Team:EPF-Lausanne/Todo
From 2011.igem.org
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{{:Team:EPF-Lausanne/Templates/Header|title=Todo}} | {{:Team:EPF-Lausanne/Templates/Header|title=Todo}} | ||
- | == | + | == General == |
- | + | ||
- | == Preparing the parts == | + | * Edit the Google doc inventory, SPECIFY THE NAMES you put on the tubes if they are not straightforward! |
+ | * Keep an eye on the SOC and LB medium stocks to be sure they are not contaminated | ||
+ | * Keep an eye on the stocks of agar plates | ||
+ | |||
+ | == Supplies == | ||
+ | |||
+ | * <s> Pick up new ladder and Hifi PLUS enzyme from magasin, when they are received. </s> | ||
+ | |||
+ | == <s>Preparing the parts</s> == | ||
* <s>Sequence the lysis cassette</s> | * <s>Sequence the lysis cassette</s> | ||
- | * Double-check lysis cassette sequence | + | * <s>Double-check lysis cassette sequence</s> |
+ | |||
+ | All the parts are verified, we can now assemble them! | ||
== Assembly == | == Assembly == | ||
+ | === 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> | ||
- | * Design Gibson primers to assemble the | + | * <s>Design Gibson primers to assemble the different plasmids.</s> |
- | * | + | * Think of new assemblies we want to make (pTet with RFP, for example) |
+ | |||
+ | Reporter plasmids: | ||
+ | * J61002 plasmid: | ||
+ | ** <s>add pTet: OK, colony PCR works</s> | ||
+ | ** add <s>Plac-RFP</s> or Plac-lysis | ||
+ | ** add Ptet-LacI subsequently: not needed anymore | ||
+ | |||
+ | TetR plasmid | ||
+ | * J23019 plasmid: <s>PCR failed so far => test new plasmids for the TetR plasmid </s> | ||
+ | * pSB3C5 plasmid: <s> Gibson transformation failed => try with pSB3K1</s> | ||
+ | * pSB3K1 plasmid: | ||
+ | ** <s>add Pconst-TetR </s> | ||
+ | ** <s> cut out RFP and religate </s> | ||
+ | ** <s>sequence</s> | ||
+ | ** <s>add Ptet-LacI</s> | ||
+ | ** <s>sequence</s> | ||
+ | |||
+ | Specifically: | ||
+ | * Run PCRs and gels on existing sequences (plasmid backbone, TetR gene, RFP) to prepare the parts | ||
+ | * extract the correct parts from the gel and purify (or purify directly PCR products) | ||
+ | * Make a Gibson reaction | ||
+ | * Transform cells -> plates -> liquid cultures | ||
+ | * From the plates, make a colony PCR | ||
+ | * Miniprep the plasmids from cultures, send for sequencing | ||
+ | |||
+ | === TetR mutants === | ||
+ | |||
+ | * <s>determine required sequences</s> | ||
+ | * <s>order primers</s> | ||
+ | * 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) | ||
+ | * [Mutation-inducing extension PCR for MITOMI: ] <-- Temporarily left to the side, in favour of site-specific mutagenesis | ||
+ | ** <s>Possibly rerun PCR; until decent results are obtained for all 6 mutations</s> | ||
+ | ** <s>Extract by gel purification</s> | ||
+ | * Site-specific mutagenesis: | ||
+ | ** <s>Receive primers</s> | ||
+ | ** <s>Run mutagenesis</s> | ||
+ | ** <s>Transform cells</s> | ||
+ | ** <s>Finish preparing media</s> | ||
+ | ** Redo mutagenesis with a new kit (Alina has it) | ||
+ | |||
+ | == MITOMI == | ||
+ | |||
+ | For wtTetR | ||
+ | |||
+ | * <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> | ||
+ | * experiment with de Brujin library spotted on His-wtTetR or/and wtTetR-GFP (this will yield PWM) | ||
+ | experiment planned on July 26 | ||
+ | * 1-off library on wtTetR linear template | ||
+ | |||
+ | Further, check the ordered muTetRs (determine position weight matrix) | ||
+ | |||
+ | * determine position weight matrix for muTetRs, compare with de Brujin results | ||
+ | |||
+ | == Microfluidics and chemostat chip == | ||
+ | |||
+ | * Continue alignment training | ||
+ | |||
+ | * Repeat experiments to check design | ||
+ | * Grow E. Coli from spotted arrays | ||
+ | * <s>[No microfluidics] Setup a plate and test tween concentrations</s> | ||
