Team:Brown/Notebook Meetings/7-6-09

From 2009.igem.org

iGEM Meeting Minutes- 7/6/09

9:05 am

Update from Team 1
- Failed digests and PCR- could it be poor technique, or insert not in pBluescript?
- Drop off fedex at Biomed fr¬ont desk for sequencing; get package out to Yale right away
- Hope to isolate EV131 insert at least by the end of the week, if not earlier

- Suggestions:
  - Run lower percentage gel to get resolution between 3000 and 3500, usually run 1% gel
  - Run a couple of individual digests with individual enzymes

- PCR
  - Amplify any other template alongside as a control

Update from Team 2
- Helped team 3 with project a bit
- Looked at quorum sequences
- Two genes with a DNA sequence in between both; both genes have been annotated, but not the promoters that lie in the region in between
- Team 2 wants to amplify the region from one of the promoters, including genes downstream
- Entire gene strand is approx. 3 kb
- Once amplified, they want to ligate death gene in front
- Plan on using electroporation protocol

- Suggestion: be careful with ribosome binding site, make sure that you do have a good ribosome binding site
  - Amplify everything up to the ATG codon at 5’ end
  - Make biobrick ends before P1, after P2, and individual ones in between genes downstream of promoter
  - Adrian question: do you foresee any problems of the quorum promoter being on constitutively? Is there any negative feedback? If there’s 50-100 copies, then the constitutive low level becomes a high level of conscription

- A Plan B?: secretion tag DNA stretch that signals secretion through the sec pathway, 30 amino acids on 5’ end

Update from Team 3
- Paper on computational design of histadine receptors
- Will contacted author of paper, who has done lots of research on altering cell receptors
- Response: wants a formal project proposal before he will actually help the team; which proteins we’re working with, what we want to do
- We were looking at TrigEZ protein→ will active the promotion of whatever genes we will put in, it binds to sugar binding protein which binds to sugars, followed by a phosporylation cascade
- Modeled with ribose, but ribose is too different from histamine→ author suggested to look for amino acid receptor
- Found “Tar” receptor→ aspartate binding
- If it works, then we’ll perform directed mutagenesis
- Hope to combine Tar and ENVZ
- Transmembrane domain is with the Tar
- Two component system
- Hope to change it to a histamine binding site
- Do fusion and mutagenesis in parallel?
- Do not do mutagenesis on fused proteins; do it only on the binding domain
- Wessel: You’ll need to design a selection method so that cells will die unless in presence of aspartate; way to test aspartate binding
- 1. System for screening positive binding: Fuse Tar and ENVZ, design some system that enables cell survival; could easily be lactamase or some ampicillin resistance. Looking for GFP is fair, but then you have to screen through visually (could be a second choice to above).
- 2. Get screening to work; take aspartate binding module Tar and mutagise. Re-insert into plasmid that contains signaling gene. Screen- survival of cells.
- 3. Insert downstream elements that allow the histamine to activate.
- Binding interaction sites lie between 1-6 and 38-188; need to sit down and look at sequence and identify exactly which sites
- How to make sure that you can mutate DNA?
- Author suggested using Histadine protein, and then worrying about signaling later; seems like a good strategy
- Chose this family of proteins in particular because they are highly conserved and stable, assuming that mutated variants will also be stable