Team:Caltech/Week 5

Monday 7/12

• Group meeting today!
• Sequencing results were really crappy (quality < 30% confidence), so began 2 5mL LB cultures of the light-lysis construct (picked from the original transformation plate).

Tuesday 7/13

• Sent off two new samples of the light-lysis construct for sequencing using the VF2 and VR primers. Results should be available tonight or tomorrow.
• Used remaining cells to inoculate new cultures of each and to extract DNA for a test digest:
  • Digested each (ideally identical) sample with EcoRI-HF and PstI, both for 1hr and for 10min.
  • The digest appears inconclusive, as a smear is visible from the top to about 3Kb, while little or no DNA is visible in the insert range (~1.5Kb, see below).

Gel image for the light-lysis test digest. (7/13)

• • Will run another gel tonight with other bricks to check efficacy of digests and will look forward to sequencing results soon.

Wednesday 7/14

• The light-lysis sequencing results could not be returned due to insufficient DNA concentration. Picked two new colonies from the original plate. Also made cultures of the previous ligations (PR aka Ligation1 and lyT aka Ligation2). Will prep tomorrow for sequencing.
• Met with the Caltech Office of Technology Transfer (Drs. Hannah Dvorak-Carbone & Jennifer Hodas)
  • Discussed patent law and IP issues relevant to our project
• Began designing primers for making glutamine & lysine peptides to experiment with transglutaminase (TGase).
• Transformed pBAD18 into both DH5\alpha and V1012 to test competence & troubleshoot transformation failures. (Used 2uL DNA and 60uL cells.)
• Prepared SOC from SOB and made 10mL aliquots to freeze.

Thursday 7/15

• The V1012 transformation was successful, but the V1012 was not. Will troubleshoot V1012 further, but it appears that the DH5\alpha cells are competent.
  • May want to make more V1012 cells competent this weekend.
  • It appears that using at least 50uL of comp cells and 2uL of DNA works most effectively. We will also use SOC to rescue the transformations from now on.
• The PR (Ligation1) and PRlyT (Ligation3) cultures grew, but the lyT (Ligation2) culture did not. This could indicate an issue with the lyT product (perhaps the colonies on the plate are contamination).
  • Will reperform the Ligation3 reaction and see if that helps. (The gel of the Ligation2 digest (above) looks correct, so there shouldn't be a problem with the ligation product.)
  • Prepped the cultures (to send for sequencing)
    • PRlyT1 = 50.1 ng/uL; PRlyT2 = 34.5 ng/uL (will send 20uL of each for seq.)
    • PR1 = 197.0 ng/uL; PR2 = 201.0 ng/uL (will send 10uL of each for seq.)
• Team signed the OTT Invention Disclosure form and submitted a project description to the OTT. Provisional patent application will be filed tomorrow.
• Designed primers for BBb_K112400 to change from Berkley standard to the correct BBa standard.
  • Will order these tomorrow so they are ready when the heat-shock promoter arrives.
• Prepared 500mL 2YT media, 1L diH2O, 2 250mL Erlenmeyer flasks and 50mL 10% glycerol. Inoculated 1 5mL culture of 2YT with DH5alpha cells.
  • Will make more competent cells tomorrow.

Friday 7/16

• Lab clean-up scheduled for today.

Weekend 7/17-18