Revision as of 18:41, 21 October 2019

luxpR-fus100:

This hybrid promoter has higher expression level when induced by luxR-AHL complex triggered by quorum sensing ( QS ) in some Gram-negative bacteria, meanwhile it’s leakage is also higher when not induced.

Usage and biology:

This fused promoter is designed for the situation that QS effect and high expression level are required at the same time in a circuit if leakage is acceptable to some extent. We applied this enhanced promoter to improve lacZ expression when bacteria reached a high density.

Design:

As the figure shows, we substituted the -35 to -10 region of the original promoter luxpR with the one of J23100, a strong constitutive promoter. This region is rarely concerned as it’s rather conservative in promoters with the same function, but it has crucial structural effect on σ factor binding and other events in transcription regulation. Fortunately the change proved to be effective on adjusting the behavior of the regulatory promoter.

Characterization:

Measurement Protocol

1. Transform a control plasmid containing luxI and luxR ( BBa_K3245002) with pUC ori ( high copy number ) and an effect plasmid containing luxpR-fus100 and GFP behind the promoter with p15A ori (medium copy number ) into DH10B.

2. Pick a single colony by a sterile tip from each of the LB plates for all the experimental and control groups. Add the colony into 3 ml LB with ampicillin at 100 μg/ml. Incubate overnight at 37℃ in a shaker.

3. Measure and keep all groups OD600 reach 1. Inoculate each group with with 1/5000 concentration. Incubate 12 hours at 37℃ in a shaker.

4. Add 100 µl bacteria culture medium into each well of a 96-well plate. One well of LB as blank, one group of wild type DH10B as control.

5. Measure OD600 and fluorescence continuously every 30 minutes with a microplate reader.

Fig.1 This figure shows luxpR-fus100 expression level induced by luxR-luxI box. The MEFL/particle changes as the OD goes up after dilution.

Reference

<p>Antunes, L. C., et al. "A mutational analysis defines Vibrio fischeri LuxR binding sites." Journal of Bacteriology 190.13(2008):4392-4397.

de Boer HA, Comstock LJ, Vasser M. The tac promoter: a functional hybrid derived from the trp and lac promoters. Proc Natl Acad Sci U S A. 1983;80(1):21–25. doi:10.1073/pnas.80.1.21

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 [[File:T--Fudan--Part51.png|400px]]
 <p><strong>Fig.1</strong> This figure shows luxpR-fus100 expression level induced by luxR-luxI box. The MEFL/particle changes as the OD goes up after dilution.
+<h2>Reference</h2>
+<p>Antunes, L. C., et al. "A mutational analysis defines Vibrio fischeri LuxR binding sites." Journal of Bacteriology 190.13(2008):4392-4397. </p>
+<p>de Boer HA, Comstock LJ, Vasser M. The tac promoter: a functional hybrid derived from the trp and lac promoters. Proc Natl Acad Sci U S A. 1983;80(1):21–25. doi:10.1073/pnas.80.1.21</p>

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