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. P. aeruginosa is a common Pseudomonas sps attach to the microplastics and contribute to microplastic-associated AMR. In our project, we make use of the complete quorum sensing regulatory circuit LasI-LasR in P. aeruginosa to engineer 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. Below are the important findings of our projects.
1. Confirmation of the presence of DNA inserts in plasmid constructs
We integrate the LasR sensing module and the reporting module that expressed EGFP to demonstrate the sensitivity of the biosensor in response to the quorum-sensing molecules of the P. aeruginosa. The transcription factor LasR was expressed under the control of the constitutive T7 promoter in the plasmid pET 21b and pET 23b. Two pET plasmids were used in order to compare if the presence of lac operator in pET 21a can prevent leaky expression of the LasR and thus give less background fluorescence signals in the uninduced state. Besides, we used two LasR inducible promoters, pLasRL and pLasR3 in the reporting module in order to compare the two promoter strengths. The two modules were transformed into DH5α individually. The extracted DNA of the two modules were then co-transformed into the BL21 C41(DE3)pLysS. The resulting engineered 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. PCR was performed to confirm the presence of LasR, pLasRL and pLasR3 in the transformed colonies.
Figure 1 and Figure 2 showed that PCR products with the correct size of 566 bp in LasR, 274 bp in pLasRL and 307 bp in pLasR3 were amplified in five colonies of all constructs confirming the presence of LasR, pLasRL and pLasR3 in the expression vector pET 21b, pET 23b and pUC57.
Figure 3 showed that PCR products with the correct size of 566 bp in LasR and 307 bp in pLasR3 were amplified in five colonies of the construct confirming the presence of LasR and pLasR3 in the pET23b-LasR-pUC57-pLasR3-EGFP plasmid.
Figure 4 showed that PCR products with the correct size of 566 bp in LasR were amplified in four and eight colonies of the constructs confirming the presence of LasR in the pET21b-LasR-pUC57-pLasRL-EGFP and pET23b-LasR-pUC57-pLasRL-EGFP plasmids.
Figure 6 showed that PCR products with the correct size of 566 bp in LasR and 307 bp in pLasR3 were amplified in seven colonies of the construct confirming the presence of LasR and pLasR3 in the pET21b-LasR-pUC57-pLasR3-EGFP plasmid.
2. EGFP production of the three different ratios of pET21b-LasR-pUC57-pLasRL-EGFP
The engineered biosensor is composed of two modules: the sensing module and the reporting module. In the LasR quorum-sensing system, the sensing module expresses the transcription factor, LasR which can bind to the AHL moleucles. The formation of LasR-AHL complex can then bind to an inducible promoter, pLasRL or pLasR3 which lead to the activation of the reporting module to produce green fluorescence readout. Since the green fluorescence signals emitted by the biosensor cells were weak (data not shown), we tried different ratio of LasR sensing module to pLasRL-EGFP reporting module as one of the modifications in the protocol. We hypothesized that the sensing module in the expression plasmids need to be in excess so that more AHL molecules can bind and drive the expression of reporting module. We transformed three different ratios of the LasR sensing module and the EGFP reporting module in pET21b-LasR-pUC57-pLasRL-EGFP construct including 1:1, 2:1 and 3:1. The biosensor cells were incubated with a cocktail of 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) at a concentration of 1x10-6M over 3 hours. 1x10-6M AHL falls within the relevant concentrations of AHL concentrations (1x10-8 to 5x10-6 for 3OC12-HSL) in water environments as reported in the literature[1].
(i) 1:1 ratio
(ii) 2:1 ratio
(iii) 3:1 ratio
Readings of OD600 were shown to indicate the normal cell growth over 3h of AHL cocktails incubation. 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 similar and it ranged from 0.2138 RFU to 0.4387 RFU.
3. Fluorescence images of the three different ratios of pET21b-LasR-pUC57-pLasRL-EGFP
Since the fluorescence signals were weak after 3h incubation with AHL cocktails, fluorescence images were taken after 15h incubation with AHL cocktails.
(i) 1:1 ratio
(ii) 2:1 ratio
(iii) 3:1 ratio
Figure 8 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. Also, there was no significant difference between the different ratios of the sensing module and the reporting module and EGFP production.
