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Team:EPF-Lausanne/Todo
From 2011.igem.org
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=== Plasmids === | === 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> | ||
| - | * <s>Design Gibson primers to assemble the | + | * <s>Design Gibson primers to assemble the different plasmids.</s> |
| - | + | ||
* Determine which sequence on LacI-plasmid and Lysis-plasmid should be used to create the "mega-plasmid" (?). | * Determine which sequence on LacI-plasmid and Lysis-plasmid should be used to create the "mega-plasmid" (?). | ||
* 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) | ||
| + | Reporter plasmids: | ||
* J61002 plasmid: | * J61002 plasmid: | ||
| - | ** <s> | + | ** <s>add pTet: OK, colony PCR works</s> |
| - | ** <s> | + | ** <s>add tetR with const promoter: troubles with amplifying the parts => left aside for the moment </s> |
| - | ** | + | ** <s>add LacI under Ptet + RFP under Plac: Gibson failed -> dropped</s> |
| - | ** | + | ** <s>add LacI under Ptet + lysis cassette under Plac: Gibson failed -> dropped</s> |
| - | + | ** add Plac-RFP or Plac-lysis | |
| - | * | + | ** add Ptet-LacI subsequently |
| - | * pSB3K1 plasmid: | + | |
| + | 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> | ||
| + | ** sequence | ||
| + | ** add Ptet-LacI | ||
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 use directly PCR products | + | * extract the correct parts from the gel and purify (or use purify directly PCR products) |
* Make a Gibson reaction | * Make a Gibson reaction | ||
* Transform cells -> plates -> liquid cultures | * Transform cells -> plates -> liquid cultures | ||
Revision as of 18:25, 14 August 2011
Todo
Contents |
General
- Make competent cells
-
Prepare antibiotic aliquotes - Edit the Google doc inventory, SPECIFY THE NAMES you put on the tubes if they are not straightforward!
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. - Determine which sequence on LacI-plasmid and Lysis-plasmid should be used to create the "mega-plasmid" (?).
- Think of new assemblies we want to make (pTet with RFP, for example)
Reporter plasmids:
- J61002 plasmid:
-
add pTet: OK, colony PCR works -
add tetR with const promoter: troubles with amplifying the parts => left aside for the moment -
add LacI under Ptet + RFP under Plac: Gibson failed -> dropped -
add LacI under Ptet + lysis cassette under Plac: Gibson failed -> dropped - add Plac-RFP or Plac-lysis
- add Ptet-LacI subsequently
-
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
-
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 use 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, check if Gibson is ok by doing a digestion
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
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!
- Upload "chemostat" protocols
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 registration- Complete Project Summary form
Team 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.
