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Team:EPF-Lausanne/Todo
From 2011.igem.org
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* <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> | ||
* <s>Design Gibson primers to assemble the different plasmids.</s> | * <s>Design Gibson primers to assemble the different plasmids.</s> | ||
| - | |||
* Think of new assemblies we want to make (pTet with RFP, for example) | * Think of new assemblies we want to make (pTet with RFP, for example) | ||
| Line 28: | Line 27: | ||
* J61002 plasmid: | * J61002 plasmid: | ||
** <s>add pTet: OK, colony PCR works</s> | ** <s>add pTet: OK, colony PCR works</s> | ||
| - | |||
| - | |||
| - | |||
** add <s>Plac-RFP</s> or Plac-lysis | ** add <s>Plac-RFP</s> or Plac-lysis | ||
| - | ** add Ptet-LacI subsequently | + | ** add Ptet-LacI subsequently: not needed anymore |
TetR plasmid | TetR plasmid | ||
| Line 42: | Line 38: | ||
** <s>sequence</s> | ** <s>sequence</s> | ||
** <s>add Ptet-LacI</s> | ** <s>add Ptet-LacI</s> | ||
| + | ** <s>sequence</s> | ||
Specifically: | Specifically: | ||
* Run PCRs and gels on existing sequences (plasmid backbone, TetR gene, RFP) to prepare the parts | * 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 | + | * extract the correct parts from the gel and purify (or purify directly PCR products) |
* Make a Gibson reaction | * Make a Gibson reaction | ||
* Transform cells -> plates -> liquid cultures | * Transform cells -> plates -> liquid cultures | ||
* From the plates, make a colony PCR | * From the plates, make a colony PCR | ||
| - | * Miniprep the plasmids from cultures, | + | * Miniprep the plasmids from cultures, send for sequencing |
=== TetR mutants === | === TetR mutants === | ||
| Line 90: | Line 87: | ||
== 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 === | === General === | ||
Make sure we comply with the [[Requirements]]! | Make sure we comply with the [[Requirements]]! | ||
| - | * Make a banner. | + | * <s>Make a banner</s>. |
| - | * Write-up team presentation | + | * <s>Write-up team presentation</s> |
* Upload our initial research about transcription factors | * 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... | ** 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... | ||
| Line 102: | Line 107: | ||
* Create '''data''' page | * 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. | + | 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 === | === Assembly === | ||
| Line 114: | Line 119: | ||
** '''Include an easy printing option''' seriously work on printing template. | ** '''Include an easy printing option''' seriously work on printing template. | ||
* Write a new protocol! | * Write a new protocol! | ||
| - | |||
=== Front Page === | === Front Page === | ||
| Line 134: | Line 138: | ||
<ol id="criterialist"> | <ol id="criterialist"> | ||
<li><s>Team registration</s></li> | <li><s>Team registration</s></li> | ||
| - | <li>Complete Project Summary form</li> | + | <li><s>Complete Project Summary form</s></li> |
<li><s>Team Wiki</s></li> | <li><s>Team Wiki</s></li> | ||
<li>Present a poster and a talk at the iGEM Jamboree</li> | <li>Present a poster and a talk at the iGEM Jamboree</li> | ||
| - | <li>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.</li></ol> | + | <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> | <b>Silver</b>: In addition to the Bronze Medal requirements...<br> | ||
<ol id="criterialist"> | <ol id="criterialist"> | ||
| - | <li>Demonstrate that at least one new BioBrick Part or Device of your own design and construction works as expected</li> | + | <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> | <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> | ||
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.
