Team:Exeter/lab book/3gip/wk8

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ExiGEM2012 Lab Book 3GP wk6


Protocols \| Single Gene Plasmids and Enzyme Characterisation \| Showcasing Polysaccharide Production \| The 3-Gene Inducible Plasmid Operon Construction \| Glycobase
9th - 13th July - 16th - 20th July - 23rd - 27th July - 30th July - 3rd August - 6th - 10th August - 13th - 17th August - 20th - 24th August - 27th - 31st August - 3rd - 7th September - 10th - 14th September - 17th - 21st September
	The 3-Gene Inducible Plasmid: 27th - 31st August 2012 Tuesday 28th August Morning •3A assembly BBa_B0034 + wbnJ in pSB1K3 BBa_B0034 + wbnK in pSB1K3 BBa_B0034 + wfcA in pSB1K3 BBa_B0034 + wbbC(1) in pSB1K3 BBa_B0034 + wbbC(2) in pSB1K3 BBa_B0034 + wbbC(3) in pSB1K3 BBa_B0034 + wclY in pSB1K3 BBa_B0034 + wbiP in pSB1C3 BBa_B0034 + wbiP 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. Afternoon •Transformation: Chloramphenicol plasmid backbone pSB1C3, 2012 Distribution Kit Plate 1 3A Tetracycline plasmid backbone pSB1T3, 2012 Distribution Kit Plate 1 7A Kanamycin plasmid backbone pSB1K3, 2012 Distribution Kit Plate 1 5A Ampicillin plasmid backbone pSB1A3, 2012 Distribution Kit Plate 1 1G Invitrogen TOP10 competent cells split were split between 2 eppendorfs for 2 x 25μl. Spread on the relevant antibiotic plates for each transformation using 150μls. Wednesday 29th August Morning •Gel Digest of DNA from 21/08/2012 Lanes 1, 2, 3, 4 ompA Lanes 6, 7, 8, 9 BBa_J23119 + BBa_B0034 wbnJ Lanes 10, 11 BBa_J13002 + wbnK BBa_B0014v Planned another 3A assembly but had run out of plasmid backbone. Afternoon •Adding cultures Cells transformed with pSB1C3, pSB1T3, pSB1K3, pSB1A3 were added into liquid medium and incubated overnight. Protocol was followed using the relevant antibiotic for the BioBrick part as the selection antibiotic. Thursday 30th August Morning •Mini-Prepping of pSB1C3, pSB1T3, pSB1K3, pSB1A3 pSB1C3, pSB1T3, pSB1K3 and pSB1A3 were nanodropped and recorded very good concentrations. Afternoon •Transformation The ligations were transformed using the Competent Cell protocol and using the competent cells made in the lab. •Adding cultures Cells transformed with amyA in pSB1C3, hasA in pSB1C3, amyA + BBa_B0014 in pSB1C3, hasA + BBa_Boo14 in pSB1C3, sacB + BBa_Boo14 in pSB1C3 were added into liquid medium and incubated overnight. See Showcasing Polysaccharide Production: 27th - 31st August 2012, Thursday 30/08/12 Protocol was followed using chloramphenicol for the BioBrick part as the selection antibiotic. Friday 31st August Morning •3A assembly BBa_J13002 + wbnJBBa_B0014 in pSB1K3 BBa_J13002 + wbnKBBa_B0014 in pSB1K3 BBa_K764001+ wfcA BBa_B0014 in pSB1A3 BBa_K764001+ wclY BBa_B0014 in pSB1A3 BBa_K026000 + BBa_B0034 in pSB1K3 BBa_K026001 + BBa_B0034 in pSB1K3 BBa_J23119 + BBa_B0034 in pSB1K3 BBa_I0050 + BBa_B0034 in pSB1K3 BBa_B0034 wbbC obtained from PCR into pSB1C3 BBa_B0034 in pSB1A3 BBa_J13002 in pSB1A3 Performed with the NEB protocol with some adaptions 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 ligated constructs were transformed using the Competent Cell protocol and using the competent cells made in the lab.