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

(→‎Applications of BBa_K649104)
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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 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.
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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.
  
  
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[sample]
 
[sample]
  
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pSB1A2 Ptet-gfp(JD22597)
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Ptet-gfp on pSB1A2(JD22597)
  
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pSB6A1 promoterless-gfp(JD22597)
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Promoterless-gfp on pSB6A1(JD22597)
  
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pSB1A2 PlsrA-gfp(BBa_K649104)(JD22597)
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PlsrA-gfp on pSB1A2(BBa_K649104)(JD22597)
  
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pSB1A2 PlsrA-gfp(BBa_K11702-GFP)(JD22597)
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PlsrA-gfp on pSB1A2(BBa_K11702-GFP)(JD22597)
  
 
[Method]
 
[Method]

Revision as of 16:33, 3 October 2011

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

K649104 OD1.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 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

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