IGEM:MIT/2005/Friday 8/12 Meeting Agenda and Minutes

General Updates

 * Biobricks request: please have up on wiki by end of today.
 * Today: clean up in Kate's lab after lunch; spend rest time learning about lab space, doing lab duties, and organizing our stuff. Visualize a white tornado!
 * Lets talk lab-use

SubTeam Updates
Format: what is the progress? what are the issues?

Input: Maxine

 * Subtilis
 * Advantages
 * Is naturally competent (saves us time!)
 * Disadvantages
 * According to "The Permeability of the Wall Fabric of E. Coli and Bacillus subtilis,"
 * E. coli and Subtilis pore size: 25kDa
 * E. coli pore size (according to many other sources) 6Da
 * Our fluorescein constructs: ~8-16 kDa
 * The Com system in Subtilis degrades double stranded DNA and then resynthesizes one strand, so our fluorescein construct would not work
 * We need to relearn all lab techniques, i.e. plating, etc. because of its different properties, such as pore production
 * We need to remake much of the ToxR system (different promoters and ribosome binding sites) so that it can fit into subtilis
 * Conclusion: advantages not enough to outweight disadvantages at this point
 * Most of the week was spent on helping FecA system

Receiver/Transmitter 1 - ToxR : Wiki-Will
-J07011 (ctx::gfp) might be correct, c/o gel test. Then again, it might not be. (see picture) -J07009 is here! Initial assembly attempts - J13002::J07009, J07009::J07006, J07009::J07043, J07009::J07044, J07009::J07045 promotor, rbs :: ToxR    ToxR::malE      ToxR::scFv(i)   ToxR::scFv(ii)  ToxR::scFv(iii) Every single plate of assemblies showed no growth this morning. I wonder what went wrong! can we back-track? my women's intuition tells me it was the transformation. -Some troubling news shows up in our Gel Purify of J07009. The picture implies J07009 (cut x/p) is ~1kb, and it should bee ~650kb
 * Activity:

- Retry Assemblies / retry transformation - At the very least: miniprep large amounts of each biobrick being used right now.
 * This Weekend (team effort):

Receiver/Transmitter 2 - FecA : Annie
-QuikChange done on FecR, PhoA --> preparing to get them sequence FecA didn't get any colonies -BB FecA Promoter However, gel on digest shows lots of other bands -Recieve all primers reordered -Ligating RBS & FecI -Primers for PCR Tetracycline and Chloramphenicol resistance form hairpins and dimers Consistent with gel bands -Discuss the FecA Promoter gel bands with an advisor -Only 1 internal primer or both forward & reverse for sequencing? -Design new primers for PCR Tetracycline and Chloramphenicol? Any other ways?
 * Updates
 * Questions

Actuator
No new updates.

Issues/Discussion

 * 1) Subtilis seems not particularly useful for us, although it might still be an option.
 * 2) Registry might want to think about subtilis as another organism to pay attention to
 * 3) Jenny's stuff needs to be passed on. Probably to Jen.
 * 4) Will: need to figure out what is happening with ToxR. Need assemblies to work!
 * 5) FecA System: Can do quick change on subparts of the FecA gene (2.3 kb). Can do not quickchange method, talk to Kate, she did this before.
 * 6) Change digest protocol... in general, lets look over our protocols
 * 7) Use program that helps design primers
 * 8) Issues with adding in the signal processing device:
 * 9) How many plasmids can stay in the cell?
 * 10) *Drug markers
 * 11) *compatibility of replication origins
 * 12) Levels of expression on plasmids
 * 13) Plasmids wont support infinitely long insert -- not longer than 10 kb
 * 14) Issue of load
 * 15) Lets do a System Integration Meeting