Team:UNIPV-Pavia/Notebook/Week1Sep

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

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.

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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 (the right) 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.

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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.

pH sensor

 * We inoculated a single colony from K116002 streaked plate in 5 ml of LB + Amp.
 * Overnight incubation at 37°C, 220 rpm.

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September, 3rd

 * 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 K116002 overnight culture. We incubated the plate (LB + Amp) with transformed bacteria overnight at 37°C.
 * 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 plated transformed bacteria and incubated overnight at 37°C.
 * We also sent purified DNA to BMR Genomics for sequencing.

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September, 4th

 * A14L plate showed colonies.


 * 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 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

 * 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).
 * Glycerol stock.

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September, 5th

 * Fermentation experiment setup
 * Wiki updating

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September, 6th

 * Fermentation experiment
 * Cloning
 * Wiki updating

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