Reporter

Part:BBa_K649104:Experience

Designed by: Hiroki Yoshise   Group: iGEM11_Tokyo_Tech   (2011-09-26)
Revision as of 11:33, 3 October 2011 by Notothenia (Talk | contribs) (→‎Applications of BBa_K649104)

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Applications of BBa_K649104

K649104 OD.png
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 promoter lsrA-gfp(BBa_649104) was much higher than that of promoterless-gfp(negative control), showing that our new promoter lsrA(BBa_649100) works. On the other hand, fluorescence intensity of PlsrA(BBa_117002)-gfp was almost the same as a promoterless-gfp(negative control), showing that BBa_K117002 does not work properly.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(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]

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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