Team:EPF-Lausanne/Todo

From 2011.igem.org

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== Microfluidic Chips ==
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== General ==
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* Continue alignment training
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== Preparing the parts ==
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* Edit the Google doc inventory, SPECIFY THE NAMES you put on the tubes if they are not straightforward!
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* Investigate failure of lysis device sequencing.
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* Keep an eye on the SOC and LB medium stocks to be sure they are not contaminated
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* Keep an eye on the stocks of agar plates
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Next steps:
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== Supplies ==
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* <s>Make competent cells</s>
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* <s>'''Test competent cells'''</s> ~10^5CFU/µg not very good
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* <s> Pick up new ladder and Hifi PLUS enzyme from magasin, when they are received. </s>
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* <s>Transform in E. Coli.</s>
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* Glycerol Stocks
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== <s>Preparing the parts</s> ==
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* <s>Plasmid preps</s>
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* Sequence the lysis cassette
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* <s>Sequence the lysis cassette</s>
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* <s>Double-check lysis cassette sequence</s>
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All the parts are verified, we can now assemble them!
== Assembly ==
== Assembly ==
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=== Plasmids ===
* <s> Research plasmid backbones. Is there one that already contains Tet-repressed LacI or GFP? </s>
* <s> Research plasmid backbones. Is there one that already contains Tet-repressed LacI or GFP? </s>
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* Design Gibson primers to assemble the three different plasmids.
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* <s>Design Gibson primers to assemble the different plasmids.</s>
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* Determine which sequence on LacI-plasmid and Lysis-plasmid should be used to create the "mega-plasmid".
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* Think of new assemblies we want to make (pTet with RFP, for example)
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== Wiki ==
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Reporter plasmids:
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* J61002 plasmid:
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** <s>add pTet: OK, colony PCR works</s>
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** add <s>Plac-RFP</s> or Plac-lysis
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** add Ptet-LacI subsequently: not needed anymore
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TetR plasmid
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* J23019 plasmid: <s>PCR failed so far => test new plasmids for the TetR plasmid </s>
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* pSB3C5 plasmid: <s> Gibson transformation failed => try with pSB3K1</s>
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* pSB3K1 plasmid:
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** <s>add Pconst-TetR </s>
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** <s> cut out RFP and religate </s>
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** <s>sequence</s>
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** <s>add Ptet-LacI</s>
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** <s>sequence</s>
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=== Protocols ===
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Specifically:
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* Run PCRs and gels on existing sequences (plasmid backbone, TetR gene, RFP) to prepare the parts
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* extract the correct parts from the gel and purify (or purify directly PCR products)
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* Make a Gibson reaction
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* Transform cells -> plates -> liquid cultures
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* From the plates, make a colony PCR
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* Miniprep the plasmids from cultures, send for sequencing
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For somebody who's made some cell cultures, please provide a sentence or two on how they're done, in the Miniprep protocol:
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=== TetR mutants ===
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* Describe cell cultures in [[Team:EPF-Lausanne/Protocols/Miniprep|miniprep protocol]]
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* <s>determine required sequences</s>
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* <s>order primers</s>
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* Amplify linear template TetR-His to get a clean starting material for mutagenesis or extension PCR strategies (previous amplification result contains an additional band over 200bp it might have influenced the results)
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* [Mutation-inducing extension PCR for MITOMI: ] <-- Temporarily left to the side, in favour of site-specific mutagenesis
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** <s>Possibly rerun PCR; until decent results are obtained for all 6 mutations</s>
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** <s>Extract by gel purification</s>
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* Site-specific mutagenesis:
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** <s>Receive primers</s>
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** <s>Run mutagenesis</s>
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** <s>Transform cells</s>
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** <s>Finish preparing media</s>
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** Redo mutagenesis with a new kit (Alina has it)
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== MITOMI ==
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For wtTetR
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* <s> repeat MITOMI with wtTetR His-tagged (linear template) and wtTetR GFP-tagged (plasmid), for consensus and negative control sequence. DNA spotted in different concentrations </s>
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* experiment with de Brujin library spotted on His-wtTetR or/and wtTetR-GFP  (this will yield PWM)
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experiment planned on July 26
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* 1-off library on wtTetR linear template
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Further, check the ordered muTetRs  (determine position weight matrix)
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* determine position weight matrix for muTetRs, compare with de Brujin results
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== Microfluidics and chemostat chip ==
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* Continue alignment training
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* Repeat experiments to check design
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* Grow E. Coli from spotted arrays
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* <s>[No microfluidics] Setup a plate and test tween concentrations</s>
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* <s>Determine growth rate as function of tween concentration (say 0.075% +- 0.7, as many increments as will fit on the chip)</s>
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* Adapt design of "chemostat" chip for e-coli
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== Wiki ==
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=== requirements ===
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# <s>The team's project must be documented on the iGEM Wiki by the deadline.</s>
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# <s>All team pages on the iGEM Wiki must be in the team namespace.</s>
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# The team's project description must be documented on their iGEM Team Wiki by the deadline. The project description should be 1-3 paragraphs and only needs to include an up-to-date explanation of the project.
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# <s>The team's wiki must include a Safety page and an attributions section.</s> Safety in "Considerations" and Attributions in "Our project"
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# <s>All iGEM Team Wikis must include on the front page the iGEM logo and a visible link back to the iGEM 2011 Wiki Main Page</s>.
=== General ===
=== General ===
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Make sure we comply with the [[Requirements]]!
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* <s>Make a banner</s>.
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* <s>Write-up team presentation</s>
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* Upload our initial research about transcription factors
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** Document our choice. We spend a few weeks on this, show how we decided. One criteria for tetR was the amount of research already done for it. Another was low cost...
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*
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* Fill-in '''attributions and contributions''' and decide where it should go on the wiki
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* Create '''data''' page
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It might make sense to merge the "resources" and "tools" menus into "Background", where we discuss our choices of transcription factor, and present our tools: Microfluidics, Gibson assembly, etc.
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=== Assembly ===
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Make a page that explains the assembly strategy, and sequence of assembly: what plasmids were made in what sequence, and where all the components come from. For example, when we make J61002-LacI-Lysis, how many parts are we assembling? Where were those parts taken from in the PCR step? Use copious illustrations.
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=== Protocols ===
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* <s>Describe cell cultures in [[Team:EPF-Lausanne/Protocols/Miniprep|miniprep protocol]]</s>
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* <s>Create protocol Template, with "Back to protocols" link at top</s>
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** '''Include an easy printing option''' seriously work on printing template.
* Write a new protocol!
* Write a new protocol!
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* Write-up team presentation
 
