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]

III 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.

IV 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).

Contribution

Group: Valencia_UPV iGEM 2018
Author: Adrián Requena Gutiérrez, Carolina Ropero
Summary: We adapted the part to be able to assemble transcriptional units with the Golden Gate assembly method
Documentation: In order to create our complete [http://2018.igem.org/Team:Valencia_UPV/Part_Collection part collection] of parts compatible with the Golden Gate assembly method, we made the part BBa_K2656003 which is this part adapted to the Golden Gate technology.

Results

Sequence and Features

>Internal Priming Screening Characterization of BBa_R0062: Has no possible internal priming sites between this BioBrick part and the VF2 or the VR primer.

The 2018 Hawaii iGEM team evaluated the 40 most frequently used BioBricks and ran them through an internal priming screening process that we developed using the BLAST program tool. Out of the 40 BioBricks we evaluated, 10 of them showed possible internal priming of either the VF2 or VR primers and sometime even both. The data set has a range of sequence lengths from as small as 12 bases to as large as 1,210 bases. We experienced the issue of possible internal priming during the sequence verification process of our own BBa_K2574001 BioBrick and in the cloning process to express the part as a fusion protein. BBa_K2574001 is a composite part containing a VLP forming Gag protein sequence attached to a frequently used RFP part (BBa_E1010). We conducted a PCR amplification of the Gag-RFP insert using the VF2 and VR primers on the ligation product (pSB1C3 ligated to the Gag + RFP). This amplicon would serve as template for another PCR where we would add the NcoI and BamHI restriction enzyme sites through new primers for ligation into pET14b and subsequent induced expression. Despite gel confirming a rather large, approximately 2.1 kb insert band, our sequencing results with the VR primer and BamHI RFP reverse primer gave mixed results. Both should have displayed the end of the RFP, but the VR primer revealed the end of the Gag. Analysis of the VR primer on the Gag-RFP sequence revealed several sites where the VR primer could have annealed with ~9 - 12 bp of complementarity. Internal priming of forward and reverse primers can be detrimental to an iGEM project because you can never be sure if the desired construct was correctly inserted into the BioBrick plasmid without a successful sequence verification.

HZAU-China 2019's Improvement

The improved part can be found here:https://parts.igem.org/Part:BBa_K3205005

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-4G12Tand 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 OD₆₀₀. The standard fluorescence by AHL of different concentrations after 8 hours of incubation was plotted (Figure 1).

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).

Figure 2. The diagram of the fluorescence intensity over time with 10⁰ nmol/L AHL stimulation.

From this graph we could see that at the low concentration of AHL (10⁰nmol/L)，the mutant’s expression of sfGFP was very low and stable, but the fluorescence intensity of wild type was increasing significantly over time.

Figure 3. The diagram of the fluorescence intensity over time with 10¹ nmol/L AHL stimulation.

When we increased the concentration of AHL to 10¹nmol/L, we could conclude that the mutant began to express sfGFP according to the increasing fluorescence intensity on this graph.

Figure 4. The diagram of the fluorescence intensity over time with 10² nmol/L AHL stimulation.

When the concentration of AHL increased to 10²nmol/L, the fluorescence intensity of the mutant and the wild type changed over time similarly.

III Discussion

According to the analysis above, we could clearly find that when we didn’t add any inducer or just added a low concentration of inducer, the mutant expressed sfGFP very low. Compared with the wild type the mutant had the advantage of low leakage. When the wild-type promoter began to express sfGFP significantly, the expression intensity of sfGFP of the mutant remained low and nearly unchanged, which showed that the threshold of the mutant was higher than the wild type. As the concentration of the inducer increased to 10²nmol/L, the fluorescence intensity of the wild type and the mutant were the same over time, which showed their similar intensity of the transcription initiation.

From what has been discussed above, the mutant had the advantages of very low leakage, high threshold, and unchanged maximum intensity. Compared to the wild type, the mutant was more suitable for our project. The biggest problem of our project was that the memory module would start itself due to the leakage of the promoter. Since the leakage of the mutant was very low and the threshold to activate this promoter was high, the mutant needed more time and a higher concentration of inducer to active the memory module than the wild type.

To sum up, the mutant not only met the requirements of our project, but also had a very wide range of application.

IV Protocol

1.Molecular cloning are conducted as the illustration in the Molecular Cloning section. https://2019.igem.org/Team:HZAU-China/Protocol

2.Transform two different plasmids with low copy ampicillin resistance into E. coli DH5α, which are named luxPR-4G12T and luxPR respectively. Two base pairs are mutated in luxPR-4G12T. And luxPR is the plasmid with the original part BBa_R0062. Then spread them onto two LB plates with ampicillin, respectively.

3.Pick four isolated colonies with a sterile tip from each of the two LB plates and add each tip into 5ml LB medium with ampicillin (20μg/mL). Incubate for 6-8 h at 37℃ in a shaker with 200rpm.

4.Add each kind of bacterial fluid into different lines of a sterile, black-coated 96-well plate. Add shaken bacterial solution and fresh medium with ampicillin to a total of 100μL, and make sure the OD₆₀₀ of each well is close to 0.1, measured by the plate reader.

5.Add 1μL AHL diluted in DMSO into each well. The final concentration range of AHL is from 10^-2 to 10⁶ nmol/L. Add 1μL DMSO without AHL served as positive controls. Blank controls are fresh LB medium, Place the 96-well plate into the automatic microplate reader (Synergy H1 hybrid multi-mode reader). Incubate at 37℃ overnight and measure the fluorescence value（excitation: 485nm, emission: 528nm）and OD₆₀₀ of each well every 30 minutes.

6.Data taken from the plate reader are exported to Excel and imported to Prism for analysis.

7.All experiments above are carried out in 3 biological replications.

V Reference

[1]Egland K A, Greenberg E P. Quorum sensing in Vibrio fischeri: elements of the luxI promoter[J]. Molecular microbiology, 1999, 31(4): 1197-1204.

[2]Bao S H, Li W Y, Liu C J, et al. Quorumsensing based small RNA regulation for dynamic and tuneable gene expression[J]. Biotechnology letters, 2019, 41(10): 1147-1154.

[3]Grant P K, Dalchau N, Brown J R, et al. Orthogonal intercellular signaling for programmed spatial behavior[J]. Molecular systems biology, 2016, 12(1).

[4]Zeng W, Du P, Lou Q, et al. Rational design of an ultrasensitive quorum-sensing switch[J]. ACS synthetic biology, 2017, 6(8): 1445-1452.