Week from July 1st, to July 5th, 2009

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July, 1st

• Sequencing results for BOL1 BioBrick: sequence was correct!

• Miniprep for:
  • A8 (for dilution/transformation)
  • A9 (for dilution/transformation)

• We transformed 10 pg of A8 and A9 miniprepped DNA in TOP10, after a proper quantification with Nanodrop and dilution. We plated transformed bacteria and incubated them overnight at 37°C. (we will call these transformations "10 pg plates")

• We prepared 0.5 l of LB + Amp.

• We infected 5 ml of LB + Amp with 10 ul of T9002 glycerol stock. We incubated the culture overnight at 37°C, 220 rpm.

• We also picked a random colony from A10 plate and infected 1 ml of LB + Amp. We incubated this inoculum at 37°C, 220 rpm for 5 and 1/2 hours. Then we prepared a glycerol stock and re-filled the remaining 250 ul of culture with 5 ml of LB + Amp to grow an overnight culture (for sequencing).

Preparation of experiment with Tecan F200

• We diluted 1:1000 the cultures of:
  • J23100
  • J23101
  • J23118
  • A1
  • A2
  • A7
  • E0240

• We incubated the diluted cultures at 37°C, 220 rpm for 3 hours.

Experiment with Tecan F200

• Description
• Purpose:
• Materials & Methods
• Protocol
• Results

July, 2nd

• Miniprep for A10 and T9002. We sent purified DNA to BMR Genomics for sequencing.

• A8 and A9 (10 pg plates) plates showed colonies!(a few, as expected) We picked 3 colonies for A8 plate and 3 colonies for A9 plate to infect 5 ml of LB + Amp. We incubated the six inocula at 37°C, 220 rpm overnight. Tomorrow they will be miniprepped and screened with digestion to check if the extra band had been lost!

Preparation of experiment with Tecan F200

• aTc stock preparation.

• We diluted 1:1000 the cultures of:
  • A8
  • A9 (X2)
  • E0240

• We incubated the diluted cultures at 37°C, 220 rpm for 3 hours. After 2 hours, one of the two A9 culture was induced with 75 ng/ml aTc and incubated again under the same conditions as before.

Experiment with Tecan F200

• Description
• Purpose:
• Materials & Methods
• Protocol
• Results

July, 3rd

• Miniprep for the overnight cultures (taken from the two 10 pg plates).

• Digestion E-P for the purified plasmids (under the same conditions as the experiment of June 30th).

Digestion results for A8 and A9 (10 pg plates): A8-3 and A9-1 colonies were kept.

• Gel results: the plasmids seemed to be pure (except from A8-1 in which the unwanted insert is easily visible). We decided to keep:
  • A8-3
  • A9-1
• glycerol stocks and to validate them with sequencing. We will call them "A8pg" and "A9pg".

• Tecan F200 data analysis.

• Team meeting.

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