Team:UNIPV-Pavia/Notebook/Week3

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Latest revision as of 21:25, 26 October 2008

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Notebook

Week 3: 06/03/08 - 06/08/08

06/03/08

• We transformed 60 µl of TOP10 with 2 µl of the 6 parts (DNA + glycerol) we received from IGEM HQ:

• NOTES: We noticed that IGEM 2007 teams which used luxI or lasR parts choosed BBa_C0061 instead of BBa_C0161 (for luxI) and BBa_C0079 instead of BBa_C0179 (for lasR). So, we decided in addition to amplify BBa_C0061 and BBa_C0079; we shall see later which one to choose for our project between the two luxI and the two lasR.

• So, we cut paper spots for BBa_C0061 and BBa_C0079 and resuspended them in 10 µl of warmed TE buffer.

• We transformed these 2 parts using 4 µl of DNA in TE.

• We plated transformed bacteria and incubated them at 37°C overnight.

06/04/08

• After overnight incubation, the following 5 plates showed colonies:

BBa_C0061 BBa_R0051 BBa_I14032 BBa_I15008 BBa_I15010

BBa_R0051 plate: very high yield from IGEM HQ DNA + glycerol

BBa_C0061 plate: medium yield from paper spot

• while the following 3 plates did not:

• We repeated the transformation for BBa_C0079, BBa_C0179 and C0161. We used 6 µl of DNA in TE for BBa_C0079, while we used 3 µl of DNA + glycerol for BBa_C0179 and BBa_C0161.

• We plated transformed bacteria and incubated them at 37°C overnight.

• While we were preparing our 5 ml cultures for the 5 working plates, we noticed that LB + Amp was contaminated! We decided to prepare a big quantity of LB + Amp and also of LB + Kan: we prepared 0.5 l LB + Amp and 0.5 l LB + Kan for liquid cultures; 0.5 l LB + Amp and 0.5 l LB + Kan for plates.

A lot of just prepared LB plates

• We picked up one colony from BBa_C0061, BBa_R0051, BBa_I14032, I15008 and I15010 plates to grow 5 ml cultures of transformed bacteria overnight.

• We also infected 5 ml of LB + Amp with 15 µl of BBa_B0030 glycerol stock. We incubated the 5 ml culture overnight at 37°C.

• We received QIAGEN QIAprep Spin Miniprep Kit!!! We will inaugurate it tomorrow on these 5 ml cultures;)

06/05/08

• We received Euroclone Antarctic Phosphatase: we are going to use it in the afternoon before ligation reaction.

• We prepared 6 glycerol stocks taking 800 µl from 5 ml cultures containing:

• We performed miniprep for these 6 parts with our new fantastic kit;) Plasmid quantification confirmed a higher yield than our previous QIAGEN kit.

• We performed plasmid digestion for these parts (20 of 30 µl).

• We had to insulate excided fragments for:

• While we had to insulate opened plasmids for:

• Due to the different dimension of the DNA we had to insulate, we ran 3 different gels:
  • one for BBa_R0051 (S-P)
  • one for BBa_I14032 (X-P) and BBa_B0030 (X-P)
  • one for BBa_C0061 (X-P), BBa_I15008 (X-P) and BBa_I15010 (X-P).

• We could see and cut all the bands we wanted, except for BBa_I14032, for which we couldn't see any band.

• We performed gel extraction for:

• All the 3 overnight plates worked (low yield for all).

• We picked up one colony from BBa_C0161, BBa_C0179 and BBa_C0079 plates to grow 5 ml cultures of transformed bacteria overnight. We did the same thing for BBa_I14032 6/3/08 plate; we didn't use glycerol stock for BBa_I14032 because we thought there was something wrong with the colony we picked on 6/4/08.

• Antarctic Phosphatase for BBa_E0240 (E-X), BBa_R0051 (S-P), BBa_B0030 (S-P), BBa_B0030 (E-X).

• We calculated the amount of vector/insert for ligation reaction to have a molar ratio of 2:1 (insert:vector).

