1. Sensitivity and Specificity of the Engineered Biosensor pSB1C3-LasR-pLasRL
The engineered biosensor pSB1C3-LasR-pLasRL is composed of the sensing module and reporting module. It is consisting of the LasR transcription factor under a constitutive promoter in the sensing module and the pLasRL inducible promoter upstream of a lacZ coding region in the reporting module. The presence of LasR and LasRL in the engineered biosensor were confirmed by DNA sequencing, PCR and RE digestion.
Three synthetic AHLs: N-butyryl L-homoserine lactone (C4-HSL), N-3-oxo-decanoyl L-homoserine lactone (C10-HSL) and N-3-oxo-dodecanoyl L-homoserine lactone (3OC12-HSL) were used to determine the sensitivity and specificity of the engineered biosensor.
After incubation of 2 mL biosensor cells with different concentration of synthetic AHLs for 2 h, the engineered biosensor was physically immobilized in the alginate hydrogel and placed in the 96-well plate for blue colour development after incubation with X-gal solution.
To reduce background expression of β-galactosidase activity in the absence of AHLs, the engineered biosensor was genetically optimized by removing the sensing module so that no LasR-AHL complex formed and no complex will bind on the reporting module and thus no β-galactosidase expression. It acts as a negative control of the biosensor, pSB1C3-LasR-pLasRL.
This genetically optimized biosensor is consisting of the pLasRL inducible promoter upstream of a lacZ coding region. It was named pSB1C3-pLasRL.
Figure 1. Photographs of the biosensor pSB1C3-LasR-pLasRL incubated with different concentrations of synthetic molecules C4-HSL at a time point of 0 min (A) and 40 min (B) after incubation with X-gal solution. Genetically optimized biosensor pSB1C3-pLasRL was used as the negative control spot. Biosensor incubated with water only labelled as -ve and it was used as the blank. The intensity of the blue colour was analysed using the ImageJ plugin and corrected by subtracting the blank value. The photographs are the typical results of triplicate quantitative X-gal assay.
As shown in Figure 1, no blue colour development was observed in both biosensor spots and negative control spots at a time point at 0 min of X-gal induction (Figure 1A). However, an increase in blue colour intensity was observed with increasing concentration of C4-HSL in biosensor spots but not in negative control spots at 40 min of X-gal induction. It was noted that blue colour intensity was low and comparable to that of the blank. The visual results were then confirmed by quantifying the colour intensity using ImageJ software.
Figure 2. β-galactosidase activity in the biosensor pSB1C3-LasR-pLasRL in response to different concentrations of synthetic molecules C4-HSL at a time point of 0 min (A) and 40 min (B) after incubation with X-gal solution. Genetically optimized biosensor pSB1C3-pLasRL was used as the negative control. The results are the means of triplicate experiments; error bars indicating standard deviations.
Figure 2 showed that β-galactosidase activity of the biosensor was similar to that of the negative control in which no significant β-galactosidase activity was observed. It suggested that the engineered bionsensor was not responsive to C4-HSL.
Figure 3. Photographs of the biosensor pSB1C3-LasR-pLasRL incubated with different concentrations of synthetic molecules C10-HSL at a time point of 0 min (A) and 35 min (B) after incubation with X-gal solution. Genetically optimized biosensor pSB1C3-pLasRL was used as the negative control spot. Biosensor incubated with water only labelled as -ve and it was used as the blank. The intensity of the blue colour was analysed using the ImageJ plugin and corrected by subtracting the blank value. The photographs are the typical results of triplicate quantitative X-gal assay.
As shown in Figure 3, no blue colour development was observed in both biosensor spots and negative control spots at a time point at 0 min of X-gal induction (Figure 3A). However, a high blue colour intensity was observed in the engineered biosensor incubated with 1.0 X 10-8 M to 1.0 X 10-5 M C10-HSL. An increase in blue colour intensity was observed with increasing concentration of C10-HSL in biosensor spots but not in negative control spots at 35 min of X-gal induction. It was noted that the development of the blue colour intensity in biosensor was higher than that of C4-HSL incubation. The visual results were then confirmed by quantifying the colour intensity using ImageJ software.
Figure 4. β-galactosidase activity in the biosensor pSB1C3-LasR-pLasRL in response to different concentrations of synthetic molecules C10-HSL at a time point of 0 min (A) and 35 min (B) after incubation with X-gal solution. Genetically optimized biosensor pSB1C3-pLasRL was used as the negative control. The results are the means of triplicate experiments; error bars indicating standard deviations.
