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

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Contents

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_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
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