Part:BBa_K3245017

luxpR-HS106

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

Usage and biology:

This fused promoter is designed for the situation that QS effect and low leakage are required at the same time in a circuit if a little expression loss is acceptable to some extent. We applied this improved promoter to link upstream QS regulation with tetR suppression effect downstream. It’s a successful trial since tetR-ptetR system is rather strict and luxpR ( WT ) is unsuitable due to its high leakage.

Design:

As the figure shows, we substituted the -35 to -10 region of the original promoter luxpR with the one of J23106, 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:

1. Transform a control plasmid containing luxI and luxR ( BBa_K3245002 ) with pUC ori ( high copy number ) and an effect plasmid containing luxpR-HS106 and GFP behind the promoter ( BBa_K3245015 ) 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.