- Transform tk's competent cells with yesterday's 3A assembly results (F2620+B0034 and CPX insert into vector pSB1AC3)
- Make LB+Cm and LB+Cm+Kan plates
- Liquid cultures of eight tubes made and put in warm room -- for making glycerol stocks
- 2 tubes of DB3.1 (Kan+)
- 2 tubes of transformed E1010 from resistance test plate (Kan+)
- 2 tubes of transformed F2620 from resistance test plate (Amp+)
- 2 tubes of transformed B0034 from resistance test plate (Amp+)
- All should be taken out tomorrow morning and used to make glycerol stocks
- 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
- 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?
- 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.
- Negative control
- EBS, salt conditions
- Absorption on Spec
- 1 OD of cells
- Take 2 mL