Team:Fudan/Improve
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Improve
Introduction
Last year team Tsinghua designed the lux pR-HS promoter that provides high expression level and low leakage in quorum sensing systems. They combined two mutations together which shows improved features caused by affinity changes between the -10 site and the RNA polymerase along with the -35 site and the luxR-AHL complex ( regulators in QS system ).
Here we give a new way to improve the properties of the pivotal promoters in QS for those who wish to apply this widely used system to their work. We substituted the -35 to -10 region of the original promoters, lux pR ( BBa_R0062) and lux pR-HS with the one from constitutive promoter family members ( J23100), according to the knowledge of lux box1 and the method of constructing hybrid promoters2. Finally we picked luxpR-fus100 and luxpR-HS100 and deployed them in our circuit.
Fig.1 Hybrid promoter diagram. 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.
For further details, please refer to... luxpR-fus100 luxpR-HS100 luxpR luxpR-HS
Protocol
1. Transform a control plasmid containing luxI-luxR ( BBa_K3245002 ) with pUC ori ( high copy number ) and an effect plasmid containing luxpR-fus100 / luxpR-HS100 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.
Result
Figure 2. Microplate reader data of quorum sensing promoters ( time as X-axis ). It shows the overall status of the promoters.
Figure 3. Microplate reader data of quorum sensing promoters ( OD600 as X-axis ). The downward curve before OD600 <0.1 is due to the parental fluorescence and the dilution effect after proliferation. It is shown that luxpR-fus100 has a higher expression level and luxpR-HS100 has low leakage at the cost of a lower expression level.
Reference
[1] Antunes, L. C., et al. "A mutational analysis defines Vibrio fischeri LuxR binding sites." Journal of Bacteriology 190.13(2008):4392-4397.
[2] 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