Part:BBa_K649104:Experience

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

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. 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)

[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.

User Reviews

UNIQf5a5ded503f77d17-partinfo-00000000-QINU UNIQf5a5ded503f77d17-partinfo-00000001-QINU