IGEM:MIT/2005/Friday 7/15 Meeting Agenda

Overview

We're pursuing two main receiver paths, one that is located on the inner membrane and works by dimerization (ToxR) and the other that is located on the outer membrane and signals by a conformational change (FecA). Today we're hoping to make sure everyone knows where we are, and then to ask for advice on how to move forward.

SubTeam Updates

(see individual wiki pages for details)

1. Input Update (Jen, for Maxine)
2. Head Receiver Unit Update (Jenny)
3. Receiver 1 Update (Will)
4. Receiver 2 Update (Annie)
5. Signal Processor Update (Ray)
6. Actuator Update (Jessica)

Experimental Schedule

1. Look at general schedule, perhaps specifics?

Discussion of Main Issues

(make list as we go along. add an issue if you want).

note: we're working on making our biobricks usable, input welcome!

1. Experimental testing of input ligand (see update to expt. page)
2. test scFv by itself? in context of our system
3. do we want to talk to ToxR people to make gene ourself? other ways of getting our hands?
   • clone it
   • order oligos and build it
   • call mekalanos lab/ michelle
4. Level of expression of fusion -- inducible promoter? different rbs strengths? -- can we get some input one which promoters are best
   • be careful of strain if using pBad promoter
   • iptg
5. dealing with having two plasmids? same plasmid?
   • 10-12 kb is max plasmid size
   • having mult. plasmid might mean more modularity -- brainstorming
6. meta issue: is there another system, a way to make toxR taller?
   • kate's paper: scFv fused to outer membrane protein -- targeting signal
7. in general, how do we want to depict our devices?
8. Annie's issues:
   • Fur? -- working with Ray -- regulates FecI.FecR,FecA promoter -- don't want the cell doing this. FecA -- can't cut out Fur box. Can we cut out Fur itself? Maybe! Issue: can the cells survive. Can we just control amount of iron?
     • Can we make point mutation? its in -35, so might disrupt polymerase binding.
     • Find out threshold of Fur system?
   • do we want a polycistronic message for the FecA system?
     • levels of expression: either on diff. plasmid, under diff. promoter. Worry about polycistronic message: if any part has problem, creates problem for all. Natural system, however, does have polycistronic message.
     • need to know what levels are naturally expressed
   • internal fusion of scFv into FecA -- talk to TK
   • internal fusion of scFv into FecA -- circular permutation
   • deleting extra loops?
9. Naive thresholding possible. -- lambda system? co-express complementary RNA
10. characterization and debug actuator -- how are we going to read out our system?
11. What is cool? What is hip? Wiki iki iki.
12. make a further issues link
13. ecocyc
14. What strain do we build ToxR in? How do we choose the promoter? Long term vs. short term
15. Mechanics of getting into lab
    • Primers here today/monday