Summary
Microplastics has been identified as hubs for microbial pathogens and their antimicrobial resistance (AMR) due to hydrophobicity, greater surface area and pit forming nature of microplastics. These pathogenic bacteria form biofilms on the microplastics. Biofilm formation is governed by the quorum sensing (QS) genes expressed as a result of the accumulation of a critical cell density. Pseudomonas species has been reported to have the abilities of attaching to the microplastics and are contributing to microplastic-associated AMR. It employs N-acyl homoserine lactone (AHL) as their command language to coordinate population behaviour during invasion and colonization. There is a complete quorum sensing regulatory circuit LasI-LasR in P. aeruginosa . In our project, we engineered a whole cell biosensor based on the LasI-LasR system to detect and quantitatively measure the presence of quorum sensing molecules secreted from P. aeruginosa for detection of microplastic pollution levels in water samples. We went through a cycle of engineering (Research → Design → Build → Test → Learn → Improve).
From literature reviews, we found that the engineering of whole-cell biosensors based on quorum sensing systems is mainly for biomedical application while the potential of biosensors for detection of microplastic level is unexplored. Thus we aimed to apply P. aeruginosa LasI-LasR circuit to engineer the proof-of-concept biosensor for the detection of specific AHL molecules. The biosensor will ultimately use to quantitatively measure the presence of AHL molecules in water samples for monitoring microplastic pollution.
The engineered biosensor is composed of two modules: the sensing module and reporting module. The sensing module expresses the transcription factor LasR which can bind to the AHL molecule that are secreted from P. aeruginosa. Then the formation of LasR-AHL complex can bind to an inducible promoter, which lead to the activation of the reporting module. A green fluorescent protein-based system is used as the reporting module, in which the expression of GFP would be induced by the LasR-AHL complex. The highly efficient expression and easy detection of the green fluorescence signal could facilitate the characterization and optimization of the biosensor system.
The transcription factor LasR was expressed under the control of the constitutive T7 promoter in the plasmid pET 21b and pET 23b. pLasR3 and pLasRL promoters were used as inducible promoters to control the expression of EGFP in pUC57 plasmid. The plasmids pET21b-LasR/ pET23b-LasR and pUC57-pLasRL-EGFP/pUC57-pLasR3-EGFP were transformed into the BL21 C41(DE3)pLysS. The resulting biosensors were named as pET21b-LasR-pUC57-pLasRL-EGFP, pET21b-LasR-pUC57-pLasR3-EGFP, pET23b-LasR-pUC57-pLasRL-EGFP and pET23b-LasR-pUC57-pLasR3-EGFP. An AHL cocktails containing three synthetic AHL molecules, N-butyryl L-homoserine lactone (C4HSL), N-3-oxo-decanoyl L-homoserine lactone (C10HSL) and N-3-oxo-dodecanoyl L-homoserine lactone (3OC12-HSL) were used to determine the specificity and sensitivity of the engineered biosensor.
Several induction tests of the four biosensors with AHL cocktails at published relevant concentrations in water environments from 1x10-5 to 1x10-10 were performed. The EGFP production rates were low as indicated by both weak fluorescence images shown by the fluorescence microscope and fluorescence intensity measured by the microplate reader readings. Stronger fluorescence signals were obtained after several modifications in AHL induction protocol. In the process, we learned that OD600 of 0.6 in starter cell culture was better than 0.3 as proposed by published protocol as the bacteria were at exponential phase when OD600 reached 0.6. Besides, we found that the presence of dimethyl sulfoxide (DMSO) used to make AHL stock solutions and subsequent dilutions caused high autofluorescence signal. Thus, DMSO was used to make AHL stock solutions and distilled water was used in making the subsequent serial dilutions of AHL solutions. To find out if the presence of more pET21b-LasR would allow the formation of more LasR-AHL complex which in turn facilitate EGFP transcription, pET21b-LasR-pUC57-pLasRL-EGFP was chosen to test the effect of transformation of different ratio of pET21b-LasR plasmid and pUC57-LasRL-EGFP plasmid into BL21 strain. The tested ratios were 1:1, 2:1 and 3:1. We found that 3:1 ratio of pET21b-LasR to pUC57-LasRL-EGFP gave the highest fluorescence signals. In the end, all four biosensors showed moderate green fluorescence signal after 15h as shown below.
(A) OD600 and EGFP production of the three different ratios of the LasR sensing module and the EGFP reporting module in pET21b-LasR-pUC57-pLasRL-EGFP incubated with a cocktail of synthetic AHL molecules at a concentration of 1x10-6 over 3 hours
(i) 1:1 ratio
(ii) 2:1 ratio
(iii) 3:1 ratio
The EGFP fluorescence intensity measured by the microplate reader was expressed in arbitrary unit as relative fluorescence unit (RFU). The PBS buffer was used to determine the background fluorescence intensity. The results showed that the EFGP production increase with time in all tested ratios of the LasR sensing module and the EGFP reporting module in pET21b-LasR-pUC57-pLasRL-EGFP. The fluorescence intensity of biosensors was ranged from 0.2138 RFU to 0.4387 RFU. Since the fluorescence signals were weak after 3h incubation with AHL cocktails, fluorescence images were taken after 15h incubation with AHL cocktails.
