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Revision as of 10:46, 3 August 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
We made restriction digests of the minipreps and annealed R0011. B0032 oligos. After we ran the gel electrophoresis and extracted fragments from the gel. We left ligations overnight and made overnight cultures for making electrocompetent cells.
PCR was done to amplify UirR and lux genes (three separate reactions were done) and attempted to make electrocompetent cells. We ligated G-blocks of PhlF and SrpR into pSB1C3.
We made 2 other PCR reactions and tested all PCR products on gel. We found out that none of the amplifications worked except for the one of luxB. We attempted to transform electrocompetent with ligations and we made overnights for making of chemically competent cells in case our transformations fail.
We redid UirS PCR and checked it on gel. This time our reaction successfully yielded a product of the expected size. (What did we change, was it primer concentrations?). We attempted to transform cells again, only this time with chemically competent cells. The large centrifuge was broken, so we divided the work and used a smaller centrifuge in the cold room. We redid digests of PhlF and SrpR G-block, checked them on gel, and redid ligations into pSB1C3.
The transformation of chemically competent cells also failed (no growth of positive controls, no growth of anything we hoped to grow) and we do not know why. The plan for today is to insert UirS in pCR2.1TOPO. We are also planning to check gel purified PhlF and SrpR on gel. PLSIR G-block is to be resuspended and digested.
We ran the PCR products of UirR and luxA-G on a gel to check if our PCR worked. We also ligated PLSIR into pSB1C3. We made competent cells and set up overnight cultures and made buffer for the ligation of UirS into TOPO TA.
We made competent cells and transformed ligations (which ligations?). We ligated the purified UirS into TOPO TA and redid the luxE PCR. We ran digested UirR, luxA,B,C,D,G on a gel and extracted them. The digests were then ligated into pSB1C3. We set up overnights of registry order
We ran a gel of the PCR product extract and set up ligations (which ligations?). We made minipreps of the overnight cultures and made competent cells and transformed ligations. The transformants were set up as overnights.
We made minipreps from the overnight cultures: TOPO TA, control, lig. 17, lig. 18, lig. 20) and we ordered primers for site-directed mutagenesis. We digested (our minipreps?), ran them on a gel, extracted them, and set up ligations. We transformed cells with the luxE ligation and set up overnights of transformants.
We did minipreps and digested ligations 21, 22, 24, and ran them on a gel. We checked yesterday’s extractions on a gel.
We digested and ran a gel to check for inversion of the UirS PCR product in the TOPO TA plasmid. We made TOP10 overnights to make competent cells for UirS/TOPO TA, PLSIR.I13500/pSB1C3, PLSIR.E5501/pSB1C3, R0011N.Phlf/pSB1C3, R0011N. SrpR/pSB1C3, and controls (lig. 36, 38, 41, and 43. We also made multiple overnights of UirR/pSB1C3, LuxA/pSB1C3, LuxB/pSB1C3, LuxC/pSB1C3, LuxD/pSB1C3, LuxE/pSB1C3, LuxG/pSB1C3. We ran luxA-G PCR products on a gel and purified them.
We checked ligations with digests and transformed ligations of R0011N with Phlf and SrpR in pSB1C3 into TOP10 (these are ligations 35, 36, 37, and 38)