Difference between revisions of "Team:Glasgow/Notebook"
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Revision as of 14:26, 29 July 2015
We went to Dundee University to attend the Scotland IGEM team's meeting. We had so much fun in the meeting, sharing different ideas with each team.
The 4 strains of E. coli used are: MGI655ZI (which has tetR, and lacIq, DS941 (recF-), TOP10 (recA-), DH5α (recA-). We designed Phlf and SrpR promoters as BioBricks to be ordered from IDT as if their prefix and suffix were cut with EcoRI and SpeI.
We grew bacteria from a single colony from an agar plate in liquid culture to prepare for the dilution series. We designed the Phlf and SrpR respressor sequences with a prefix, B0032 ribosome binding site, TACTAG scar sequence, the repressor itself, TACTAGAG scar sequence, B0010 extended terminator with the help of Steve, and suffix.
We made TOP10 (which lacks methylation and restriction system, which makes it difficult to ‘cross’ with strains that have the systems) and DH5α (which lacks a restriction system, but has a modification system, ergo, DNA from it can be transferred to other cells) competent for transformation. Since we needed to have the cells in exponential growth phase, we pipetted 4 mL of overnight culture into 20 mL of fresh broth. We made chloramphenicol agar plates. We are also tested our dilution series on non-selective agar plates.
For TOP10 we had a transformation efficiency of 1.26 x 106 colonies/µg of DNA. For DH5α the control was not transformed. DH5α can be 40 times less competent than TOP10, which would explain our result. TOP10 is mutant in the repressor for lacI, while DH5α has one copy, so they cannot fully repress the (multiply copied) plasmid. Therefore with IPTG there is no difference in GFP expression in TOP10, and there may be some difference in DH5α.
We did MiniPrep of the BioBrick transformants and GFP without a promoter. We kept a gel over the weekend in the refrigerator.
LuxD encodes a transferase, luxE is a synthetase, and luxC is the reductase. If the association map on photobiology of the DEG complex is correct, the whole tetrameric complex may be temperature-sensitive.
We transformed bacteria that glowed (F1 and F3 are glowing, while F2 and F4 are controls). We are going to measure their glow tomorrow with a more appropriate exposure configuration.We made competent cells.We were going over primer design.
We transformed cultures with BioBricks (for us and for the master’s lab) and ligations. To make bacteria glow, we have repeated the same experiment as yesterday. After 1h in the 25°C incubator we did not observe bacteria to glow. After 5h in the incubator bacteria were glowing bright. We ordered primers for uirR, uirS and all of the lux genes: LuxA, LuxB, LuxC, LuxD, LuxE, LuxG. For the forward primers we included B0032 ribosome binding site and scar. In addition, we ordered a g-block for plsiR. Sean gave us recipes for PCR and Restriction.
We made overnight cultures.
We made glycerol stocks from the BioBricks as well as MiniPreps