Difference between revisions of "Team:Fudan/Protocols"
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table { border-collapse: collapse; border-spacing: 0; } | table { border-collapse: collapse; border-spacing: 0; } | ||
</style> | </style> | ||
| + | <title>2018 iGEM Team:Fudan - Protocols</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="./Demonstrate">Demonstration</a></li> |
| − | <li><a href=" | + | <li><a href="./Antigen_Receptors">Antigen, Receptors</a></li> |
| − | <li><a href=" | + | <li><a href="./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="./Addon#ribo">Addon: ribo</a></li> |
| − | <li><a href=" | + | <li><a href="./Addon#TALE">Addon: TALE</a></li> |
| − | <li><a href=" | + | <li><a href="./Addon#T2">Addon: T2</a></li> |
| − | <li><a href=" | + | <li><a href="./Model#Transcriptional_Amplifer">Model: transcriptional amplifer</a></li> |
| − | <li><a href=" | + | <li><a href="./Model#NotchLigandKinetics">Model: Notch-ligand kinetics</a></li> |
| − | <li><a href=" | + | <li><a href="./Software">Software</a></li> |
</ul> | </ul> | ||
<ul id="dropdown3" class="dropdown-content"> | <ul id="dropdown3" class="dropdown-content"> | ||
| − | <li><a href=" | + | <li><a href="./InterLab">iGEM interLab</a></li> |
| − | <li><a href=" | + | <li><a href="./Notebook">Our notebook</a></li> |
| − | <li><a href=" | + | <li><a href="./Primers">Primers used</a></li> |
| − | <li><a href=" | + | <li><a href="./Protocols">Protocols</a></li> |
| − | <li><a href=" | + | <li><a href="./Safety">Safety</a></li> |
</ul> | </ul> | ||
<ul id="dropdown4" class="dropdown-content"> | <ul id="dropdown4" class="dropdown-content"> | ||
| − | <li><a href=" | + | <li><a href="./Basic_Parts">Basic parts</a></li> |
| − | <li><a href=" | + | <li><a href="./Composite_Parts">Composite parts</a></li> |
| − | <li><a href=" | + | <li><a href="./Optimization">Optimization</a></li> |
| − | <li><a href=" | + | <li><a href="./Parts_Collection">Parts collection</a></li> |
| − | <li><a href=" | + | <li><a href="./Improve">Parts improvement</a></li> |
| − | <li><a href=" | + | <li><a href="./Measurement">Quantification</a></li> |
</ul> | </ul> | ||
<ul id="dropdown5" class="dropdown-content"> | <ul id="dropdown5" class="dropdown-content"> | ||
| − | <li><a href=" | + | <li><a href="./Bio-Art">Bio-Art display</a></li> |
| − | <li><a href=" | + | <li><a href="./Collaborations">Collaborations</a></li> |
| − | <li><a href=" | + | <li><a href="./Design_Intention">Design intention</a></li> |
| − | <li><a href=" | + | <li><a href="./Human_Practices">Human practices</a></li> |
| − | <li><a href=" | + | <li><a href="./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="./Acknowledgement">Acknowledgement</a></li> |
| − | <li><a href=" | + | <li><a href="./Attributions">Attributions</a></li> |
| − | <li><a href=" | + | <li><a href="./Heritage">Heritage</a></li> |
| − | <li><a href=" | + | <li><a href="./Team">Members</a></li> |
| − | <li><a href=" | + | <li><a href="./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="./Demonstrate">Demonstration</a></li> |
| − | <li><a href=" | + | <li><a href="./Antigen_Receptors">Antigen, Receptors</a></li> |
| − | <li><a href=" | + | <li><a href="./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="./Addon#ribo">Addon: ribo</a></li> |
| − | <li><a href=" | + | <li><a href="./Addon#TALE">Addon: TALE</a></li> |
| − | <li><a href=" | + | <li><a href="./Addon#T2">Addon: T2</a></li> |
| − | <li><a href=" | + | <li><a href="./Model#Transcriptional_Amplifer">Model: transcriptional amplifer</a></li> |
| − | <li><a href=" | + | <li><a href="./Model#NotchLigandKinetics">Model: Notch-ligand kinetics</a></li> |
| − | <li><a href=" | + | <li><a href="./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="./InterLab">iGEM interLab</a></li> |
| − | <li><a href=" | + | <li><a href="./Notebook">Our notebook</a></li> |
| − | <li><a href=" | + | <li><a href="./Primers">Primers used</a></li> |
| − | <li><a href=" | + | <li><a href="./Protocols">Protocols</a></li> |
| − | <li><a href=" | + | <li><a href="./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="./Basic_Parts">Basic parts</a></li> |
| − | <li><a href=" | + | <li><a href="./Composite_Parts">Composite parts</a></li> |
| − | <li><a href=" | + | <li><a href="./Optimization">Optimization</a></li> |
| − | <li><a href=" | + | <li><a href="./Parts_Collection">Parts collection</a></li> |
| − | <li><a href=" | + | <li><a href="./Improve">Parts improvement</a></li> |
| − | <li><a href=" | + | <li><a href="./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="./Bio-Art">Bio-Art display</a></li> |
| − | <li><a href=" | + | <li><a href="./Collaborations">Collaborations</a></li> |
