Part:BBa_K786001:Experience

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Characterization of BBa_K786001

1. Promoter efficiency for BBa_K786001. BBa_K786002, BBa_K786003

To test the expression of sensory rhodopsin triggered by constitutive promoter BBa_J23100 to sense light,

we need to test the effect conferred by different E. coli strains to expression of red fluorescence protein reporter downstream of BBa_J23100 to different bacterial strains. It allows us to select the suitable strain(s) for this constitutive promoter for expressing sensory rhodopsin.

Florescence plate reader was used to take readings of fluorescence emission of 635nm and absorbance at 600nm (OD600) between time intervals of 12 hours on each strain. The measurements were started when the cultures reached a OD600 of around 0.4 that represents log phase of active proliferation. Growth curve and fluorescence intensity against time were plotted to compare cell growth and protein expression on different strains.

Three independent experiments were conducted. No significant difference was observed on the growth curves, indicating a similar growth rate among the three bacterial strains with BBa_J23100 transformed. It implies the promoter does not cause cell toxicity or growth inhibition of these three bacterial strains.

For the protein expression, the results showed that the fluorescence intensity of reporter in DH5α was significantly lower compared with TOP10 and BL21(DE3).

 

To conclude, DH5α is not an optimal strain to utilize promoter BBa_J23100, while TOP10 and BL 21(DE3) can effectively express the reporter. Therefore, in downstream application of our light sensing biobricks (BBa_K786001, BBa_K786002, BBa_K786003) in which BBa_J23100 was used, DH5α are not used.

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