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

• Activity:

-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

• This Weekend (team effort):

- Retry Assemblies / retry transformation
- At the very least: miniprep large amounts of each biobrick being used right now.

Receiver/Transmitter 2 - FecA : Annie

• Updates

-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

• Questions

-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?

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:
   1. How many plasmids can stay in the cell?
      • Drug markers
      • compatibility of replication origins
   2. Levels of expression on plasmids
   3. Plasmids wont support infinitely long insert -- not longer than 10 kb
9. Issue of load
10. Lets do a System Integration Meeting