Part:BBa_K649101:Experience

Applications of BBa_K649101

To confirm LsrR represses lsrA promoter, we constructed BBa_K649105.

We characterized BBa_K649105 in E.coli MG1655.

Fluorescence intensity is decreased by LsrR repression.
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.

After four hours from OD590 reaching 0.15, we measured OD.
This work is done by Hiroki Yoshise.

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