Part:BBa_K2057006:Experience

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Applications of BBa_K2057006

As promoter BtI-BtII (BtI-BtIIp) was obtained from a gene found in B. thuringiensis, we want to test whether it drives the expression of a gene in E.coli. Our purpose was to demonstrate if promoters BtI-BtII and chiA74 could be used as constitutive promoters to express the LasR in our biological circuit. E. coli Top10/pHT3101-chiA74p, E. coli Top10/pHT3101-BtI-BtIIp, and E. coli Top10/pHT3101-BtI-BtIIp-gfp were grown at 37ºC to reach approximately 1 X 108 cells/mL. Cells were centrifuged, dissolved in 150 mM NaCl, 100 mM Tris, pH8, and sonicated. Protein concentration was determined by the Bradford method (BioRad). Fluorescence was determined using the same amount of protein. As was expected, the higher fluorescence, due to the GFP, was observed with E. coli Top10/pSB1C3-BtI-BtIIp-gfp (approx. 30000 UFR), indicating that dual promoter BtI-BtII from B. thuringiensis is functional in E. coli. No fluorescence was detected with E. coli lacking of the gfp.

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