Revision as of 03:12, 21 October 2019

Modified pLux

Quorum sensing activated promoter that responds natively to the Lux pathway (activated by LuxR + 3OC6HSL). This specific instance of this promoter has a mutation on nucleotide 18 of the consensus sequence, which reportedly diminishes genetic crosstalk with the Las system.

Usage and Biology

XHD-WS-Wuhan-A characterization

Aim of experiment

Based on K2771000 part, a genetic circuit LuxR-pLux-GFP was constructed to characterize the function of this modified pLux promoter.

Methods

1. Construction of pSB1C3-pLuxR-pLux-GFP We designed our genetic circuit based on the synthetic biology principle in Fig 1. The circuit was then constructed into plasmid backbone pSB1C3 by DNA synthesis. This plasmid was identified by PCR and enzyme digestion.

Fig1.Gene circuit of pLuxR-plux-GFP combinations

2. GFP expression of E.coli under the control of K2771000 induced by different concentration of AHL (3OC6HSL)

The plasmid (pSB1C3-pLuxR-pLux-GFP) containing K2771000 part was transformed into E.coli DH5α strain. Single colony was selected to inoculate LB broth containing chloramphenicol and cultured overnight. Then overnight culture was inoculated in fresh LB broth containing chloramphenicol at 1:50 to expand the culture. When OD600 reached 0.4-0.6, we performed the experiment. Different concentrations of 3OC6HSL solutions were added into the test tube. The final concentration of 3OC6HSL was 0 nM, 0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 1,000 nM, 10,000 nM, and 100,000 nM respectively. Samples were collected every one hour. GFP fluorescence intensity (485 nm excitation/ 528 nm emission) and OD600 value were measured at the same time.

Results

We used the constructed plasmid as template and then took PCR to obtain the corresponding fragment pLux-GFP. As shown in Fig2, the size of the PCR product was as expected. The constructed plasmid was then double digested with Sph I/Pst I. the results indicate that enzyme digestion was as expected.

Fig 2. Gel electrophoresis of pLux + GFP fragment after PCR and pSB1C3-pLuxR-pLux-GFP double-digested with Sph I and Pst I.

The standard fluorescence intensity (FL/OD600) was obtained by dividing the fluorescence intensity by OD600. As show in Fig.3, there is no difference from 0 h to 6 h in DH5α treated with different 3OC6HSL concentrations. The fluorescence intensity was increased compared to the control group after 7 h treatment (0nM).

From Fig.3 we know that the fluorescence intensity is strongest at 8 hours. So 8h was chosen for further study.As shown in Fig.4, pLux promoter is very sensitive to 3OC6HSL and requires only 1nm to detect fluorescence signals. The threshold for pLux detection is low, while the background of the control is very high, indicating the leak of this promoter.

Fig 3. The value of GFP fluorescence in DH5α treated with different 3OC6HSL concentrations.

As show in Fig.3,  the fluorescence intensity is strongest at 8 h. So 8 h was chosen for further study. As shown in Fig.4, pLux promoter is very sensitive to 3OC6HSL. And 1 nm of 3OC6HSL was required to detect fluorescence signals. The threshold for pLux detection is low and the background of the control is very high, which indicats the leak of this promoter.  

Fig.4 GFP expression of E.coli under the control of pLux (K2771000) in different concentrations of 3OC6HSL.

Summary

The plasmid constructed based on K2771000 is sensitive to 3OC6HSL. The threshold of the pLux promoter is 1nM. However, the background is very high. Our results indicated the pLux promoter has high thresholds and low leakage of this promoter need to be developed.

Reference:

Grant et al. Orthogonal intercellular signaling for programmed spatial behavior. Mol Syst Biol. (2016) 12: 849

Sequence and Features

Functional Parameters