><td><i>FACS.pdf</i></td>
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                                    <td>Cell Sorting</td><td><i>FACS.pdf</i></td>
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<td><a href="https://2018.igem.org/File:T--Fudan--FACS.pdf" target="_blank"><i class="fa fa-download"></i></a></td>
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                                    <td><a href="https://2018.igem.org/File:T--Fudan--FACS.pdf" target="_blank"><i class="fa fa-download"></i></a></td>
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<td>Cell Staining</td><td><i>FixStain.pdf</i></td>
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                                    <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>
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                                    <td><a href="https://2018.igem.org/File:T--Fudan--FixStain.pdf" target="_blank"><i class="fa fa-download"></i></a></td>
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<td>Time-lapse Live-cell Imaging</td><td><i>TimeLapseImaging.pdf</i></td>
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                                    <td>Time-lapse Live-cell Imaging</td><td><i>TimeLapseImaging.pdf</i></td>
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<td><a href="https://2018.igem.org/File:T--Fudan--TimeLapseImaging.pdf" target="_blank"><i class="fa fa-download"></i></a></td>
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                                    <td><a href="https://2018.igem.org/File:T--Fudan--TimeLapseImaging.pdf" target="_blank"><i class="fa fa-download"></i></a></td>
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</div>
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                        </div>
 
                         <p style="color:grey">
 
                         <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>
 
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                 </main>
 
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                     <h2 style="margin: 0;padding: 10px 0;">Abstract</h2>
 
                     <h2 style="margin: 0;padding: 10px 0;">Abstract</h2>
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                     <p style="margin:0;">Contact-dependent signaling is critical for multicellular biological
+
                     <p style="margin: 0">Contact-dependent signaling is critical for multicellular biological
−
                        events, yet customizing contact-dependent signal transduction between
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                            events, yet customizing contact-dependent signal transduction between
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                        cells remains challenging. Here we have developed the ENABLE toolbox, a
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                            cells remains challenging. Here we have developed the ENABLE toolbox, a
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                        complete set of transmembrane binary logic gates. Each gate consists of
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                            complete set of transmembrane binary logic gates. Each gate consists of
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                        3 layers: Receptor, Amplifier, and Combiner. We first optimized synthetic
+
                            3 layers: Receptor, Amplifier, and Combiner. We first optimized synthetic
−
                        Notch receptors to enable cells to respond to different signals across the
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                            Notch receptors to enable cells to respond to different signals across the
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                        membrane reliably. These signals, individually amplified intracellularly by
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                            membrane reliably. These signals, individually amplified intracellularly by
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                        transcription, are further combined for computing. Our engineered zinc finger-based
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                            transcription, are further combined for computing. Our engineered zinc finger-based
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                        transcription factors perform binary computation and output designed products.
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                            transcription factors perform binary computation and output designed products.
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                        In summary, we have combined spatially different signals in mammalian cells,
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                            In summary, we have combined spatially different signals in mammalian cells,
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                        and revealed new potentials for biological oscillators, tissue engineering,
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                            and revealed new potentials for biological oscillators, tissue engineering,
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                        cancer treatments, bio-computing, etc. ENABLE is a toolbox for constructing
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                            cancer treatments, bio-computing, etc. ENABLE is a toolbox for constructing
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                        contact-dependent signaling networks in mammals. The 3-layer design principle
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                            contact-dependent signaling networks in mammals. The 3-layer design principle
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                        underlying ENABLE empowers any future development of transmembrane logic circuits,
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                            underlying ENABLE empowers any future development of transmembrane logic circuits,
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                        thus contributes a foundational advance to Synthetic Biology.
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                            thus contributes a foundational advance to Synthetic Biology.
 
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Revision as of 11:03, 17 October 2018

Protocols

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Protocols

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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 a an inverted epifluorescence microscope (IX-81, Olympus) and an sCMOS camera (pixel size = 0.3222 μm; Zyla 5.5, Andor; 20x objective N.A. 0.75) and 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.

Our protocols as PDF files  Download
Be a Good Lab MemberGoodLabPractices.pdf
Molecular CloningMolecularCloning.pdf
Tissue CultureCellCulture.pdf
Make a Stable Cell LineMakeStableCellLine.pdf
Cell SortingFACS.pdf
Cell StainingFixStain.pdf
Time-lapse Live-cell ImagingTimeLapseImaging.pdf

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.