"
Team:EPF-Lausanne/Todo
From 2011.igem.org
(Difference between revisions)
(→Wiki) |
(→Plasmids) |
||
| Line 22: | Line 22: | ||
* J61002 plasmid: | * J61002 plasmid: | ||
** <s>adding pTet: OK</s> | ** <s>adding pTet: OK</s> | ||
| - | ** adding tetR with const promoter: troubles with amplifying the parts | + | ** adding tetR with const promoter: troubles with amplifying the parts => left aside for the moment |
** adding LacI under Ptet + RFP under Plac: only one colony, but has a strange plasmid size | ** adding LacI under Ptet + RFP under Plac: only one colony, but has a strange plasmid size | ||
** adding LacI under Ptet + lysis cassette under Plac: no colonies for the moment | ** adding LacI under Ptet + lysis cassette under Plac: no colonies for the moment | ||
| - | * <s>J23019 plasmid: PCR failed so far | + | * <s>J23019 plasmid: PCR failed so far => test new plasmids for the LacI plasmid </s> |
| - | * pSB3C5 plasmid: glycerol stock, pconst-TetR added, | + | *<s>pSB3C5 plasmid: glycerol stock, pconst-TetR added, transformation failed => try with pSB3K1</s> |
| - | * pSB3K1 plasmid: glycerol stock | + | * pSB3K1 plasmid: glycerol stock, primers ordered |
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 | + | * extract the correct parts from the gel and purify (or use directly PCR products) |
* Make a Gibson reaction | * Make a Gibson reaction | ||
* Transform cells -> plates -> liquid cultures | * Transform cells -> plates -> liquid cultures | ||
Revision as of 09:23, 20 July 2011
Todo
Contents |
General
- Prepare antibiotic aliquotes
- Edit the Google doc inventory, SPECIFY THE NAMES you put on the tubes if they are not straightforward!
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 three different plasmids. -
Receive said primers - 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)
- J61002 plasmid:
-
adding pTet: OK - adding tetR with const promoter: troubles with amplifying the parts => left aside for the moment
- adding LacI under Ptet + RFP under Plac: only one colony, but has a strange plasmid size
- adding LacI under Ptet + lysis cassette under Plac: no colonies for the moment
-
-
J23019 plasmid: PCR failed so far => test new plasmids for the LacI plasmid pSB3C5 plasmid: glycerol stock, pconst-TetR added, transformation failed => try with pSB3K1- pSB3K1 plasmid: glycerol stock, primers ordered
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 directly PCR products)
- Make a Gibson reaction
- Transform cells -> plates -> liquid cultures
- Miniprep the plasmids from cultures, check if Gibson is ok by doing a digestion
TetR mutants
-
determine required sequences -
order primers - [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
- Finish preparing media
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 6
- 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
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
General
Make sure we comply with the Requirements!
- Write-up team presentation
- Upload our initial research about transcription factors
- Fill-in attributions and contributions and decide where it should go on the wiki
- Create data page
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