(B) Fluorescence images of the three different ratios of the LasR sensing module and the EGFP reporting module in pET21b-LasR-pUC57-pLasRL-EGFP incubated with a cocktail of synthetic AHL molecules at a concentration of 1x10-6after 15 hours
Incubation with distilled water was used as the negative control. The bright-field images showed the cell morphology of the biosensor cells and the fluorescence images showed green fluorescence signals emitted from the cells.
(i) 1:1 ratio
(ii) 2:1 ratio
(iii) 3:1 ratio
The results showed that moderate green fluorescence signals were emitted from the cells in all tested ratios of the LasR sensing module and the EGFP reporting module in pET21b-LasR-pUC57-pLasRL-EGFP.
(C) OD600 and EGFP production of the four biosensors incubated with a cocktail of synthetic AHL molecules at a concentration of 1x10-6M over 1.5 hours
(i) pET21b-LasR-pUC57-pLasRL-EGFP
(ii) pET21b-LasR-pUC57-pLasR3-EGFP
(iii) pET23b-LasR-pUC57-pLasRL-EGFP
(iv) pET23b-LasR-pUC57-pLasR3-EGFP
The results showed that the EFGP production increase with time in all four biosensors. The fluorescence intensity of biosensors was ranged from 0.1344 RFU to 0.1780 RFU. Since the fluorescence signals were weak after 3h incubation with AHL cocktails, fluorescence images were taken after 15h incubation with AHL cocktails.
(D) Fluorescence images of the biosensors incubated with a cocktail of synthetic AHL molecules at a concentration of 1x10-6M after 15 hours
Incubation with distilled water was used as the negative control. The bright-field images showed the cell morphology of the biosensor cells and the fluorescence images showed green fluorescence signals emitted from the cells.
(i) pET21b-LasR-pUC57-pLasRL-EGFP
(ii) pET21b-LasR-pUC57-pLasR3-EGFP
(iii) pET23b-LasR-pUC57-pLasRL-EGFP
(iv) pET23b-LasR-pUC57-pLasR3-EGFP
The results showed that moderate green fluorescence signals were emitted from the cells in all four biosensors afte 15h incubation with AHL cocktails.
A sensitive biosensor should allow a rapid detection and monitoring of a relevant range of AHLs within samples. Our results indicated that the developed biosensors showed obvious fluorescence signals only after 15h and they were not sensitive enough to detect the presence of AHL molecules. Therefore, we adopted another approach to construct a new biosensor by integrate LasR and pLasRL-EGFP in a single plasmid (pSB1C3) instead of two separate plasmids. The resulting biosensor, named pSB1C3-LasR-pLasRL-EGFP showed high EGFP production rates in the presence of AHL cocktails within 3 hours which demonstrate the successful engineering of the biosensor.
(E) OD600 and EGFP production of pSB1C3-LasR-pLasRL-EGFP incubated with a cocktail of synthetic AHL molecules at a concentration of 1x10-5M and 1x10-11M after 16 hours
(i) 1x10-5M
(ii) 1x10-11M
The results showed that the EFGP production increase significantly with time. The fluorescence intensity of biosensors was ranged from 4.743 RFU to 48.74 RFU when incubated with 1x10-5M AHL cocktails and from 3.346 RFU to 16.16 RFU when incubated with 1x10-11M AHL cocktails after 16h. The highest fluorescence intensity of biosensors in the previous study was ranged from 0.2138 RFU to 0.4387 RFU when using different tested ratio of the LasR sensing module and the EGFP reporting module incubated with 1x10-6M AHL cocktail after 3h. In this study, the fluorescence intensity of biosensors was 5.959 RFU when incubated with 1x10-5M AHL cocktail after 2.5h. Thus, the new biosensors exhibited at least 10-fold (13.5-fold) higher fluorescence intensity compared with biosensors made in the previous study.
(F) Fluorescence images of pSB1C3-LasR-pLasRL-EGFP incubated with a cocktail of synthetic AHL molecules at a concentration of 1x10-11M after 3 hours
These results demonstrated that a biosensor for the detection of a P. aeruginosa-specific quorum sensing molecules was successfully engineered and a strong GFP expression was developed within 3 hours after incubation with AHL molecules as low as 1x10-11M.
Future prospect
This engineering cycle has led us to further characterize the specificity of the engineered biosensor towards the three AHL molecules in the cocktail. Besides, we will study the ability of the biosensor to detect native AHL molecules produced by P. aeruginosa. Ultimately, we will replace the EGFP expression system in the reporting module with a reporter system based on pigment production such as red pigment lycopene as a visible readout for the development of paper-based device for microplastic detection.
