(Difference between revisions)

• Introduction
  • Home
  • The Team
  • Photo Gallery
• Our Project
  • Results Summary
  • Selection System
  • In-Vitro Characterization
  • In-Vivo Characterization
  • Microfluidics
  • Data
  • Attributions
• Tools
  • Gibson Assembly
  • MITOMI
• Notebook
  • Protocols
  • May
  • June
  • July
  • August
  • September
  • October
• Considerations
  • Human Practices
  • Safety
• Acknowledgements
  • Sponsors
  • Partners

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 requirements 7.2 General 7.3 Assembly 7.4 Protocols 7.5 Front Page 8 Clean room 9 Judging requirements (copied from 2011.igem.org)

General

• Edit the Google doc inventory, SPECIFY THE NAMES you put on the tubes if they are not straightforward!
• Keep an eye on the SOC and LB medium stocks to be sure they are not contaminated
• Keep an eye on the stocks of agar plates

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.
• Think of new assemblies we want to make (pTet with RFP, for example)

Reporter plasmids:

• J61002 plasmid:
  • add pTet: OK, colony PCR works
  • add Plac-RFP or Plac-lysis
  • add Ptet-LacI subsequently: not needed anymore

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
  • sequence

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 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, send for sequencing

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

requirements

1. The team's project must be documented on the iGEM Wiki by the deadline.
2. All team pages on the iGEM Wiki must be in the team namespace.
3. The team's project description must be documented on their iGEM Team Wiki by the deadline. The project description should be 1-3 paragraphs and only needs to include an up-to-date explanation of the project.
4. The team's wiki must include a Safety page and an attributions section. Safety in "Considerations" and Attributions in "Our project"
5. All iGEM Team Wikis must include on the front page the iGEM logo and a visible link back to the iGEM 2011 Wiki Main Page.

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)