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  • Results Summary
  • Selection System
  • In-Vitro Characterization
  • In-Vivo Characterization
  • Microfluidics
  • Data
  • Attributions
• Tools
  • Gibson Assembly
  • MITOMI
• Notebook
  • Protocols
  • May
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• Considerations
  • Human Practices
  • Safety
• Acknowledgements
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Todo

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
  • 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, transformed
• pSB3K1 plasmid: glycerol stock

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
• 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