Difference between revisions of "Part:BBa K649104:Experience"
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[sample] | [sample] | ||
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pSB1A2 Ptet-gfp(JD22597) | pSB1A2 Ptet-gfp(JD22597) | ||
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pSB6A1 promoterless-gfp(JD22597) | pSB6A1 promoterless-gfp(JD22597) | ||
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pSB1A2 PlsrA-gfp(BBa_K649104)(JD22597) | pSB1A2 PlsrA-gfp(BBa_K649104)(JD22597) | ||
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pSB1A2 PlsrA-gfp(BBa_K11702-GFP)(JD22597) | pSB1A2 PlsrA-gfp(BBa_K11702-GFP)(JD22597) | ||
[Method] | [Method] | ||
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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. | 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. | ||
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2. After their OD590 reached 0.15, the fresh cultures were diluted 1:100. | 2. After their OD590 reached 0.15, the fresh cultures were diluted 1:100. | ||
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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 | 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 | ||
Revision as of 10:00, 3 October 2011
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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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