Difference between revisions of "Part:BBa K649101:Experience"
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===Applications of BBa_K649101=== | ===Applications of BBa_K649101=== | ||
− | To confirm LsrR represses lsrA promoter, we constructed BBa_K649105. | + | To confirm LsrR represses lsrA promoter, we constructed BBa_K649105. |
+ | |||
+ | We characterized BBa_K649105 in E.coli MG1655. | ||
+ | |||
+ | [[Image:Lsrr.png|thumb|center|500px|Fluorescence intensity is decreased by LsrR repression.<br>This work is done by Hiroki Yoshise.]] | ||
+ | |||
+ | |||
+ | We confirmed that LsrR represses lsrA promoter. We measured LsrR repression activity by using a plasmid (BBa_K649105) which have a lsrR gene downstream of lsrA promoter-gfp.By the LsrR repression, the fluorescence intensity decreased 3-fold. This result shows that LsrR successfully repressed lsrA promoter. 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 pSB6A1(JM2.300)(positive control) | ||
+ | |||
+ | Promoterless-gfp on pSB6A1(JM2.300)(negative control) | ||
+ | |||
+ | PlsrA-gfp on pSB3K3(MG1655) | ||
+ | |||
+ | PlsrA-gfp-PlsrR-lsrR on pSB3K3(MG1655) | ||
+ | |||
+ | [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 1:10. | ||
+ | |||
+ | |||
+ | 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:LsrOD.png|thumb|center|500px|After four hours from OD590 reaching 0.15, we measured OD.<br>This work is done by Hiroki Yoshise.]] | ||
+ | |||
===User Reviews=== | ===User Reviews=== |
Revision as of 13:29, 28 October 2011
Applications of BBa_K649101
To confirm LsrR represses lsrA promoter, we constructed BBa_K649105.
We characterized BBa_K649105 in E.coli MG1655.
We confirmed that LsrR represses lsrA promoter. We measured LsrR repression activity by using a plasmid (BBa_K649105) which have a lsrR gene downstream of lsrA promoter-gfp.By the LsrR repression, the fluorescence intensity decreased 3-fold. This result shows that LsrR successfully repressed lsrA promoter. 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 pSB6A1(JM2.300)(positive control)
Promoterless-gfp on pSB6A1(JM2.300)(negative control)
PlsrA-gfp on pSB3K3(MG1655)
PlsrA-gfp-PlsrR-lsrR on pSB3K3(MG1655)
[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 1:10.
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
UNIQf7e147c781c528eb-partinfo-00000000-QINU UNIQf7e147c781c528eb-partinfo-00000001-QINU