4. EGFP production of the four engineered biosensors
The engineered biosensors were incubated with a cocktail of synthetic AHL molecules at a concentration of 1x10-6M for 1.5 hours. Readings of OD600 were shown to indicate the normal cell growth over 1.5h of AHL cocktails incubation. 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.
(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 similar and it ranged from 0.1344 RFU to 0.1780 RFU.
5. Fluorescence microscope images of the four engineered biosensors
Since the fluorescence signals were weak after 1.5h incubation with AHL cocktails, fluorescence images were taken after 15h incubation with 1x10-6M AHL cocktails.
(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 after 15h incubation with 1x10-6M AHL cocktails. Among the four biosensors, pET21b-LasR-pUC57-pLasRL-EGFP showed more obvious green fluorescence signals with less autofluorescence as shown in Figure 9(i). However, our results showed that there was no significant difference in EGFP production among the four biosensors indicating that the uses of either pET 21b and pET 23b and either pLasRL and pLasR3 in the expression plasmid exhibit no differences in the functionality of engineered biosensors.
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 moderate fluorescence signals after 15h and they were not sensitive enough to detect the presence of AHL molecules. Therefore, we adopted another strategy to construct a new biosensor by integrate the LasR sensing module and the pLasRL-EGFP reporting module in a single plasmid (pSB1C3) and then transform into BL21 strain instead of transform two separate plasmids into a BL21 strain. After confirmation of the presence of insert DNA, the resulting biosensor, named pSB1C3-LasR-pLasRL-EGFP was tested for the specificity and sensitivity.
6. Confirmation of the presence of DNA inserts in pSB1C3-LasR-pLasRL-EGFP
Figure 11 showed that PCR products with the correct size of 566 bp in LasR and 274 bp in pLasRL were amplified in five colonies of the construct confirming the presence of LasR and pLasRL in the pSB1C3-LasR-pLasRL-EGFP plasmid.
7. Fluorescence microscope images of three colonies of pSB1C3-LasR-pLasRL-EGFP
Three colonies of the biosensor were incubated with a cocktail of synthetic AHL molecules at a concentration of 1x10-11M for 3 hours and the fluorescence microscope images were taken.
(i) pSB1C3-LasR-pLasRL-EGFP - 1
(ii) pSB1C3-LasR-pLasRL-EGFP - 3
(iii) pSB1C3-LasR-pLasRL-EGFP - 4
These results demonstrated that a more sensitive 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. Among the three colonies, pSB1C3-LasR-pLasRL-EGFP-4 showed the highest fluorescence signals with the least background, thus it was used for subsequently studies.
8. EGFP production of pSB1C3-LasR-pLasRL-EGFP
A. EGFP production of pSB1C3-LasR-pLasRL-EGFP incubated with a cocktail of synthetic AHL molecules at a concentration of 1x10-5M and 1x10-11M in 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.
B. EGFP production of pSB1C3-LasR-pLasRL-EGFP incubated with individual synthetic AHL molecules at a concentration of 1x10-7M in 4 hours
(i) Distilled water (negative control)
(ii) N-butyryl L-homoserine lactone (C4HSL)
(iii) N-3-oxo-decanoyl L-homoserine lactone (C10HSL)
(iv) N-3-oxo-dodecanoyl L-homoserine lactone (3OC12-HSL)
EGFP production of biosensor cells were incubated with 1x10-7M N-butyryl L-homoserine lactone (C4HSL) was similar to that of negative control suggesting that the biosensor was unable to detect C4HSL. However, when the biosensor cells were incubated with 1x10-7M N-3-oxo-decanoyl L-homoserine lactone (C10HSL) and N-3-oxo-dodecanoyl L-homoserine lactone (3OC12-HSL), EGFP production increased up to 14.86 RFU. The results suggested that the biosensor was able to detect C10HSL and 3OC12-HSL.
9. Fluorescence microscope images of pSB1C3-LasR-pLasRL-EGFP after incubation with individual AHLs
In the presence of the 1x10-7 C10HSL and 3OC12-HSL, the engineered biosensor cells produced strong green fluorescence signals while in the presence of C4HSL and in the control set-up, there was no green fluorescence signals observed. With strong GFP fluorescence signals produced by biosensor cells in the presence of C10HSL and 3OC12-HSL, the results suggested that the biosensor was able to detect C10HSL and 3OC12-HSL.
10. Response of pSB1C3-LasR-pLasRL-EGFP toward three synthetic AHLs
The biosensor was incubated in the presence of each AHL at 1x10-6M and 1x10-7M for 3h. The results are the means of triplicate experiments. The biosensor without the addition of AHL molecules was used as the negative control. The results demonstrated that the EGFP production rate was significantly higher in 1x10-7M C10HSL than that induced by the concentration of 1x10-6M C10HSL and induced by the addition of 3OC12-HSL. There was no EGFP production when the biosensor was induced by the addition of C4HSL.
11. EGFP production of pSB1C3-LasR-pLasRL-EGFP
The biosensor cells were incubated with 1x10-7M individual AHLs for 4h. The results are the means of triplicate experiments. It demonstrated that EGFP production rate of the biosensor was the highest when it was incubated with 3OC12-HSL followed by C10HSL. The biosensor did not show response when it was incubated with C4HSL.
12. EGFP production rates of pSB1C3-LasR-pLasRL-EGFP at different concentrations of synthetic AHL molecules 3OC12-HSL
We then characterized the sensitivity of the biosensor by quantifying the EGFP production rate in response to a range of AHL molecules, 3OC12-HSL from 1x10-7 to 1x10-14M for 19h. The results are the means of triplicate experiments.
The result demonstrated that the EGFP production rates of the biosensor started to increase when the concentration of 3OC12-HSL increased from 1x10-12M and peaked at a steady rate when the concentration of 3OC12-HSL reached 1x10-7M.
13. Quantitative relationship between the EGFP production rate and 3OC12-HSL concentration
An empirical mathematical model, the Hill equation was used to model EGFP production rate (y) as a function of the initial concentration of the synthetic AHL molecule, 3OC12-HSL. In the Hill equation[2]:
Four parameters (A, B, C and n) were used to make the non-linear best fit curve using the experimental results. The parameter A is the baseline of EGFP production rate when AHL initial concentration is 0 M. The parameter B is the maximum EGFP production rate. The parameter C is called EC50 which represents the AHL concentration that can induce a half-maximal EGFP production rate.
14. Quantitative relationship between the EGFP production rate and C10HSL concentration
The Hill equation showed that the biosensor was highly sensitive to the 3OC12-HSL with EC50 values as low as 2.823 x 10-11M 3OC12-HSL. The biosensor also sensitive to the C10HSL with EC50 values as low as 6.841 x 10-9M. It was shown that the concentration of 3OC12-HSL detected in the effluent from P. aeruginosa biofilm was 1.4 x 10-8M and its concentration within the biofilm was over 600 μM[3]. Also the concentration of 3OC12-HSL detected in the supernatant of planktonic cultures are estimated to be between 1 x 10-8 and 5 x 10-6M[4]. Our engineered biosensor with an EC50 value of 2.823 x 10-11M indicate the feasibility of the biosensor in detecting the 3OC12-HSL in both biofilm and water samples. Besides, the engineered biosensor had an EC50 value which was lower than the whole-cell biosensor that had an EC50 value of 5.91 x 10-9M for 3OC12-HSL used for detection of water contamination by P. aeruginosa[2]. This indicates the high sensitivity of our engineered biosensor in nanoscale in the detection of 3OC12-HSL.
