IGEM:MIT/2007/Notebook/2007-7-18

Agenda

 * 1) Transform tk's competent cells with yesterday's 3A assembly results (F2620+B0034 and CPX insert into vector pSB1AC3)
 * 2) Make LB+Cm and LB+Cm+Kan plates
 * 3) Liquid cultures of eight tubes made and put in warm room -- for making glycerol stocks
 * 4) 2 tubes of DB3.1 (Kan+)
 * 5) 2 tubes of transformed E1010 from resistance test plate (Kan+)
 * 6) 2 tubes of transformed F2620 from resistance test plate (Amp+)
 * 7) 2 tubes of transformed B0034 from resistance test plate (Amp+)
 * 8) All should be taken out tomorrow morning and used to make glycerol stocks
 * 9) Meeting with TK and Drew

Results from Transformations

 * BBa_I13500 (B0034+GFP), 200 µl: 50 colonies
 * BBa_I13500, 100 µl: 10 colonies
 * PICSS8, 200 µl, #2: 5 colonies
 * PICSS8, 100 µl, #2: 1 colony
 * pHIE6, 100 µl, #2: 25 colonies
 * pHIE6, 200 µl, #2: 45 colonies
 * CPX, 50 µl: lawn (as expected)
 * CPX, 100 µl: lawn (as expected)
 * CPX, 200 µl: lawn (as expected)

Transformed tk's competent cells

 * Aliquoted one of the original vials into eight eppendorfs, 100µL each.
 * Did 3 sets of transformations:
 * 2 of yesterday's 3A assembly product (plasmid contains: F2620+B0034+CPX in pSB1AC3 backbone)
 * 1 negative control (pSB1AC3 digested with EcoRI and PstI)
 * Followed our Transformation protocol (tk's modified protocol)


 * Transformations were put into 37C room at 11:30 AM and incubated for 2 hrs

Poured Plates, again

 * Used stringent working concentrations:
 * 10µg of Kanamycin per mL of LB
 * 25µg of Chloramphenicol per mL of LB
 * Poured 5 LB+Cm+Kan plates
 * Poured 29 LB+Cm plates
 * Cm plates have green labels
 * Used stringent/relaxed concentrations for other plates:
 * 25 plates with 30µg/mL of Kan
 * 20 plates with 35µg/mL of Amp

Meeting Notes

 * Run a sensitivity easy experiment


 * HgCl2 MSDS
 * Ask tom for HgCl2, or even chemical room


 * Reshman has the DNA for Leucine zipper
 * CPX to display ?


 * 'Transcriptional Terminator'


 * B0010 and B0012
 * B0015 is the double terminator and works very well.


 * Get T7 antibody, order
 * Think of getting fluorescent version of the body, secondary version that is fluorescent
 * Endy lab or Brian's lab. Has origin antibody.


 * Could use FLAG tag in FhuA, or HA tag, needs to work internally; CMiC


 * Get EC20 display, and CCG?
 * LPP-OMP-A


 * First Lorenzo, fused a single chain antibody. Antibody, can get complement;


 * Make Cell maps
 * Input then output.


 * Cysteines: will not export proteins that have internal cysteine bonds. Periplasm is where they farm.  No export through that system.


 * Washing:
 * Negative control
 * EBS, salt conditions


 * Absorption on Spec
 * 1 OD of cells
 * Take 2 mL