Difference between revisions of "Part:BBa K2558001"

Figure.3. Leakage and 10^-10 mM AHL stimulation of of lux pR promoter WT, Mutant 5, Mutant 10 and HS. The fluorescence strengths and OD values are measured by microplate reader at 510 nm and 600 nm wavelength. Plasmids with lux pR promoter WT, Mutant 5 and Mutant 10 are transferred into E.coli DH5α. A The bacteria was treated without AHL inducing. The higher the ratio (Fluorescence/OD) is, the more leakage the promoter has. B The bacteria was treated with 10-10 mM AHL induction. The higher the ratio (Fluorescence/OD) is, the more sensitive the promoter is.

It is observed that the higher leakage lux pR mutant has, the more sensitive this promoter mutation is. The wild type of lux pR shown with black strand in figures has the highest level of both leakage and sensibility. The next three are Mutant 5 (G-36T), Mutant 6 (G-36C), Mutant 9 (T-35G). Mutant 5 has almost the same sensitivity to wild type, but the leakage is much smaller than the wild type. Mutant 6 and Mutant 9 has a little bit less leakage and medium sensitivity. We have also done the experiment with 10-9 M and 10-8 M AHL, the all promoters show high expression levels. (Data not shown) We repeated AHL stimulation test on Mutant 5 and Mutant 10 (Figure.2) and decided to combine the two mutations together to form a new promoter with high sensitive and small leakage. We named it lux pR-HS. We tested lux pR-HS under the same conditions as those mutations. We can see that the lux pR-HS has similar leakage to Mutant 10, while it has much higher sensitivity. (Figure.3) The statistics of relative induction and leakage intensity of the 10 Mutants and lux pR-HS are shown in Table.1

Table.1. Results of lux pR mutation analysis.

As for why the mutation happened on -35 to -37 sites would decrease the leakage of lux pR without severely influencing the expression level or sensibility, we have some hypothesis and explanations. It is reported that -35 to -37 sites are last three bases of lux box which is upstream of the lux promoter and bound specifically by luxR, one of regulatory protein involved in lux expression system.[1] G19T mutation (-36 G to T mutation upstream of lux pR promoter) has less influence than other mutation on sites -35 to -37, which is same to our result. Besides, the G19T is not involved in the two important regions of nucleotides 3 to 5 and 16 to 18 which directly contacted with luxR protein. Therefore, this single change of nucleotide would decrease the leakage of lux promoter without influencing the expression level too much.[2]

Protocol

1. one Transform the plasmids into E. coli DH5α.
2. two 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 5ml LB medium with ampicillin at 100 ng/µl. Incubate for 6-8 h at 37℃ in a shaker.
3. three Measure OD600 of the culture medium with photometer. Dilute the culture medium until OD600 reaches 0.6.
4. four Add 100 µl bacteria culture medium into a sterile 96-well plate. Add AHL to final concentrations of 0, 10-10,10-9,10-8 M. Fresh LB medium serves as blank control. Positive control is colony constantly expressing sfGFP and negative control is colony without sfGFP expression. Place the 96-well plate into an automatic microplate reader. Incubate at 16℃ overnight and record the fluorometric value at 510 nm and OD600 for each well every 30 minutes.
5. five Each group is repeated for at least 3 times.

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] Zeng, Weiqian, et al. "Rational design of an ultrasensitive quorum-sensing switch." Acs Synthetic Biology 6.8(2017).

Sequence and Features