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  • Gibson Assembly
  • MITOMI
• Notebook
  • Protocols
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Todo

Contents 1 General 2 Supplies 3 Preparing the parts 4 Assembly 4.1 Plasmids 4.2 TetR mutants 5 MITOMI 6 Microfluidics and chemostat chip 7 Wiki 7.1 General 7.2 Assembly 7.3 Protocols 7.4 Front Page 8 Clean room

General

• 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 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, colony PCR works
  • 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 PCR, but failed
  • adding LacI under Ptet + lysis cassette under Plac: colony PCR failed -> need to de Gibson again
• 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: colony PCR worked

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, after purification)
• 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