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
   1. 2 tubes of DB3.1 (Kan+)
   2. 2 tubes of transformed E1010 from resistance test plate (Kan+)
   3. 2 tubes of transformed F2620 from resistance test plate (Amp+)
   4. 2 tubes of transformed B0034 from resistance test plate (Amp+)
   5. All should be taken out tomorrow morning and used to make glycerol stocks
4. Meeting with TK and Drew

Results from Transformations

• BBa_L13500, 200 µl: 50 colonies
• BBa_L13500, 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

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