Week from October 12th, to October 18th, 2009

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October, 12th

• We repeated the digestion for F2620TOP10(S-P) and B5new2-3(X-P-ClaI).

• Gel run/cut for the two plasmids: bands were good.

• Gel extraction for:
  • F2620TOP10(S-P)
  • B5new2-3(X-P-ClaI)
  • B3(X-P)
  • B4(X-P)
  • A19-1(S-P)

• Unfortunately we had a very low DNA yield for almost all of them: A19-1(S-P) and F2620(S-P) could not be used :'(

• We decided not to repeat A19-1 extraction (and then the A21 re-cloning) and to assay our A21 construct for ethanol production.

• F2620 DNA was over, so we inoculated 8 ul of F2620MIT1 glycerol stock in 7 ml of LB + Amp. We incubated this inoculum overnight (37°C, 220 rpm).

• We inoculated 12 of our assemblies in 3 ml of LB + Amp in order to prepare the DNA to send to iGEM HQ:
  • A1
  • A2
  • A3
  • A4
  • A5
  • A6
  • A7
  • A8pg
  • A9pg
  • A11-3
  • A12-2
  • A15-3

• We ordered a gas chromatography for a sample of the supernatants (taken the previous day).

October, 13th

• Miniprep for the 12 inocula to be sent.

• Miniprep, cut S-P, gel run/cut/purification for F2620MIT1. This time we had a good yield;)

• Ligations:
  • B14 = F2620(S-P) + B3(X-P) in pSB1A2
  • B15 = F2620(S-P) + B4(X-P) in pSB1A2
• We incubated them at 16°C overnight.

October, 14th

• We transformed the two ligations (after 1:20 dilution) and incubated the plated bacteria overnight (37°C).

• We inoculated all the remaining assemblies (12) in order to send purified DNA to iGEM HQ (3 ml of LB + Amp).

• We inoculated 8 ul of B5new2-3, F2620TOP10 and B0015 glycerol stocks in 8 ml of LB + Amp + 2% glucose. We incubated them at 37°C, 220 rpm in the morning.

• Team meeting

October, 15th

• We diluted 25 ul of A14S-3 in 3 ml of LB + Amp and in 3 ml of LB + Amp + 1mM IPTG. We incubated the two cultures at 37°C, 220 rpm for 6 hours. Then we measured OD600 and fluorescence, but unfortunately their GFP levels were the same.

• Miniprep for the remaining BioBricks.

• pH measurement for B5new2-3, F2620TOP10 and B0015 24 hour cultures. We diluted 1:100 these three cultures in LB + Amp + 10% glucose and aliquoted them in the microplate reader (anaerobic).

• We inoculated 4 colonies of B14 plate and 3 colonies of B15 plate in 5 ml of LB + Amp and incubated them at 37°C, 220 rpm overnight. Tomorrow they will be screened!

• We received gas chromatography results.

October, 16th

• Cloning
• Fermentation experiment setup
• Wiki updating

October, 17th

• Fermentation experiment
• Wiki updating

October, 18th

• Wiki updating

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