+ | * <s>Determine growth rate as function of tween concentration (say 0.075% +- 0.7, as many increments as will fit on the chip)</s> | ||
+ | * Adapt design of "chemostat" chip for e-coli | ||
== Wiki == | == Wiki == | ||
+ | === requirements === | ||
+ | |||
+ | # <s>The team's project must be documented on the iGEM Wiki by the deadline.</s> | ||
+ | # <s>All team pages on the iGEM Wiki must be in the team namespace.</s> | ||
+ | # 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. | ||
+ | # <s>The team's wiki must include a Safety page and an attributions section.</s> Safety in "Considerations" and Attributions in "Our project" | ||
+ | # <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 === | ||
+ | |||
+ | Make sure we comply with the [[Requirements]]! | ||
+ | * <s>Make a banner</s>. | ||
+ | * <s>Write-up team presentation</s> | ||
+ | * Upload our initial research about transcription factors | ||
+ | ** 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... | ||
+ | * | ||
+ | * Fill-in '''attributions and contributions''' and decide where it should go on the wiki | ||
+ | * Create '''data''' page | ||
+ | |||
+ | 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. | ||
+ | |||
+ | === Assembly === | ||
+ | |||
+ | 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. | ||
=== Protocols === | === Protocols === | ||
- | |||
* <s>Describe cell cultures in [[Team:EPF-Lausanne/Protocols/Miniprep|miniprep protocol]]</s> | * <s>Describe cell cultures in [[Team:EPF-Lausanne/Protocols/Miniprep|miniprep protocol]]</s> | ||
- | + | * <s>Create protocol Template, with "Back to protocols" link at top</s> | |
- | + | ** '''Include an easy printing option''' seriously work on printing template. | |
* Write a new protocol! | * Write a new protocol! | ||
- | |||
- | == Clean | + | === Front Page === |
+ | |||
+ | Eventually (i.e when the project is approaching completion), the following should be present on the front page: | ||
+ | * Project abstract | ||
+ | * Link to the Data Page | ||
+ | * Sponsors | ||
+ | * Pretty layout... | ||
+ | |||
+ | == Clean room == | ||
* Order lab notebook | * Order lab notebook | ||
- | * Order storage box | + | * <s>Order storage box</s> |
+ | |||
+ | |||
+ | == Judging requirements (copied from 2011.igem.org)== | ||
+ | <div id="regional"> | ||
+ | <b>Bronze</b>: <br> | ||
+ | <ol id="criterialist"> | ||
+ | <li><s>Team registration</s></li> | ||
+ | <li><s>Complete Project Summary form</s></li> | ||
+ | <li><s>Team Wiki</s></li> | ||
+ | <li>Present a poster and a talk at the iGEM Jamboree</li> | ||
+ | <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> | ||
+ | |||
+ | <b>Silver</b>: In addition to the Bronze Medal requirements...<br> | ||
+ | <ol id="criterialist"> | ||
+ | <li><s>Demonstrate that at least one new BioBrick Part or Device of your own design and construction works as expected</s></li> | ||
+ | <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> | ||
+ | |||
+ | <b>Gold</b>: In addition to the Bronze and Silver Medal requirements, any one or more of the following: <br> | ||
+ | <ol id="criterialist"> | ||
+ | <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> | ||
+ | <li>Help another iGEM team by, for example, characterizing a part, debugging a construct, or modeling or simulating their system</li> | ||
+ | <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> | ||
+ | </div> | ||
+ | |||
{{:Team:EPF-Lausanne/Templates/Footer}} | {{:Team:EPF-Lausanne/Templates/Footer}} |
Latest revision as of 01:06, 21 September 2011
Todo
Contents |
General
- Edit the Google doc inventory, SPECIFY THE NAMES you put on the tubes if they are not straightforward!
- Keep an eye on the SOC and LB medium stocks to be sure they are not contaminated
- Keep an eye on the stocks of agar plates
Supplies
-
Pick up new ladder and Hifi PLUS enzyme from magasin, when they are received.
