Difference between revisions of "Part:BBa K649104:Experience"

Revision as of 21:42, 3 October 2011

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Applications of BBa_K649104

Median fluorescence intensity(MFI) of BBa_K649104 was much higher than that of promoterless-gfp(negative control).
This work is done by Takuya Tsubaki.

Fluorescence intensity of our lsrA promoter-gfp(BBa_649104) was much higher than that of promoterless-gfp(negative control), showing that our new lsrA promoter(BBa_649100) works. On the other hand, fluorescence intensity of lsrA promoter-gfp((BBa_117002)-gfp) was almost the same as promoterless-gfp(negative control), showing that lsrA promoter(BBa_K117002) does not work properly.The difference between lsrA promoter(BBa_K117002) and lsrA promoter(BBa_649100) is whether promoter contains CRP binding site or not. Our lsrA promoter(BBa_649100) contains this site but lsrA promoter(BBa_K117002) does not. According to Wang(2005) 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]

Ptet-gfp on pSB1A2(JD22597)

Promoterless-gfp on pSB6A1(JD22597)

PlsrA-gfp on pSB1A2(BBa_K649104)(JD22597)

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

JD22597 lacks lsrR.

[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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