Team:UNIPV-Pavia/Notebook/Week3

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Notebook

Week 1	Week 2	Week 3	Week 4	Week 5	Week 6	Week 7
Week 8	Week 9	Week 10	Week 11	Week 12	Week 13	Week 14
Week 15	Week 16	Week 17	Week 18	Week 19	Week 20	Week 21
Week 22	Week 23	Week 24

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:

BBa_C0179	BBa_C0161	BBa_R0051	BBa_I14032
BBa_I15008	BBa_I15010

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:

BBa_C0079	BBa_C0179
BBa_C0161

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:

BBa_R0051	BBa_I15008	BBa_I15010	BBa_I14032
BBa_C0061	BBa_B0030

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:

BBa_I15008 (X-P)	BBa_I15010 (X-P)	BBa_I14032 (X-P)	BBa_C0061 (X-P)
BBa_B0030 (X-P)

While we had to insulate opened plasmids for:

BBa_R0051 (S-P)

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:

BBa_R0051 (S-P)	BBa_B0030 (X-P)	BBa_C0061 (X-P)	BBa_I15008 (X-P)
BBa_I15010 (X-P)

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):

BBa_J23100-BBa_E0240 (Pcon(E-S)-assGFP(E-X))
BBa_J23100-BBa_B0030 (Pcon(E-S)-RBS(E-X))
BBa_R0051-BBa_B0030 (Plam(S-P)-RBS(X-P))
BBa_B0030-BBa_E0040 (RBS(S-P)-GFP(X-P))
BBa_B0030-BBa_C0051 (RBS(S-P)-cI(X-P))
BBa_B0030-BBa_E1010 (RBS(S-P)-RFP(X-P))
BBa_B0030-BBa_C0061 (RBS(S-P)-luxI_LVA(X-P))
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:

BBa_C0161

BBa_C0179

BBa_C0079

BBa_I14032

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.

Wiki updating.