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

'''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