| − | <li><a href=" | + | <li><a href="./Design_Intention">Design intention</a></li> |
| − | <li><a href=" | + | <li><a href="./Human_Practices">Human practices</a></li> |
| − | <li><a href=" | + | <li><a href="./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="./Acknowledgement">Acknowledgement</a></li> |
| − | <li><a href=" | + | <li><a href="./Attributions">Attributions</a></li> |
| − | <li><a href=" | + | <li><a href="./Heritage">Heritage</a></li> |
| − | <li><a href=" | + | <li><a href="./Team">Members</a></li> |
| − | <li><a href=" | + | <li><a href="./Sponsors">Sponsors</a></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<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 an inverted epifluorescence microscope (IX-81, Olympus) and a sCMOS camera (pixel size = 0.3222 | + | <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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<!--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> | ||
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<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"> | ||
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<span>Project</span> | <span>Project</span> | ||
<ul> | <ul> | ||
| − | <li><a href=" | + | <li><a href="./Demonstrate">Demonstration</a></li> |
| − | <li><a href=" | + | <li><a href="./Antigen_Receptors">Antigen, Receptors</a></li> |
| − | <li><a href=" | + | <li><a href="./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 383: | Line 385: | ||
<span>Dry lab</span> | <span>Dry lab</span> | ||
<ul> | <ul> | ||
| − | <li><a href=" | + | <li><a href="./Addon#ribo">Addon: ribo</a></li> |
| − | <li><a href=" | + | <li><a href="./Addon#TALE">Addon: TALE</a></li> |
| − | <li><a href=" | + | <li><a href="./Addon#T2">Addon: T2</a></li> |
| − | <li><a href=" | + | <li><a href="./Model#Transcriptional_Amplifer">Model: transcriptional amplifer</a></li> |
| − | <li><a href=" | + | <li><a href="./Model#NotchLigandKinetics">Model: Notch-ligand kinetics</a></li> |
| − | <li><a href=" | + | <li><a href="./Software">Software</a></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 394: | Line 396: | ||
<span>Wet lab</span> | <span>Wet lab</span> | ||
<ul> | <ul> | ||
| − | <li><a href=" | + | <li><a href="./InterLab">iGEM interLab</a></li> |
| − | <li><a href=" | + | <li><a href="./Notebook">Our notebook</a></li> |
| − | <li><a href=" | + | <li><a href="./Primers">Primers used</a></li> |
| − | <li><a href=" | + | <li><a href="./Protocols">Protocols</a></li> |
| − | <li><a href=" | + | <li><a href="./Safety">Safety</a></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<span>Toolbox</span> | <span>Toolbox</span> | ||
<ul> | <ul> | ||
| − | <li><a href=" | + | <li><a href="./Basic_Parts">Basic parts</a></li> |
| − | <li><a href=" | + | <li><a href="./Composite_Parts">Composite parts</a></li> |
| − | <li><a href=" | + | <li><a href="./Optimization">Optimization</a></li> |
| − | <li><a href=" | + | <li><a href="./Parts_Collection">Parts collection</a></li> |
| − | <li><a href=" | + | <li><a href="./Improve">Parts improvement</a></li> |
| − | <li><a href=" | + | <li><a href="./Measurement">Quantification</a></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<span>Outreach</span> | <span>Outreach</span> | ||
<ul> | <ul> | ||
| − | <li><a href=" | + | <li><a href="./Bio-Art">Bio-Art display</a></li> |
| − | <li><a href=" | + | <li><a href="./Collaborations">Collaborations</a></li> |
| − | <li><a href=" | + | <li><a href="./Design_Intention">Design intention</a></li> |
| − | <li><a href=" | + | <li><a href="./Human_Practices">Human practices</a></li> |
| − | <li><a href=" | + | <li><a href="./Public_Engagement">Public engagement</a></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 427: | Line 429: | ||
<span>Team</span> | <span>Team</span> | ||
<ul> | <ul> | ||
| − | <li><a href=" | + | <li><a href="./Acknowledgement">Acknowledgement</a></li> |
| − | <li><a href=" | + | <li><a href="./Attributions">Attributions</a></li> |
| − | <li><a href=" | + | <li><a href="./Heritage">Heritage</a></li> |
| − | <li><a href=" | + | <li><a href="./Team">Members</a></li> |
| − | <li><a href=" | + | <li><a href="./Sponsors">Sponsors</a></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<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 | |
| − | + | </div><div class="col s12 m6 l4"><i class="fa fa-twitter"></i> <i class="fa fa-wechat"></i> Fudan_iGEM | |
| − | + | </div> | |
| − | + | </div> | |
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</div> | </div> | ||
</footer> | </footer> | ||
Revision as of 18:09, 6 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.