Team:Caltech/Week 10

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iGEM 2010



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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.
 
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