• We performed ligation reaction (30 µl final volume) for (vectors are in bold type):

1. BBa_J23100-BBa_E0240        (Pcon(E-S)-assGFP(E-X))
2. BBa_J23100-BBa_B0030        (Pcon(E-S)-RBS(E-X))
3. BBa_R0051-BBa_B0030        (Plam(S-P)-RBS(X-P))
4. BBa_B0030-BBa_E0040        (RBS(S-P)-GFP(X-P))
5. BBa_B0030-BBa_C0051        (RBS(S-P)-cI(X-P))
6. BBa_B0030-BBa_E1010        (RBS(S-P)-RFP(X-P))
7. BBa_B0030-BBa_C0061        (RBS(S-P)-luxI_LVA(X-P))
8. BBa_B0030-BBa_C0078        (RBS(S-P)-lasI(X-P))

• We incubated ligation overnight at 16°C.

06/06/08

• We prepared 4 glycerol stocks taking 800 µl from 5 ml cultures containing:

• Miniprep for BBa_C0161, BBa_C0179, BBa_C0079 and BBa_I14032.

We performed plasmid digestion for these 4 parts (20 of 30 µl). We ran two different gels: one for BBa_C0161 (X-P), BBa_C0179 (X-P) and BBa_C0079 (X-P) one for BBa_I14032 (X-P), that is smaller than the other 3 parts For the second time we couldn't see any band for BBa_I14032. We performed gel extraction for: BBa_C0161 (X-P) BBa_C0179 (X-P) BBa_C0079 (X-P)

Mattia cutting BBa_C0161 (X-P), BBa_C0179 (X-P) and BBa_C0079 (X-P) gel

• We decided to sequence BBa_I14032 to check if the sequence is correct. Unfortunately we had not enough plasmid and so we picked another colony from 6/3/08 plate, infected 9 ml LB + Kan and incubated it overnight at 37°C. We decided to use 9 ml instead of 5 ml in order to verify if we could increment our yield without saturating the QIAGEN spin column and without performing a midiprep.

• We transformed the whole ligation volume (30 µl) for all the 8 overnight ligations.

• We plated transformed bacteria and incubated at 37°C overnight.

06/07/08

We took 800 µl of BBa_I14032 overnight culture to prepare a glycerol stock. Miniprep for BBa_I14032 9 ml culture: absorbance spectrum was very good, so we decided to use 9 ml cultures even the next times. We sent BBa_I14032 sample and VF2 primer to Primm for sequencing.

NanoDrop output for BBa_I14032 9 ml miniprep

• Only BBa_J23100-BBa_B0030 (Pcon(E-S)-RBS(E-X)) plate showed colonies. We put it at 4°C.

• NOTES: obviously we were not happy with this result. So, how could we do to debug this situation?
  • Our hypothesis was that we had to focus on plasmid digestion process: we approximately checked the length of every opened plasmid/excided fragment we wanted to insulate on gel and they were right.
  • We were sure that restriction enzymes cut both the restriction sites (E-S or X-P) for excided fragments, but we could not be so sure whether opened plasmids had been cut (E-X or S-P) in both restriction sites or only in one site.
  • In previous digestion protocols to open plasmids, we performed 2 cutting cycles: one for the first enzyme (1 hour and 30 min) and one for the second enzyme (1 hour and 30 min).
  • We supposed there was something wrong with that digestion protocol, so we decided to perform plasmid digestion again for our 4 plasmids to open, following the digestion protocol (read Protocols section for details).
  • We also noticed that digestion protocol for excided fragments worked very well for BBa_B0030, whose fragment length is 15 bp, so we hoped that this protocol might actually work to open plasmids.
  • In addition, we decided to check if our only working plate was a false positive: next week we will perform PCR/electrophoresis to check for contaminating plasmids (we could not check immediately for insert length by electrophoresis because BBa_J23100 and BBa_B0030 are small parts).

• Wiki updating.

06/08/08

• We picked up one colony from BBa_J23100-BBa_B0030 plate to grow a 9 ml culture of transformed bacteria overnight.

• We also infected 9 ml of LB + Amp with 15 µl of BBa_B0030, BBa_B0030 (another time), BBa_R0051, BBa_E0240 glycerol stocks. We incubated 9 ml cultures overnight at 37°C.

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