Team:Exeter/lab book/3gip/wk9

Operon Construction  |  Glycobase

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Monday 3rd September

Morning

•3A Assembly

BBa_J13002 + amyA BBa_B0014 in pSB1K3

BBa_J13002 + hasA BBa_B0014 in pSB1K3

ompA + sacB BBa_B0014 in pSB1K3

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.

Afternoon

•Transformation

The ligations were transformed using the Competent Cell protocol using our Competent Cells made in the lab.

•Transferring colonies to liquid medium

Time restraints, weekend and talks had meant there was a backlog of 3A transformations to be miniprepped. All transformations from the 21/08/2012 onwards were added into liquid medium and incubated overnight. Four colonies from each plate. 10μl of broth was used to ensure a good DNA concentration.

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

Tuesday 4th September

Morning

•Miniprepping

Following the growth of our cultures from the night before, 89 minipreps needed to be performed this morning. Using two teams we took on the task, 24 mini-preps at a time. Problems with labelling and timing did occur.

Most of the mini-preps were nanodropped with some showing very good concentrations and some not. Some of the tubes were centrifuged by mistake before being lysed and neutralised seriously affecting the final concentration.

•Transferring colonies to liquid medium

Cells transformed with BBa_J13002 + Cyc_BBa_B0014 in pSB1K3, BBa_J13002 + has_BBa_B0014 in pSB1K3, ompA + sacB BBa_B0014 in pSB1K3 were all added into liquid medium and incubated overnight. See, Showcasing Polysaccharide Production: 27th - 31st August 2012, Wednesday 05/09/12

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

Wednesday 5th September

Today was a day for helping out on other projects, tidying up the lab and tying up loose ends.

Thursday 6th September

Morning

•Gel Digest of DNA from 21/08/2012

A digestion using EcoR1 and PstI was run on all of the miniprepped DNA from Tuesday. The tubes were set up and mastermixes made and added. There would not be enough time to set the gel up and run it so the digest was left in the incubator overnight at 37°C. The gel was wrapped and stored in the fridge and the prepared DNA was stored at -20°C.

There wasn't enough EcoR1 left for the digestion so instead we used Fermentas enzymes. The protocol remained the same but Fermentas Buffer H was used instead of the NEB Buffer 2.

Afternoon

•Protein Expression

The gel showed positive for BBa_J13002 + wbnK BBa_B0014 and BBa_K764001+ wclY BBa_B0014. We grew some in 10μl of liquid broth and incubated. When the OC600 reached 0.5A on the spectrophotometer we induced the cultures with 0.5mM of IPTG with the placI promoter and 50ng/ml, 100ng/ml and 5mg/ml with the Tetr promoter. The samples were placed back in the incubator. 1ml samples were taken at 15.30 and 16.30 and frozen. The remaining samples were placed back in the incubator.

Friday 7th September

Morning

•SDS-PAGE

A final sample for the protein expression was taken at 9.00am this morning and frozen.

The Biggest Gel We Have Ever Run.

Undergraduate iGEM Lesson for the week – don’t take on too much!

The frozen samples for protein expression were unfrozen and the 9.00am samples were run on an SDS-page gel. It showed no unique proteins. In future we would always tag proteins to eliminate many of the identification problems.