Taken together, we first design and engineered four biosensors based on LasI-LasR quorum sensing regulatory circuit LasI-LasR in P. aeruginosa. The plasmids expressing the LasR sensing module and the pLasRL/pLasR3-EGFP reporting module were transformed into BL21 strain to yield the whole-cell biosensor with green fluorescence signal. With several modifications in the protocol, moderate green fluorescence signals were obtained in all four biosensors after 15h incubation with 1x10-6M AHL cocktails. No significant difference in the EGFP production in all biosensors. In an attempt to engineer a more sensitive biosensor with fast detection of AHL molecules in a sample, the strategy of integrating both the LasR sensing module and the pLasRL-EGFP in a single expression plasmid was used. The engineered biosensor with both modules integrated in a single expression plasmid was shown to induce a higher EGFP production rate at a lower AHL concentration and within a shorter time than the four biosensors with the two modules expressed in different plasmids. To conclude, 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 in the presence of quorum sensing molecules of P. aeruginosa, 3OC12-HSL and C10HSL as low as 1x10-11M. In addition, the engineered biosensor had high sensitivity in response to the quorum sensing molecules, 3OC12-HSL with an EC50 value of 2.823 x 10-11M and C10HSL with EC50 values as low as 6.841 x 10-9M. The biosensor has potential applications to provide a rapid, sensitive and quantitative detection of the microplastic pollution levels in water samples.
Future perspectives
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 very limited. For example, an E. coli biosensor which specifically detect Vibrio cholerae based on its quorum sensing molecule autoinducer-I (CAI-1) was developed to inhibit the growth of V. cholerae cells [5]. Besides, a whole-cell biosensor based on P. aeruginosa quorum sensing circuit was developed to detect short- and long-chain AHLs in sputum samples from cystic fibrosis lungs [6]. Recently, a whole-cell biosensor based on QscR quorum sensing signal system of P. aeruginosa was first developed to detect waterborne bacterial pathogens[2]. Thus, we aimed to apply P. aeruginosa LasI-LasR circuit to engineer 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.
Our engineered biosensor had high sensitivity in response to the quorum sensing molecules, 3OC12-HSL, which is the cognate signal molecule of P. aeruginosa. To test the possibility of the use of the biosensor for detection of other AHL molecules, the specificity of the engineered biosensor toward N-butyryl L-homoserine lactone (C4HSL) and N-3-oxo-decanoyl L-homoserine lactone (C10HSL) were also be studied. The results demonstrated that the engineered biosensor had no response to C4HSL. However, the biosensor had a strong response to C10HSL as shown in Figure 16, the quantitative relationship between the EGFP production rate and C10HSL concentration showed EC50 values as low as 6.841 x 10-9M. C10HSL is one of the quorum sensing molecules secreted by key biofilm-forming bacteria on microplastics in marine environment [7]. The ability of the biosensor to respond to C10HSL is important for monitoring microplastics pollution as some biofilm-forming bacteria on microplastics produce C10HSL as the main quorum sensing signals. Also, we will further characterize the ability of the biosensor to detect native AHL molecules produced by P. aeruginosa.
For detection of environmental water samples, it is important to utilize a type of sensor and detection method which can be portable, that is to be used in the field site. Bacterial whole-cell biosensors have several advantages on the use because they are relatively easy and inexpensive to prepare and store, their ability to withstand a range of environmental conditions such as pH or temperature fluctuations and can be integrated into various platforms for rapid high-throughput or on-site detection. In this study, the highly efficient expression and easy detection of the green fluorescence signal was used to facilitate the characterization and optimization of the biosensor system. 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 or colour production such as detection of blue β-galactosidase activity as a visible readout for the development of paper-based device for microplastic detection.
Whole-cell biosensors give promising results in the field with several advantages, but there are concern about the biosafety of using these systems in the field directly because this system is classified as genetically modified organisms. We have adopted the following strategies in project design to minimize the risks. First, we use plasmids as vectors to carry quorum sensing circuits rather than introducing the genome to inhibit the spread of recombinant DNA to environment and other organisms via Horizontal Gene Transfer (HGT). Besides, we add LasR regulatory elements, pLasRL and pLasR3 to the system to control the gene expression.
Engineered whole-cell biosensors with implemented quorum sensing circuit represents a good alternative for environmental monitoring of microplastics pollution. Also, the information of molecules to agonize and antagonize quorum-sensing systems provide further insights about bacterial communication.
Reference
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3. Charlton, T.S., et al., A novel and sensitive method for the quantification of N-3-oxoacyl homoserine lactones using gas chromatography-mass spectrometry: application to a model bacterial biofilm. Environ Microbiol, 2000. 2(5): p. 530-41.
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