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Team:EPF-Lausanne/Todo
From 2011.igem.org
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** adding LacI under Ptet + RFP under Plac: cells transformed | ** adding LacI under Ptet + RFP under Plac: cells transformed | ||
** adding LacI under Ptet + lysis cassette under Plac: cells transformed | ** adding LacI under Ptet + lysis cassette under Plac: cells transformed | ||
| - | * J23019 plasmid: PCR failed so far -> test new plasmids for the LacI plasmid | + | * <s>J23019 plasmid: PCR failed so far -> test new plasmids for the LacI plasmid </s> |
| - | * pSB3C5: glycerol stock, | + | * pSB3C5 plasmid: glycerol stock, ready to be transformed with assembled plasmid |
| - | * pSB3K1: glycerol stock | + | * pSB3K1 plasmid: glycerol stock |
Specifically: | Specifically: | ||
| - | * Run PCRs and gels on existing sequences (plasmid backbone, TetR gene, RFP) | + | * Run PCRs and gels on existing sequences (plasmid backbone, TetR gene, RFP) to prepare the parts |
| + | * 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 === | === TetR mutants === | ||
Revision as of 12:07, 15 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: primers ordered
- adding LacI under Ptet + RFP under Plac: cells transformed
- adding LacI under Ptet + lysis cassette under Plac: cells transformed
-
-
J23019 plasmid: PCR failed so far -> test new plasmids for the LacI plasmid - pSB3C5 plasmid: glycerol stock, ready to be transformed with assembled plasmid
- pSB3K1 plasmid: glycerol stock
Specifically:
- Run PCRs and gels on existing sequences (plasmid backbone, TetR gene, RFP) to prepare the parts
- 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<s> - <s>Extract by gel purification
-
- Site-specific mutagenesis:
- Receive primers
- Run mutagenesis
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
Protocols
-
Describe cell cultures in miniprep protocol - Create protocol Template, with "Back to protocols" link at top
- Include an easy printing option
- Write a new protocol!
- Upload "chemostat" protocols
General
- Write-up team presentation
- Upload our initial research about transcription factors
Clean room
- Order lab notebook
-
Order storage box
