Week from August 31st, to September 6th, 2009

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August, 31st

• We inoculated:
  • 10 colonies from B5new2 plate in 4 ml of LB + Kan for screening;
  • B8-5 (8 ul from glycerol stock) in 4 ml of LB + Kan (for purification process);

• We sent A15-2 purified DNA (stored at -20°C) to BMR Genomics for sequencing.

Experiment with Tecan F200

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

September, 1st

• We received sequencing results for:
  • A16-4: sequence ok!
  • A14-3: sequence had a deletion in C0012, as we expected because of A11-2 sequencing results, previously received;
  • B8-5: sequence wrong in VF2 and good in VR;
  • A17: chromatogram was good, but ligation failed (R0011 was not in the insert).

• COMMENTS after sequencing results:
  • now we have a new aTc sensor (A16) to test together with A9;
  • B8 has to be repeated or purified, we will try both the approaches;
  • A14 has to be repeated using A11-3, which has a consistent sequencing result;
  • A17 is going to be ligated today (this time using gel extraction).

• Glycerol stocks for the 10 B5new2 grown cultures.

• Miniprep for B5new2 (10 samples) and for B8-5.

• Digestion for:
  • B5new2 (10 samples) for screening (E-P cut);
  • B8-5 for purification from gel (E-P cut);
  • R0011 (stored at -20°C) for A17new ligation (S-P cut)
  • E0240 (stored at -20°C) for A17new ligation (X-P cut)

• Medium-size gel for B5new2(E-P); small-size gel for B8-5(E-P), R0011(S-P) and E0240(X-P).

• Medium gel results: B5new2 clones all showed the correct length for ligated plasmid! we decided to keep B5new2-3 to continue the assemblies and B5new2-9 as a backup copy. Both plasmids were sent to BMR Genomics for sequencing.

• Small gel results: R0011(S-P) and E0240(X-P) were cut and purified through gel-extraction, while B8-5 could not be cut because the bands were not correct: maybe the heaviest band noticed on August 24th was a non-completely digested fragment, not a correct-size insert. We decided to throw B8-5 glycerol stock away.

• Ligation: A17new = R0011(S-P) + E0240(X-P) in pSB1A2

• We incubated ligation reaction at 16°C overnight.

• We inoculated:
  • B5new2-3
  • B6-3
  • A4
  • F2620MIT1
  • A11-3

pH sensor

• We streaked a single colony LB + Amp agar plate using K116002 glycerol stock. We incubated the plate at 37°C overnight.
• Preparation of LBK (LB with potassium instead of sodium) at pH 5,5 - 6,6 - 7,5 - 8,5 for the first experiment we would have performed in the following days.

September, 2nd

• Miniprep for:
  • B5new2-3
  • B6-3
  • A11-3
  • A4
  • F2620MIT1
  • E0240 pellet (stored at -20°C)

• Digestions:
  • B5new2-3(E-X)
  • B6-3(E-X)
  • A11-3(S-P)
  • A4(E-S)
  • F2620MIT1(E-S)
  • E0240(X-P)

• Gel run, cut and band purification for all the samples.

• Ligations:
  • B7new = A4(E-S) + B5new2-3(E-X) in pSB1AK3
  • B8new = A4(E-S) + B6-3(E-X) in pSB1AK3
  • B9 = F2620MIT1(E-S) + B5new2-3(E-X) in pSB1AK3
  • B10 = F2620MIT1(E-S) + B6-3(E-X) in pSB1AK3
  • A14L = A11-3(S-P) + E0240(X-P) in pSB1A2

• We incubated the five reactions at 16°C overnight.

• Transformation/plating for A17new ligation.

• We inoculated a single colony from K116002 streaked plate in 5 ml of LB + Amp. We incubated the inoculum at 37°C, 220 rpm overnight.

pH sensor

• The plate showed grown colonies, we picked one colony and infected 1 ml of LB + Amp.
• Overnight incubation.

September, 3rd

• Miniprep for K116002 overnight culture. We used 1 ul of this sample to transform TOP10 in order to have the pH sensor in the same strain as the reference promoter (i.e. A2). We incubated the plate (LB + Amp) with transformed bacteria overnight at 37°C. We also sent purified DNA to BMR Genomics for sequencing.

• A17new overnight plate showed only one colony...easy screening for this plate! Colony PCR/electrophoresis for this sample. We stored this colony in 100ul of LB + Amp and incubated it at 37°C waiting for the end of the reaction.

• Gel results: the blank was not so clean, but the only colony was positive:) We inoculated the colony in 5 ml of LB + Amp to grow an overnight culture (37°C, 220 rpm).

• We transformed these ligations:
  • B7new 1:40 on Kan
  • B8new 1:40 on Kan
  • B9 1:40 on Kan
  • B10 1:40 on Kan
  • A14L 1:20 on Amp

• We incubated B7new, B8new, B9 and B10 at 37°C in the morning. Then, five colonies for each plate were picked after about 11 hours. We inoculated these colonies in 5 ml of LB + Kan and incubated them overnight (37°C, 220 rpm). NOTE: B7new and B8new had smaller colonies than B9 and B10.

• We incubated A14L plate overnight at 37°C.

pH sensor

• Miniprep for the culture.
• Transformation into TOP10 E.coli.
• We plated transformed bacteria and incubated overnight at 37°C.

September, 4th

• A14L plate showed colonies and K116002(TOP10) plate showed a bacterial carpet (with some single colony).

• We inoculated a single colony from K116002(TOP10) plate in 1 ml of LB + Amp and incubated this inoculum for about 6 hours (37°C, 220 rpm). Then, a glycerol stock was prepared.

• Colony PCR/electrophoresis for 6 colonies from A14L plate. Colonies were saved inoculating them in 1 ml of LB + Amp and incubating them at 37°C, 220 rpm waiting for the end of the reaction.

• Gel results: all the colonies showed the amplicon with the right size, but even some extra band...we decided to keep A14L-1 to stock all the colonies in glycerol. We also decided to keep A14L-1: we inoculated it in 5 ml of LB + Amp to grow an overnight culture to check the sequence.

• We received sequencing results for B8-5again(VF2) and it was not correct.

• Glycerol stock/miniprep for 21 cultures:
  • B7new X5 colonies
  • B8new X5 colonies
  • B9 X5 colonies
  • B10 X5 colonies
  • A17new

• We sent A17new purified DNA to BMR Genomics for sequencing.

• Screening (E-P digestion cut) for B7new, B8new, B9 and B10 samples.

pH sensor

The plate showed grown colonies.

• We picked a single colony and infected 4 ml LB + Amp.
• Incubation overnight.

September, 5th

pH sensor

• Glycerol stock for the overnight TOP10 E.coli culture containing pNhaA + GFP + Amp plasmid.

September, 6th

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