Team:Caltech/Week 10

Monday 8/16

• Group meeting!
• Ran CPCR gels on L31, L34, L43, L58 & L71
  • All negative.

Tuesday 8/17

• Took our team photo!
• Ran more CPCR of L58 & L81.
  • Gel was negative.
• Cultured HSP-LacI-GFP cells in LB + 1M glucose to try to up-regulate LacI repression of GFP production. Incubated cells at 37°C overnight.

Wednesday 8/18

• LacI appears fully repressed in L62 cells after overnight incubation at 37°C.
  • Will now spin down cells and resuspend in LB + IPTG to see if overnight incubation now activates GFP production, as predicted.
• Picked more CPCR colonies of L81 & L58 (x6) to try to find plastic production cells.
• Transformed L60 & L61. Plated on plates containing 50μL 50mM glucose (dried).
  • We hypothesize that the reason these ligations produced no colonies is due to leaky LacI repression, allowing a low level of lysis expression, eventually killing all the cells. Will see if there are cells on these plates tomorrow.
• Submitted our poster deign for printing for Sasha's SURF presentation tomorrow.
• Brian illuminated half of a plate containing a lawn of HSP+GFP cells to try some rudimentary printing. No fluorescence after 3 hours; will check tomorrow.

Thursday 8/19

• L60 & L61 grew colonies! Began CPCR to find correct inserts.
  • Gel is negative - all bands appear to be RFP.
• Gel of L58 CPCR was negative again. Will design 'ligation primers' to connect the two for L58. L81-4 was correct; miniprepped the DNA for ligation tomorrow.
• Brian's test printing attempt plates appear somewhat green, but there is no boundary between the lit and unlit regions. Will re-try test more carefully next week.
• Sasha presented our SURF poster today! People seem pretty excited about the project, but we need more data.

Friday 8/20

• Began rough HSP activation kinetics experiment.
  • With 3 cultures of HSP+GFP cells grown 2 days @ 30°C: incubated two at 42°C for {5, 10}min, leaving the third on the bench for 15 min. Took 200μL samples of each to measure the fluorescence & OD600 right after incubation and every 15 minutes thereafter for 5 hours. Incubated the tubes at 30°C while not taking aliquots.
  • Plotted the fluorescence (RFU), normalized for OD (RFU/OD600) vs time.