Team:Exeter/lab book/3gip/wk10

Operon Construction  |  Glycobase

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Monday 10th September

Morning

•3A Assembly

BBa_K206000_BBaB0034 + ompA BBa_K322921_BBa_B0014 in pSB1C3 (1)

BBa_K206000_BBaB0034 + ompA BBa_K322921_BBa_B0014 in pSB1C3 (2)

BBa_J13002 + ompA BBa_K322921_BBa_B0014 in pSB1C3 (1)

BBa_J13002 + ompA BBa_K322921_BBa_B0014 in pSB1C3 (2)

BBa_J23119 + wbnJ BBa_B0014 in pSB1C3

BBa_B0034 + wfcA BBaB0014 in pSB1C3

BBa_B0034 + wfcA BBaB0014 in pSB1K3

BBaK094120_BBa_B0034 + wfcA BBaB0014 in pSB1C3

Performed with the NEB protocol with some adaptations again. The volume of digestion was changed from 50μl to 20μl by adding less water so as to increase the DNA concentration. The enzymes and buffer concentrations were adapted as below. This was not a linearized plasmid backbone so for the plasmid digestion we used the NEB Biolabs protocol adapted to the amounts shown below.

Tuesday 11th September

Morning

•Transferring BioBricks into pSB1C3

wbnJ into pSB1C3

wbnK into pSB1C3

wfcA into pSB1C3

wbbC(1) into pSB1C3

wbbC(2) into pSB1C3

wbbC(3) into pSB1C3

wclY into pSB1C3

wbiP into pSB1C3

wbiP into pSB1C3

The genes and the destination plasmid were cut using EcoR1 and PstI. The digest was incubated for 90 minutes and then deactivated by heating at 80°C for 20 minutes. The ligation was performed as per the 3A assembly above but 4µl of plasmid DNA was used to 2µl of destination plasmid.

Afternoon

•Transformation

The ligations above and the ligations from the 12/09/2012 were transformed using the Competent Cell protocol using our competent cells made in the lab.

Wednesday 12th September

Morning

wbbC(1) into pSB1C3, wbbC(2) into pSB1C3, wbbC(3) into pSB1C3 and wbiP into pSB1C3 did not form colonies on the plates. They were repeated.

•Transferring BioBricks into pSB1C3

ompA into pSB1C3

wbbC(1) into pSB1C3

wbbC(2) into pSB1C3

wbbC(3) into pSB1C3

wbiP into pSB1C3

The genes and the destination plasmid were cut using EcoR1 and PstI. The digest was incubated for 90 minutes and then deactivated by heating at 80°C for 20 minutes. The ligation was performed as per the 3A assembly above but 4μl of plasmid DNA was used to 2μl of destination plasmid.

•3A Assembly

ompA + amyA BBa_B0014

Performed with the NEB protocol with some adaptations again. The volume of digestion was changed from 50μl to 20μl by adding less water so as to increase the DNA concentration. The enzymes and buffer concentrations were adapted as below. This was not a linearized plasmid backbone so for the plasmid digestion we used the NEB Biolabs protocol adapted to the amounts shown below.

•Two Step Phusion PCR using a 6 X 10 serial dilution

The reaction was set up using the Biolabs Phusion PCR protocol.

Two reactions were set up:

Reaction 1: Pbad Large and wbnJ using the primers supplied. See Operon Construction: 9th - 13th July 2012

Reaction 2: Reaction 1: Pbad Large and wbnJ using the primers supplied and varying the magnesium concentration. See Operon Construction: 9th - 13th July 2012

After PCR, PCR mix was dyed, loaded onto the gel and run for 20 minutes. No bands were shown on the gel so the PCR did not work.

Afternoon

•Transferring colonies to liquid medium

Cells transformed with below were added into liquid medium and incubated overnight.

BBa_E0240

BBa_K206000_BBaB0034 + ompA BBa_K322921_BBa_B0014 in pSB1C3 (1)

BBa_K206000_BBaB0034 + ompA BBa_K322921_BBa_B0014 in pSB1C3 (2)

BBa_J13002 + ompA BBa_K322921_BBa_B0014 in pSB1C3 (1)

BBa_J13002 + ompA BBa_K322921_BBa_B0014 in pSB1C3 (2)

BBa_J23119 + wbnJ BBa_B0014 in pSB1C3

BBa_B0034 + wfcA BBaB0014 in pSB1C3

BBaK094120_BBa_B0034 + wfcA BBaB0014 in pSB1C3

wbnJ into pSB1C3

wbnK into pSB1C3

wfcA into pSB1C3

wclY into pSB1C3

wbiP into pSB1C3

Protocol was followed using chloramphenicol for the BioBrick part as the selection antibiotic.

Thursday 13th. September

Morning

ompA + amyA BBa_B0014 showed no colonies on the overnight plate.

•Miniprepping

BBa_E0240

BBa_K206000_BBaB0034 + ompA BBa_K322921_BBa_B0014 in pSB1C3 (1)

BBa_K206000_BBaB0034 + ompA BBa_K322921_BBa_B0014 in pSB1C3 (2)

BBa_J13002 + ompA BBa_K322921_BBa_B0014 in pSB1C3 (1)

BBa_J13002 + ompA BBa_K322921_BBa_B0014 in pSB1C3 (2)

BBa_J23119 + wbnJ BBa_B0014 in pSB1C3

BBa_B0034 + wfcA BBaB0014 in pSB1C3

BBaK094120_BBa_B0034 + WfcA BBaB0014 in pSB1C3

wbnJ into pSB1C3

wbnK into pSB1C3

wfcA into pSB1C3

wclY into pSB1C3

wbiP into pSB1C3

All mini-preps were nanodropped showing very good concentrations.

Afternoon

•Gel Digest of DNA from 21/08/2012

A digestion using EcoR1 and PstI was run on all of the miniprepped DNA from today.

For the digestion we used Fermentas enzymes instead. The protocol remained the same but Fermentas Buffer H was used instead of the NEB Buffer 2.

Friday 14th September

Today a digestion/ligation was set up to put our remaining BioBricks into pSB1C3.

wbnJ

BBa_K094120_BBaB0034 in pSB1C3

wclY in pSB1C3

wbbc in pSB1C3

wfcA in pSB1C3

BBaB0034_wbiP in pSB1C3

BBaB0034_wbnK in pSB1C3

BBaB0034_wclY in pSB1C3

BBa_K206000_BBa_B0034 in pSB1C3

BBa_ K206001_BBa_B0034 in pSB1C3

BBa_I0500_BBa_B0034 in pSB1C3

BBa_J23119_ BBa_B0034 in pSB1C3

ompA in pSB1C3

ompA K322921_BBa_B0014 in pSB1C3

BBa_K094120_BBa_B0034_wclY BBa_B0014 in pSB1C3

BBa_J13002 _BBa_B0034_wclY BBa_B0014 in pSB1C3

BBa_J13002 amyA BBa_B0014 in pSB1C3

BBa_J13002 hasA BBa_B0014 in pSB1C3

The genes and the destination plasmid were cut using EcoR1 and PstI. The digest was incubated for 90 minutes and then deactivated by heating at 80°C for 20 minutes. The ligation was performed as per the 3A assembly above but 4μl of plasmid DNA was used to 2μl of destination plasmid.

For the digestion we used Fermentas enzymes instead. The protocol remained the same but we used Fermentas Buffer H instead of the NEB Buffer 2.

There was not enough time to transform any of these this afternoon so after the ligation they were stored at -20°C for the weekend.