Team:UNIPV-Pavia/Notebook/Week4

Week 4: 06/09/08 - 06/14/08
06/09/08
 * We prepared 4 glycerol stocks taking 800 µl from 9 ml cultures containing:


 * We performed digestion protocol to open plasmids:


 * We ran a gel with these parts.


 * We performed gel extraction.


 * Antarctic Phosphatase for these 4 parts.


 * Ligation (30 µl final volume):
 * 1) BBa_J23100-BBa_E0240       (Pcon(E-S)-assGFP(E-X))
 * 2) BBa_R0051-BBa_B0030       (Plam(S-P)-RBS(X-P))
 * 3) BBa_B0030-BBa_E0040       (RBS(S-P)-GFP(X-P))
 * 4) BBa_B0030-BBa_C0051       (RBS(S-P)-cI(X-P))
 * 5) BBa_B0030-BBa_E1010       (RBS(S-P)-RFP(X-P))
 * 6) BBa_B0030-BBa_C0061       (RBS(S-P)-luxI_LVA(X-P))
 * 7) BBa_B0030-BBa_C0078       (RBS(S-P)-lasI(X-P))
 * 1) BBa_B0030-BBa_C0078       (RBS(S-P)-lasI(X-P))


 * We incubated ligation overnight at 16°C.

06/10/08
 * We transformed the whole volume of the ligations.


 * We plated the 7 ligations.

06/11/08
 * BBa_J23100-BBa_E0240 and BBa_R0051-BBa_B0030 plates showed respectively 4 and 1 colonies, while the other plates didn't show any colony.


 * We picked up one colony from the two working plates to grow 9 ml cultures of transformed bacteria overnight.


 * We also infected 9 ml of LB + suitable antibiotic with 30 µl of:


 * glycerol stocks. We incubated all the 9 ml cultures overnight at 37°C.


 * We contacted Francesca Ceroni from Bologna team for suggestions about ligation reaction. We decided to reduce reaction volume from 30 to 20 µl, increment insert:vector molar ratio from 1:2 to 1:3.

06/12/08
 * We prepared 9 glycerol stocks taking 800 µl from 9 ml cultures containing:


 * Miniprep for the 9 parts.


 * We tested our microscope and BBa_J23100-BBa_E0240 fluorescence:
 * We infected 150 µl of LB + Amp with 30 µl of BBa_J23100 glycerol stock (positive control)
 * We took 30 µl of BBa_J23100-BBa_E0240 9 ml culture and infected 150 µl of LB + Amp.
 * We incubated these two cultures at 37°C, 220 rpm for 3 hours.


 * Then, we took the 180 µl cultures and tried to watch them respectively through red and green fluorescence channel. BBa_J23100-BBa_E0240 didn't glow, while our positive control, BBa_J23100, glowed correctly through red channel.




 * Digestion for:


 * Gel run for these parts.


 * Gel extraction for these parts.


 * Antarctic Phosphatase for BBa_E0240(E-X) and BBa_B0030(S-P)


 * Ligation (20 µl final volume):
 * 1) BBa_J23100-BBa_E0240       (Pcon(E-S)-assGFP(E-X))
 * 2) BBa_B0030-BBa_C0051       (RBS(S-P)-cI(X-P))
 * 3) BBa_B0030-BBa_E1010       (RBS(S-P)-RFP(X-P))
 * 4) BBa_B0030-BBa_C0061       (RBS(S-P)-luxI_LVA(X-P))
 * 5) BBa_B0030-BBa_C0078       (RBS(S-P)-lasI(X-P))
 * 1) BBa_B0030-BBa_C0061       (RBS(S-P)-luxI_LVA(X-P))
 * 2) BBa_B0030-BBa_C0078       (RBS(S-P)-lasI(X-P))


 * We prepared 2 eppendorf tubes for each ligation reaction. We incubated one ligation set at 4°C overnight and the other ligation set at 25°C for 3 hours.


 * We received sequencing results for BBa_I14032: the sequence was 37 bp long, but it was not PlacIQ. We contacted Meagan to try to solve this problem.

06/13/08
 * We prepared 5 glycerol stocks taking 800 µl from 9 ml cultures grown.


 * Miniprep for these cultures


 * We sent all the 5 purified plasmids to Primm for sequencing.


 * We transformed the whole volume of the ligations.


 * We plated the 10 ligations.

06/14/08
 * Only one of 10 ligation plates worked: BBa_B0030-BBa_C0078 showed one colony...Horrible result!


 * We decided to dedicate the following week to debug our protocols and to change something:
 * Reduce ligation volume to 10 µl
 * Inactivate T4 Ligase after ligation heating at 65°C for 10 min
 * Heat at 65°C for 5 min ligation mix before adding T4 Ligase and its buffer
 * Use different amounts of vector and insert
 * Don't use Antarctic Phosphatase