Figure.3. Leakage and 10^-10 mM AHL stimulation of of lux pR promotor WT, Mutant 5, Mutant 10 and HS. The fluorescence strengths and OD values are measured by microplate reader respectively at 510 nm and 600 nm wavelength. Plasmids with lux pR promotor WT, Mutant 5 and Mutant 10 are transferred into E.coli DH5α. A The bacteria were treated without AHL inducing. High (Fluorescence/OD) ratio indicates strong leakage of promotor. B The bacteria were treated with 10^-10 mM AHL induction. High (Fluorescence/OD) ratio indicates high expression intensity.

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 promotor with high intensity and small leakage. We named it lux pR-HS. We tested lux pR-HS under the same conditions. We can see that the lux pR-HS has similar leakage to Mutant 10, while it has much higher expression intensity. (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 some mutations on -35 to -37 sites would decrease the leakage of lux pR without severely influencing the expression level, we have some hypotheses and explanations. It is reported that -35 to -37 sites are last three bases of lux box which is upstream of the lux promotor and bound specifically by luxR, one of regulatory protein involved in lux expression system.[1] G-36T mutation has less influence than other mutation on sites -35 to -37, which is same to our result. Besides, G-36T is not involved in the two regions of nucleotides -52 to -50 and -39 to -37 which directly contacted with luxR protein. Therefore, this single substitution of nucleotide would decrease the leakage of lux promotor 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 measure the fluorometric value at 510 nm and OD600 of each well every 30 minutes.
5. five Each group should be 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).

SJTU-BioX-Shanghai 2019's Characterization

This lux pR-HS promoter is put upstream of repL(BBa_K2996000) to form a composite part(BBa_K2996006) that will respond to AHL, together with a constant expression of LuxR. RepL is a P1 lytic replication element that can act on mini-F origin to promote plasmid replication. Thus we quantified the effect of lux pR-HS indirectly by measuring plasmid copy number through qPCR. We tried different AHL concentration from 0 to 10-6 M and measured copy number in the time course. Both absolute copy number and relevant copy number amplification compared to non-induction were calculated. The result confirmed the small leakage of lux pR-HS and it also showed obvious responding to AHL, with the maximum effect reached at a concentration of 10-7 M at 3.5h of induction.

Protocol

AHL induction and preparation for copy number measurement. (1)Transfer pLux-trig(with luxpR-HS driven repL) and pLux-cas(with luxpR-HS driven Cas1/2 and constantly expressed LuxR) into E.coli BL21(DE3) (2)Inoculate 1% overnight bacterial culture in Erlenmeyer flask. (3)Incubate for 2h until early exponential stage (OD600 is 0.2-0.3). (4)Add AHL to final concentrations of 10-9, 10-8, 10-7, 10-6 M. (5)Take 500μL of the culture every 30min. (6)Centrifuge at 4000rpm for 5min immediately after taking out. (7)Discard the supernatant and wash with 500μL PBS. (8)Heat at 100℃ for 10min. (9)Store at -20℃ for at least 20min. (10)Centrifuge at 6500rpm for 1min. (11)Remove the supernatant into a clean 1.5mL EP tube. (12)Store at -20℃. (13)Add 3ul as qPCR template and run qPCR.

Fudan 2019’s characterization and improvement:

Background:

In our project, we hope that this promoter can trigger the regulation of the downstream tetR circuit properly without leakage and subsequent deactivation. Since we use the production of its upstream regulating genes luxI and luxR as its signal in our final circuit, our measurement continued this design and measure the curve basing on OD value. Data collected in unit of the concentration of bacteria will directly reflect if the system works properly.

Experiment design and measurement:

To test this part’s function, we transferred DH10B bacteria with plasmids carrying the promoter with another LuxI-LuxR ( BBa_K3245002 ) plasmid at the same time. LuxI-LuxR is a upstream regulation box which produces signal that can activate luxpR in QS system. And we added GFP after luxpR as the reporter of its expression level. By measuring fluorescence/OD600, the expressing level of luxpR can be known and the data are comparable with our other works.

Result: