Wild type terminator from T7 bacteriophageĬonstitutive promoter veg(BBa_K143012) coupled to the strong Ribosome Binding Site spoVG(BBa_K143021) from B. We first confirmed that the parts from the series expressed RFP differentially according to their strengths by growing overnight cultures of each and looking for the color spectrum from dark red to light red to colorless.įig.5: Burden assay results for a sample of interesting burdensome parts. Having this promoter strength information allowed us to hypothesize that if the burden monitor was indeed accurate, parts with strong promoters would express higher levels of burden and parts with weak promoters would express lower levels of burden. The measured strengths of each of these parts have been established by previous teams (iGEMBerkeley). The Anderson Series of Constitutive Promoters (parts that each contain a promoter of varying strength, an RBS of fixed strength, and an RFP whose degree of expression is dictated by promoter strength) was chosen for the first step of demonstration of validity of our burden monitor. Using these 4 methods, we were able to confirm both the validity and the replicability of the burden monitor. We decided that we would approach this in four ways: (1) By using the burden monitor to measure the burden of constructs with known measured promoter strengths, (2) By using the burden monitor to measure the burden of the calibration strains we created to confirm that the results match the burden levels we hypothesized for each one, (3), By seeing if burdensome parts from the 2018 iGEM kit reflected high burden positions on a burden graph, and (4) By sending out the burden monitor to other teams (MSU, Rice, and Texas Tech University) for them to test out on their parts so that we could verify replicability of the protocol. In order to demonstrate that our engineered system- the burden monitor- worked, we decided to test it out in a series of realistic conditions both at our own lab at UT Austin and the labs of the teams we collaborated with.
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