Difference between revisions of "Part:BBa K649104:Experience"
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===Applications of BBa_K649104=== | ===Applications of BBa_K649104=== | ||
| − | We characterized BBa_K649104 and BBa_K117002 in lsrR(-) strain of E.coli. | + | We characterized BBa_K649104 and BBa_K117002 in <i>lsrR</i>(-) strain of <i>E.coli</i>. |
| − | [[Image: | + | [[Image:PlsrA2.png|thumb|center|600px|Fluorescence intensity of BBa_K649104 was much higher than that of promoterless-gfp(negative control).<br>Strain used in this assay lacks <i>lsrR</i>.<br>This work is done by Takuya Tsubaki.]] |
| − | Fluorescence intensity of our lsrA promoter-gfp(BBa_K649104) was much higher than that of promoterless-gfp(negative control), showing that our new lsrA promoter(BBa_K649100) works. In spite of no LsR repression, gene transcription does not take place sufficiently. On the other hand, fluorescence intensity of lsrA promoter-gfp((BBa_K117002)-(BBa_J54103)) was almost the same as promoterless-gfp(negative control), showing that lsrA promoter(BBa_K117002) does not work properly. Gene transcription takes place sufficiently. The difference between lsrA promoter(BBa_K117002) and lsrA promoter(BBa_K649100) is whether promoter contains CRP binding site or not. Our lsrA promoter(BBa_K649100) 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. | + | Fluorescence intensity of our lsrA promoter-<i>gfp</i>(BBa_K649104) was much higher than that of promoterless-<i>gfp</i>(negative control), showing that our new lsrA promoter(BBa_K649100) works. In spite of no LsR repression, gene transcription does not take place sufficiently. On the other hand, fluorescence intensity of lsrA promoter-<i>gfp</i>((BBa_K117002)-(BBa_J54103)) was almost the same as promoterless-<i>gfp</i>(negative control), showing that lsrA promoter(BBa_K117002) does not work properly. Gene transcription takes place sufficiently. The difference between lsrA promoter(BBa_K117002) and lsrA promoter(BBa_K649100) is whether promoter contains CRP binding site or not. Our lsrA promoter(BBa_K649100) 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. |
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[sample] | [sample] | ||
| − | Ptet-gfp on pSB1A2(JD22597) | + | Ptet-<i>gfp</i> on pSB1A2(JD22597) |
| − | Promoterless-gfp on pSB6A1(JD22597) | + | Promoterless-<i>gfp</i> on pSB6A1(JD22597) |
| − | PlsrA-gfp on pSB1A2(BBa_K649104)(JD22597) | + | PlsrA-<i>gfp</i> on pSB1A2(BBa_K649104)(JD22597) |
| − | PlsrA-gfp on pSB1A2( | + | PlsrA-<i>gfp</i> on pSB1A2(BBa_K117002-<i>gfp</i>)(JD22597) |
| − | JD22597 is a strain lacking lsrR. | + | JD22597 is a strain lacking <i>lsrR</i>. |
[Method] | [Method] | ||
| − | 1.Overnight cultures of reporter strains grown at 37 | + | 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. | 2. After their OD590 reached 0.15, the fresh cultures were diluted 1:100. | ||
| − | 3. After 4-hour incubation at 37 | + | 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. |
| − | [[Image: | + | [[Image:Our_new_lsrA_promoter_activity%28BBa_K649104%29.png|thumb|center|400px|After four hours from OD590 reaching 0.15, we measured OD.<br>This work is done by Takuya Tsubaki.]] |
===User Reviews=== | ===User Reviews=== | ||
Latest revision as of 15:18, 28 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
We characterized BBa_K649104 and BBa_K117002 in lsrR(-) strain of E.coli.
Fluorescence intensity of our lsrA promoter-gfp(BBa_K649104) was much higher than that of promoterless-gfp(negative control), showing that our new lsrA promoter(BBa_K649100) works. In spite of no LsR repression, gene transcription does not take place sufficiently. On the other hand, fluorescence intensity of lsrA promoter-gfp((BBa_K117002)-(BBa_J54103)) was almost the same as promoterless-gfp(negative control), showing that lsrA promoter(BBa_K117002) does not work properly. Gene transcription takes place sufficiently. The difference between lsrA promoter(BBa_K117002) and lsrA promoter(BBa_K649100) is whether promoter contains CRP binding site or not. Our lsrA promoter(BBa_K649100) 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.
The working part we created allow the use of AI-2 as a signaling molecule, which is a very powerful tool to build complex Synthetic Biology systems.
[sample]
Ptet-gfp on pSB1A2(JD22597)
Promoterless-gfp on pSB6A1(JD22597)
PlsrA-gfp on pSB1A2(BBa_K649104)(JD22597)
PlsrA-gfp on pSB1A2(BBa_K117002-gfp)(JD22597)
JD22597 is a strain lacking 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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User Reviews
UNIQ9d9b691275c9f636-partinfo-00000000-QINU UNIQ9d9b691275c9f636-partinfo-00000001-QINU