Part:BBa_K649104:Experience

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Applications of BBa_K649104

To confirm that promoter lsrA(BBa_K117002) does not work properly, we introduced a gfp gene(BBa_J54103) downstream of the promoter. As a consequence, fluorescence intensity of promoter lsrA(BBa_K117002)-gfp was almost the same as promoterless-gfp(negative control), showing that promoter promoter lsrA(BBa_K117002) does not work properly. On the other hand, fluorescence intensity of our promoter lsrA-gfp(BBa_649104) was much higher than that of promoterless-gfp(negative control), showing that our new promoter lsrA(BBa_649100) works. The difference between promoter lsrA(BBa_K117002) and promoter lsrA(BBa_649100) is whether promoter contains CRP binding site or not. Our promoter lsrA(BBa_649100) contains this site. According to Wang et al, cAMP-CRP directly binds to the upstream of promoter and stimulates expression of the lsr operon.

…TGTGAtctattcgTCGGA…

CRP recognition sites are shown in capital letter.BBa_K649100 contains this sites.

[sample]

pSB1A2 Ptet-gfp(JD22597)

pSB6A1 promoterless-gfp(JD22597)

pSB1A2 PlsrA-gfp(BBa_K649104)(JD22597)

pSB1A2 PlsrA-gfp(BBa_K11702-GFP)(JD22597)

[Method]

1.Overnight cultures of reporter strains grown at 37 °C in LB medium containing appropriate antibiotics were diluted 1:100 into 3 ml of LB medium and were incubated at 37 °C as fresh cultures.

2. After their OD590 reached 0.15, the fresh cultures were diluted 1:100.

3. After 4-hour incubation at 37 °C, 1 ml of each culture was moved to 1.6ml tube and its fluorescence intensity was measured with a flow cytometer

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