Revision as of 09:36, 16 October 2019

luxPR_4G12T

The expression of this promoter can be up-regulated by the activation of LuxR activator protein which can form a complex with an autoinducer, 3-oxo-hexanoyl-HSL. This complex can bind to the upstream regulatory element of the promoter and increase the rate of the transcription. According to the theory above we can design a plasmid which constitutively expresses LuxR on the upstream of luxPR and a gene of interest expressed by luxPR. Protein of interest can be simply expressed by adding HSL into the medium.

The promoter was improved from BBa_R0062. We mutated the T on its fourth site to G, and the C on its twelfth site to T. Comparing to the original part, the new part needs a higher HSL concentration to be activated. Implementing this mutation, we gained an improved luxPR with low leakage, high threshold without losing its highest intensity.

The significant meaning of this improvement is that it can apparently delay the positive feedback of the quorum-sensing circuit, which will provide an ideal promoter for our project.

Sequence and Features

I Background

luxPR promoter is the key element to quorum-sensing circuits, for it mediates the final effects of quorum-sensing, producing proteins of interest. luxPR promoter is a σ-70 dependent promoter with an upstream regulatory element at -42.5, named lux box. luxR, playing as an activator, will bind to lux box and interact with CTD and NTD of the α subunit of RNA polymerase, resulting in the activation of the promoter.[1] So far, lots of teams have made effort on improving luxPR promoter, but they’ve all focused on mutating the -10 region and the -35 region. σ-70 factors will bind to the -10 and -35 region tightly and then form holo-RNA polymerases (holo-RNAP) with core RNA polymerases to initiate transcription. This method usually results in the change of its maximum intensity but little effect to its dynamic characteristics. Taking these into consideration, we decided to mutate its lux box to change its dynamic characteristics. Since lux box is the binding site of the activator protein luxR, the mutation to lux box will vary its binding features with luxR, thus affecting its activation characteristics. Our efforts on this hypothetical were proved, since we successfully gained a mutated luxPR with low leakage, high activation threshold without decreasing its maximum intensity.

II Results

We made nine mutants according to the theory provided by papers at first [2-4]. And then we selected a mutant from the nine mutants which met our expectation well, and named this mutant as luxPR-4G12T. The fourth and the twelfth bases of the original luxPR were mutated to G and T simultaneously.

We constructed two plasmids. One expressed sfGFP under the control of luxPR-4G12T and the other expressed sfGFP under the control of original luxPR. Then we respectively transformed them into E. coli DH5α. In theory, AHL could induce gene expression by activating luxPR promotor. We added different concentrations of AHL into the bacteria, and measured the fluorescence value and OD600 every 30 minutes. The standard fluorescence intensity was obtained by dividing the fluorescence intensity by OD. The standard fluorescence by AHL of different concentrations after 8 hours of incubation was plotted (Figure 1).

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Figure 1. The standard fluorescence by AHL of different concentrations after 8 hours of incubation. Fluorescence intensity and OD values were measured by microplate reader respectively at 528 nm and 600 nm wavelength.

It was observed that the expression intensity of sfGFP of the mutant was low at a low concentration of AHL, and the intensity remained stable. But the wild type had a certain degree of leakage without AHL. And we discovered that when the concentration of AHL reached 10-1nmol/L, the fluorescence intensity of wild type had increased, but the mutant didn’t change much. When the concentration went up to 102nmol/L, the fluorescence intensity of the mutant and the wild type became similar. In contrast, the mutant had a very low leakage, high threshold, and unchanged intensity.

Then we used different concentrations of AHL to induce the mutant and the wild type respectively. The diagrams of fluorescence intensity over time are shown below (Figure 2-4).