IGEM:MIT/2005/Friday 8/5 Meeting Agenda and Minutes
General Updates
- Monday meeting TBRun by Maxine
- Assemblies: at beginning of week, we're planning on doing plating -> ON -> miniprep of all constructs needed for upcoming assemblies.
- This turns a ~4 day process into a ~2 day process
- Updated flowchart?
- Registry problems seem to be worked out...
- Should make glycerols for registry of all parts made or assembled
- Will start transitioning to Endy lab early next week, probably.
- Look at all the green on our parts site! Wow!
SubTeam Updates
Format: what is the progress? what are the issues?
Input: Maxine
1. Fluorescein and Oligo Uptake by competent (lots of competent cells made if anyone needs them) and overnighted MC4100 culture
0-no fluorescence
5-high fluorescence
Competent cells, fluorescein: 5
Competent cells, m9: 2.5
Competent cells, oligo: 1
Overnight cells, fluorescein: 1
Overnight cells, m9: 1
Overnight cells, oligo: 2
Should I post pics online?
2. Effects of Various Fluorescein Concentrations on MC4100 Overnight Culture Experiment
a) FACS: Fluorescence increased w/increasing conc for the most part. Should I get help for more detailed analysis?
b) plating results:
control: 5.2 X 10^8 cells/ml
5M: 4.5 X 10^8 cells/ml
500mM: 4.7 X 10^8 cells/ml
50mM: 1.60 X 10^9 cells/ml
5mM: 7.3 X 10^8 cells/ml
500microM: 1.26 X 10^9 cells/ml
50microM: 1.03 X 10^9 cells/ml
5microM: 3.2 X 10^8 cells/ml
Receiver Head-Unit: Jenny
Progress: scFv Testing Materials (most?) for western blot. Promoter.RBS biobrick BBa_J13002. Primer start-codon for scFv Progress: ToxR Unit scFv.TT Biobricked. BBa_J07043, 44, 45.
Receiver/Transmitter 1 - ToxR : Wiki-Will
Progress:
- assembling ctx-promotor (J07007) :: gfp composite.
- transform failed --> plate showed no growth this morning.
- i'd like to think we'll get this piece working this weekend.
Problems:
- waiting on ToxR (drew, how about your "i can assemble it in a week, wait forget i said that." i don't want to forget.)
*want to review the system? http://openwetware.org/index.php?title=Image:SystemToxRpathway.jpg scFv, with terminator malE , negative control PhoA, positive control. Ready, but still must remove restriction site with quikchange (Hi Annie!) fusion of scFv, malE, and PhoA to ToxR. (for obvious reasons) _ have constitutive promotor :: rbs part fusion to ToxR ... _ ctx promotor :: gfp ***almost done***
Receiver/Transmitter 2 - FecA : Annie
- Progress:
-Worked out details of all building processes [[../../Annie's Notes/]] -Specified more parts J07046-J07063 composite parts for FecA system -Base parts made: FecA, FecI, FecR, attempted FecA' -Base parts plan to make next week: FecA', FecR', FecA Promoter, -Assembling: rbs with FecI and FecR, rbs with promoter -Have overnight culture and a plate of DY329, ready for making FecA- and Fur-
- Problems:
-2 of 5 primers for adding linker to scFv are wrong. Add a different aa sequence 5'RVARAVTTRSRISTRSRSG 3' instead of 5'PNSASHSGSAPNTSSAPGS 3' -Primers for fusion are wrong. If we go with the accidentally-created linker, we can use half of the already ordered primers --> reorder the other half.
- Solutions:
-reorder
Signal Processor: Ray
- Waiting on parts from registry to do own assembly
Actuator
- Have begun using E0840 (rbs:gfp:TT) in conjunction with other systems
Issues/Discussion
- Input: having trouble with repeatable results
- Do we know that our input molecules are what we think they are?
- To look at experiment reproducibility, be consistent!
- Still waiting for ToxR -- Can I call?
- Sequencing: get info from biopolymer -- need plasmid DNA and primers
- Everything we've assembled, get sequenced!!