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== Clean rooms ==
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=== Front Page ===
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Eventually (i.e when the project is approaching completion), the following should be present on the front page:
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* Project abstract
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* Link to the Data Page
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* Sponsors
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* Pretty layout...
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== Clean room ==
* Order lab notebook
* Order lab notebook
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* Order storage box
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* <s>Order storage box</s>
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== Judging requirements (copied from 2011.igem.org)==
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<div id="regional">
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<b>Bronze</b>: <br>
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<ol id="criterialist">
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<li><s>Team registration</s></li>
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<li><s>Complete Project Summary form</s></li>
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<li><s>Team Wiki</s></li>
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<li>Present a poster and a talk at the iGEM Jamboree</li>
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<li><s>At least one new submitted and well-characterized standard BioBrick Part or Device. A new application of and outstanding documentation (quantitative data showing the Part’s/ Device’s function) of a previously existing BioBrick part in the “Experience” section of that BioBrick’s Registry entry also counts.</s></li></ol>
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<b>Silver</b>: In addition to the Bronze Medal requirements...<br>
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<ol id="criterialist">
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<li><s>Demonstrate that at least one new BioBrick Part or Device of your own design and construction works as expected</s></li>
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<li>Characterize the operation of at least one new BioBrick Part or Device and enter this information in the “Main Page” section of that Part’s/Device’s Registry entry.</li></ol>
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<b>Gold</b>: In addition to the Bronze and Silver Medal requirements, any one or more of the following: <br>
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<ol id="criterialist">
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<li>Improve an existing BioBrick Part or Device and enter this information in the Registry (in the “Experience” section of that BioBrick’s Registry entry).</li>
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<li>Help another iGEM team by, for example, characterizing a part, debugging a construct, or modeling or simulating their system</li>
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<li>Outline and detail a new approach to an issue of Human Practice in synthetic biology as it relates to your project, such as safety, security, ethics, or ownership, sharing, and innovation.</li></ol>
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</div>
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{{:Team:EPF-Lausanne/Templates/Footer}}
{{:Team:EPF-Lausanne/Templates/Footer}}

Latest revision as of 01:06, 21 September 2011

Retrieved from "http://2011.igem.org/Team:EPF-Lausanne/Todo"