Week from August 17th, to August 23rd, 2009

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August, 17th

• This week we planned to perform a gel run for all the constructs of the ethanol producing operon in order to check for contaminant bands. Then, we planned to ligate a promoter upstream of the ethanol producing operon and to build up measurement systems for A11 (lactose sensor) and A12 (aTc sensor).

• We inoculated 8 ul of:
  • B1-13
  • B2-5
  • B3-5
  • B4-2
  • B5-3
  • B6-3
  • E0240
  • A11-2
• glycerol stocks in 5 ml of LB + suitable antibiotic.

• We incubated these cultures overnight (37°C, 220 rpm).

Preparation of experiment with Tecan F200

• We dissolved 22 mg of lactose in 5 ml of LB + Kan in order to have 4.5% lactose.

• We prepared lactose assay kit for testing.

Experiment with Tecan F200

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August, 18th

• Glycerol stock for E0240, A11-2, B1, B2, B3 and B4 in order to have a backup.

• Miniprep for:
  • B1-13
  • B2-5
  • B3-5
  • B4-2
  • B5-3
  • B6-3
  • E0240
  • A11-2

• Digestion with EcoRI for:
  • B1-13
  • B2-5
  • B3-5
  • B4-2
  • B5-3
  • B6-3

• Digestion with EcoRI-PstI for:
  • B1-13
  • B2-5
  • B3-5
  • B4-2
  • B5-3
  • B6-3

• Digestion with PstI for:
  • B1-13
  • B2-5
  • B3-5
  • B4-2
  • B5-3
  • B6-3

• Electrophoresis for the digested plasmids.

Digestion check on fermentation parts (1st gel).

Digestion check on fermentation parts (2nd gel).

• Gel results:
  • B1 - ok
  • B2 - ok
  • B3 - two extra-bands (consistent with previous gels)
  • B4 - ok
  • B5 - two extra bands (consistent with previous gels)
  • B6 - two extra bands (consistent with previous gels)

• We decided to try to ligate a promoter upstream of B5 and B6 anyway.

• We inoculated 8 ul of:
  • A4(X2)
  • A12
• glycerol stocks.

• We incubated these inocula at 37°C, 220 rpm overnight.

August, 19th

• Digestion for:
  • E0240(X-P)
  • A11-2(S-P)
  • B5(E-X)
  • B6(E-X)

• Miniprep for:
  • A4(X2)
  • A12

• Digestion for:
  • A4(E-S)(X2)
  • A12(S-P)

• Precipitation with sodium acetate for:
  • A11-2(S-P)
  • B5(E-X)
  • B6(E-X)
  • A4(E-S)(X2)
  • A12(S-P)

• Gel run/cut/purification for E0240.

• Ligations:
  • B7 = A4(E-S) + B5(E-X) in pSB1AK3
  • B8 = A4(E-S) + B6(E-X) in pSB1AK3
  • A14 = A11(S-P) + E0240(X-P) in pSB1A2
  • A16 = A12(S-P) + E0240(X-P) in pSB1A2

Experiment with Tecan F200

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August, 20th

• We resuspended pSB3K3 from 2009 Registry Distribution. The plasmid with ccdB was not consistent, so we resuspended a consistent brick (of ~700bp length, suggested in the Registry "Help" page: Kit Plate 2, well 15L, I714891 brick) contained in pSB3K3.

• We transformed the overnight ligations and I714891 in TOP10 and plated transformed bacteria on LB agar plates + Amp (A14 and A16) or + Kan (pSB3K3, B7 and B8). We incubated the plates at 37°C overnight.

• We sent these samples (stored at -20°C) to BMR Genomics for sequencing:
  • A15-1
  • A15-3
  • A11-2
  • A11-3
  • B5-3
  • B6-3
  • B3-5 (again, because we wanted to check for contaminants in our native stock)

August, 21st

• Colony PCR for A14 (5 colonies) and A16 (5 colonies) plates. Colonies were inoculated in 1 ml of LB + Amp and let grow waiting for the end of the reaction.

• Electrophoresis for PCR results.

• Gel results (picture not taken, sorry...):
  • A14 - 3rd colony seems the most pure and it had the expected length for ligated plasmid.
  • A16 - reaction worked only on A16-1, but it was negative.

• We decided to keep A14-3 (positive at PCR) and A16-4 (randomly chosen) for digestion screening: we prepared a glycerol stock for them and re-filled the remaining 250 ul of bacteria with 4 ml of LB + Amp.

• We incubated these cultures at 37°C, 220 rpm overnight.

• We picked 2 colonies from B7, 5 colonies from B8 and 1 colony from I714891 plates. We inoculated them in 1 ml of LB + Kan and incubated them at 37°C, 220 rpm for 5 hours and 1/2. Then we prepared glycerol stocks and re-filled the remaining 250 ul with 4 ml of LB + Kan.

• NOTE: we decided not to perform PCR on B7 and B8 plates because of the large size of positive inserts.

• We incubated the re-filled cultures at 37°C, 220 rpm overnight.

Experiment with Tecan F200

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August, 22nd

• Pellet preparation for the 10 overnight cultures, ready to be miniprepped next week! Pellets were stored at -20°C.

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