Team:UNIPV-Pavia/Notebook/Week3Aug

 = Week from August 17th, to August 23rd, 2009 =

August, 17th

 * This week we planned to perform a gel run for all the constructs of the ethanol producing operon in order to check for contaminant bands. Then, we planned to ligate a promoter upstream of the ethanol producing operon and to build up measurement systems for A11 (lactose sensor) and A12 (aTc sensor).


 * We inoculated 8 ul of:
 * B1-13
 * B2-5
 * B3-5
 * B4-2
 * B5-3
 * B6-3
 * E0240
 * A11-2
 * glycerol stocks in 5 ml of LB + suitable antibiotic.


 * We incubated these cultures overnight (37°C, 220 rpm).

Preparation of experiment with Tecan F200


 * We dissolved 22 mg of lactose in 5 ml of LB + Kan in order to have 4.5% lactose.


 * We prepared lactose assay kit for testing.

Experiment with Tecan F200


 * Download Protocol

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August, 18th

 * Glycerol stock for E0240, A11-2, B1, B2, B3 and B4 in order to have a backup.


 * Miniprep for:
 * B1-13
 * B2-5
 * B3-5
 * B4-2
 * B5-3
 * B6-3
 * E0240
 * A11-2


 * Digestion with EcoRI for:
 * B1-13
 * B2-5
 * B3-5
 * B4-2
 * B5-3
 * B6-3


 * Digestion with EcoRI-PstI for:
 * B1-13
 * B2-5
 * B3-5
 * B4-2
 * B5-3
 * B6-3


 * Digestion with PstI for:
 * B1-13
 * B2-5
 * B3-5
 * B4-2
 * B5-3
 * B6-3


 * Electrophoresis for the digested plasmids.




 * Gel results:
 * B1 - ok
 * B2 - ok
 * B3 - two extra-bands (consistent with previous gels)
 * B4 - ok
 * B5 - two extra bands (consistent with previous gels)
 * B6 - two extra bands (consistent with previous gels)


 * We decided to try to ligate a promoter upstream of B5 and B6 anyway.


 * We inoculated 8 ul of:
 * A4(X2)
 * A12
 * glycerol stocks.


 * We incubated these inocula at 37°C, 220 rpm overnight.

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August, 19th

 * Digestion for:
 * E0240(X-P)
 * A11-2(S-P)
 * B5(E-X)
 * B6(E-X)


 * Miniprep for:
 * A4(X2)
 * A12


 * Digestion for:
 * A4(E-S)(X2)
 * A12(S-P)


 * Precipitation with sodium acetate for:
 * A11-2(S-P)
 * B5(E-X)
 * B6(E-X)
 * A4(E-S)(X2)
 * A12(S-P)


 * Gel run/cut/purification for E0240.


 * Ligations:
 * B7 = A4(E-S) + B5(E-X) in pSB1AK3
 * B8 = A4(E-S) + B6(E-X) in pSB1AK3
 * A14 = A11(S-P) + E0240(X-P) in pSB1A2
 * A16 = A12(S-P) + E0240(X-P) in pSB1A2

Experiment with Tecan F200
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August, 20th

 * We resuspended pSB3K3 from 2009 Registry Distribution. The plasmid with ccdB was not consistent, so we resuspended a consistent brick (of ~700bp length, suggested in the Registry "Help" page: Kit Plate 2, well 15L, I714891 brick) contained in pSB3K3.


 * We transformed the overnight ligations and I714891 in TOP10 and plated transformed bacteria on LB agar plates + Amp (A14 and A16) or + Kan (pSB3K3, B7 and B8). We incubated the plates at 37°C overnight.


 * We sent these samples (stored at -20°C) to BMR Genomics for sequencing:
 * A15-1
 * A15-3
 * A11-2
 * A11-3
 * B5-3
 * B6-3
 * B3-5 (again, because we wanted to check for contaminants in our native stock)

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August, 21st

 * Colony PCR for A14 (5 colonies) and A16 (5 colonies) plates. Colonies were inoculated in 1 ml of LB + Amp and let grow waiting for the end of the reaction.


 * Electrophoresis for PCR results.


 * Gel results (picture not taken, sorry...):
 * A14 - 3rd colony seems the most pure and it had the expected length for ligated plasmid.
 * A16 - reaction worked only on A16-1, but it was negative.


 * We decided to keep A14-3 (positive at PCR) and A16-4 (randomly chosen) for digestion screening: we prepared a glycerol stock for them and re-filled the remaining 250 ul of bacteria with 4 ml of LB + Amp.


 * We incubated these cultures at 37°C, 220 rpm overnight.


 * We picked 2 colonies from B7, 5 colonies from B8 and 1 colony from I714891 plates. We inoculated them in 1 ml of LB + Kan and incubated them at 37°C, 220 rpm for 5 hours and 1/2. Then we prepared glycerol stocks and re-filled the remaining 250 ul with 4 ml of LB + Kan.


 * NOTE: we decided not to perform PCR on B7 and B8 plates because of the large size of positive inserts.


 * We incubated the re-filled cultures at 37°C, 220 rpm overnight.

Experiment with Tecan F200
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August, 22nd

 * Pellet preparation for the 10 overnight cultures, ready to be miniprepped next week! Pellets were stored at -20°C.

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