The result showed that β-galactosidase activity of the biosensor for C10-HSL increased from 0 arbitrary unit at 1.0 X 10-10 M to 0.09 arbitrary units at 1.0 X 10-5 M after 35 min incubation with X-gal solution. There was a weak background expression of β-galactosidase activity of the negative control from 1.0 X 10-8 M to 1.0 X 10-5 M in which the highest β-galactosidase activity was 0.025 arbitrary units. It suggested that the engineered biosensor was responsive to C10-HSL.
Figure 5. Photographs of the biosensor pSB1C3-LasR-pLasRL incubated with different concentrations of synthetic molecules 3OC12-HSL at a time point of 0 min (A) and 40 min (B) after incubation with X-gal solution. Genetically optimized biosensor pSB1C3-pLasRL was used as the negative control spot. Biosensor incubated with water only labelled as -ve and it was used as the blank. The intensity of the blue colour was analysed using the ImageJ plugin and corrected by subtracting the blank value. The photographs are the typical results of triplicate quantitative X-gal assay.
As shown in Figure 5, no blue colour development was observed in both biosensor spots and negative control spots at a time point at 0 min of X-gal induction (Figure 5A). However, a high blue colour intensity was observed in the engineered biosensor incubated with 1.0 X 10-7 M to 1.0 X 10-5 M 3OC12-HSL. An increase in blue colour intensity was observed with increasing concentration of 3OC12-HSL in biosensor spots but not in negative control spots at 40 min of X-gal induction. It was noted that the development of the blue colour intensity in biosensor was higher than that of C4-HSL and C10-HSL incubation. The visual results were then confirmed by quantifying the colour intensity using ImageJ software.
Figure 6. β-galactosidase activity in the biosensor pSB1C3-LasR-pLasRL in response to different concentrations of synthetic molecules 3OC12-HSL at a time point of 0 min (A) and 40 min (B) after incubation with X-gal solution. Genetically optimized biosensor pSB1C3-pLasRL was used as the negative control. The results are the means of triplicate experiments; error bars indicating standard deviations.
The result showed that β-galactosidase activity of the biosensor for 3OC12-HSL increased from 0 arbitrary unit at 1.0 X 10-8 M to 0.125 arbitrary units at 1.0 X 10-5 M after 40 min incubation with X-gal solution. There was no background expression of β-galactosidase activity of the negative control from 1.0 X 10-11 M to 1.0 X 10-5 M. It suggested that the engineered biosensor was responsive to 3OC12-HSL and the level was comparable to that of C10-HSL.
Taken together, C4-HSL elicit weak and delayed responses, whereas 3OC12-HSL and C10-HSL induce higher and time-dependent β-galactosidase activity in engineered biosensor. Control biosensor showed minimal activation, validating the specificity of the transcriptional response.
It is concluded that the engineered biosensor pSB1C3-LasR-pLasRL was responsive to 3OC12-HSL and C10-HSL but not C4-HSL. It exhibited sensitivity at the lowest concentrations of 1.0 X 10-7 M.
The average range of AHL concentration in water environments as reported in the literature is 1.0 X 10-8 M to 5.0 X 10-6 M for 3OC12-HSL [2]. The engineered biosensor showed a bit lower sensitivity toward 3OC12-HSL and C10-HSL and thus not sensitive enough to detect the presence of AHL molecules.
The LasR promoter family is involved in quorum sensing control of gene regulation in P. aeruginosa, in which the binding of AHL molecules to the LasR transcription factor positively up-regulates transcription of LasR responsive promoters. In an experiment to compare the relative strength of the three LasR responsive promoters, pLasR3, pLasRV and pLasRL across a range of AHL concentrations in E. coli, it was found that pLasR3 showed a higher binding efficiency than pLasRL [3]. Therefore, we engineered a new biosensor by replacing pLasR3 as the inducible promoter in the reporting module.
The new whole-cell biosensor consisting of the LasR transcription factor under a constitutive promoter in the sensing module and the pLasR3 inducible promoter upstream of a lacZ coding region in the reporting module. It was named pSB1C3-LasR-pLasR3. The genetically optimized biosensor with sensing module removed and consists of the pLasR3 inducible promoter upstream of a lacZ coding region in the reporting module was used as a negative control. It was named pSB1C3-pLasR3.
