Part:BBa_K649201:Experience

Applications of BBa_K649201

We prepared a competent cell JM2.300 into which Pbad/araC-Cre(pSB1A2, BBa_I718008) had been constructed. Subsequently, the BioBrick was constructed into the cell. The strain was grown in a 3ml liquid culture, and 75μl of 2M arabinose was added to induce Cre expression. For the control experiments we used the same strain without arabinose induction and a JM2.300 strain which was induced with arabinose and had only BioBrick. All the strains were cultured each for periods of 0.5, 1, 2, and 4 hours, and in each case the florescence levels were measured by flow cytometer and FLC.

We measured the fluorescence levels to prove that recombination only occurs in presence of Cre recombinase. On the sample with the Pbad/araC-Cre construction, we found that recombination occurred when arabinose was added. We could also observe recombination occurred when arabinose was not added, which can be explained due to a leaking in the Pbad/araC promoter. Opposite to this result, when we measured the levels of the sample without the Pbad/araC-Cre construction, we found that the GFP levels were far lower than those of the sample with the Pbad/araC-Cre construction. This clearly proves that our lox constructions respond correctly to the effects of Cre recombinase.

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We also confirmed our results optically by taking florescence photographs. Whole samples were plated and incubated in 37℃ for 10 hours. Photographs of three samples of BBa_K649201 were taken at t = 0.5 hours using red florescence filter, green florescence filter and no filter as shown below, respectively.

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