Preparing the parts
-
Sequence the lysis cassette -
Double-check lysis cassette sequence
All the parts are verified, we can now assemble them!
Assembly
Plasmids
-
Research plasmid backbones. Is there one that already contains Tet-repressed LacI or GFP? -
Design Gibson primers to assemble the different plasmids. - Think of new assemblies we want to make (pTet with RFP, for example)
Reporter plasmids:
- J61002 plasmid:
-
add pTet: OK, colony PCR works - add
Plac-RFPor Plac-lysis - add Ptet-LacI subsequently: not needed anymore
-
TetR plasmid
- J23019 plasmid:
PCR failed so far => test new plasmids for the TetR plasmid - pSB3C5 plasmid:
Gibson transformation failed => try with pSB3K1 - pSB3K1 plasmid:
-
add Pconst-TetR -
cut out RFP and religate -
sequence -
add Ptet-LacI -
sequence
-
Specifically:
- Run PCRs and gels on existing sequences (plasmid backbone, TetR gene, RFP) to prepare the parts
- extract the correct parts from the gel and purify (or purify directly PCR products)
- Make a Gibson reaction
- Transform cells -> plates -> liquid cultures
- From the plates, make a colony PCR
- Miniprep the plasmids from cultures, send for sequencing
TetR mutants
-
determine required sequences -
order primers - 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)
- [Mutation-inducing extension PCR for MITOMI: ] <-- Temporarily left to the side, in favour of site-specific mutagenesis
-
Possibly rerun PCR; until decent results are obtained for all 6 mutations -
Extract by gel purification
-
- Site-specific mutagenesis:
-
Receive primers -
Run mutagenesis -
Transform cells -
Finish preparing media - Redo mutagenesis with a new kit (Alina has it)
-
MITOMI
For wtTetR
-
repeat MITOMI with wtTetR His-tagged (linear template) and wtTetR GFP-tagged (plasmid), for consensus and negative control sequence. DNA spotted in different concentrations - experiment with de Brujin library spotted on His-wtTetR or/and wtTetR-GFP (this will yield PWM)
experiment planned on July 26
- 1-off library on wtTetR linear template
Further, check the ordered muTetRs (determine position weight matrix)
- determine position weight matrix for muTetRs, compare with de Brujin results
Microfluidics and chemostat chip
- Continue alignment training
- Repeat experiments to check design
- Grow E. Coli from spotted arrays
-
[No microfluidics] Setup a plate and test tween concentrations -
Determine growth rate as function of tween concentration (say 0.075% +- 0.7, as many increments as will fit on the chip) - Adapt design of "chemostat" chip for e-coli
Wiki
requirements
-
The team's project must be documented on the iGEM Wiki by the deadline. -
All team pages on the iGEM Wiki must be in the team namespace. - 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.
-
The team's wiki must include a Safety page and an attributions section.Safety in "Considerations" and Attributions in "Our project" -
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.
General
Make sure we comply with the Requirements!
-
Make a banner. -
Write-up team presentation - Upload our initial research about transcription factors
- 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...
- Fill-in attributions and contributions and decide where it should go on the wiki
- Create data page
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.
Assembly
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.
Protocols
-
Describe cell cultures in miniprep protocol -
Create protocol Template, with "Back to protocols" link at top- Include an easy printing option seriously work on printing template.
- Write a new protocol!
Front Page
Eventually (i.e when the project is approaching completion), the following should be present on the front page:
- Project abstract
- Link to the Data Page
- Sponsors
- Pretty layout...
Clean room
- Order lab notebook
-
Order storage box
Judging requirements (copied from 2011.igem.org)
Bronze:
Team registrationComplete Project Summary formTeam Wiki- Present a poster and a talk at the iGEM Jamboree
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.
Silver: In addition to the Bronze Medal requirements...
Demonstrate that at least one new BioBrick Part or Device of your own design and construction works as expected- 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.
Gold: In addition to the Bronze and Silver Medal requirements, any one or more of the following:
- Improve an existing BioBrick Part or Device and enter this information in the Registry (in the “Experience” section of that BioBrick’s Registry entry).
- Help another iGEM team by, for example, characterizing a part, debugging a construct, or modeling or simulating their system
- 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.