2. Sensitivity and Specificity of the Engineered Biosensor pSB1C3-LasR-pLasR3
Figure 7. Photographs of the biosensor pSB1C3-LasR-pLasR3 incubated with different concentrations of synthetic molecules C4-HSL at a time point of 0 min (A) and 40 min (B) after incubation with X-gal solution. Genetically optimized biosensor pSB1C3-pLasR3 was used as the negative control spot. Biosensor incubated with water only labelled as -ve and it was used as the blank. The intensity of the blue colour was analysed using the ImageJ plugin and corrected by subtracting the blank value. The photographs are the typical results of triplicate quantitative X-gal assay.
As shown in Figure 7, no blue colour development was observed in both biosensor spots and negative control spots at a time point at 0 min of X-gal induction (Figure 7A). A low blue colour intensity was observed in different concentrations of C4-HSL in biosensor spots but not in negative control spots at 40 min of X-gal induction (Figure 7B). The low blue colour intensity was low and comparable to that of the blank. The visual results were then confirmed by quantifying the colour intensity using ImageJ software.
Figure 8. β-galactosidase activity in the biosensor pSB1C3-LasR-pLasR3 in response to different concentrations of synthetic molecules C4-HSL at a time point of 0 min (A) and 40 min (B) after incubation with X-gal solution. Genetically optimized biosensor pSB1C3-pLasR3 was used as the negative control. The results are the means of duplicate experiments; error bars indicating standard deviations.
Figure 8 showed that β-galactosidase activity of the biosensor for C4-HSL increased from 0 arbitrary unit at 1.0 X 10-8 M to 0.04 arbitrary units at 1.0 X 10-5 M after 40 min incubation with X-gal solution and no significant β-galactosidase activity was observed in the negative control. It suggested that the engineered biosensor was not responsive to C4-HSL.
Figure 9. Photographs of the biosensor pSB1C3-LasR-pLasR3 incubated with different concentrations of synthetic molecules C10-HSL at a time point of 0 min (A) and 35 min (B) after incubation with X-gal solution. Genetically optimized biosensor pSB1C3-pLasR3 was used as the negative control spot. Biosensor incubated with water only labelled as -ve and it was used as the blank. The intensity of the blue colour was analysed using the ImageJ plugin and corrected by subtracting the blank value. The photographs are the typical results of triplicate quantitative X-gal assay.
As shown in Figure 9, no blue colour development was observed in both biosensor spots and negative control spots at a time point at 0 min of X-gal induction (Figure 9A). However, a high blue colour intensity was observed in the engineered biosensor incubated with 1.0 X 10-8 M to 1.0 X 10-5 M C10-HSL. An increase in blue colour intensity was observed with increasing concentration of C10-HSL in biosensor spots but not in negative control spots at 35 min of X-gal induction (Figure 9B). It was noted that the development of the blue colour intensity in biosensor was higher than that of C4-HSL incubation. The visual results were then confirmed by quantifying the colour intensity using ImageJ software.
Figure 10. β-galactosidase activity in the biosensor pSB1C3-LasR-pLasR3 in response to different concentrations of synthetic molecules C10-HSL at a time point of 0 min (A) and 35 min (B) after incubation with X-gal solution. Genetically optimized biosensor pSB1C3-pLasR3 was used as the negative control. The results are the means of triplicate experiments; error bars indicating standard deviations.
The result showed that β-galactosidase activity of the biosensor for C10-HSL increased from 0 arbitrary unit at 1.0 X 10-8 M to 0.075 arbitrary units at 1.0 X 10-5 M and peak at 1.0 X 10-6 M with 0.09 arbitrary unit after 35 min incubation with X-gal solution. There was no background expression of β-galactosidase activity of the negative control from 1.0 X 10-11 M to 1.0 X 10-7 M (Figure 10B). It suggested that the engineered biosensor was responsive to C10-HSL. It was noted that there was low background expression of β-galactosidase activity in engineered biosensor at start time (Figure 10A).
Figure 11. β-galactosidase activity in the biosensor pSB1C3-LasR-pLasR3 in response to different concentrations of synthetic molecules C10-HSL at a time point of 0 min (A) and 35 min (B) after incubation with X-gal solution. Genetically optimized biosensor pSB1C3-pLasR3 was used as the negative control. The results are the means of triplicate experiments; error bars indicating standard deviations. The relationship between β-galactosidase activity and the concentration of the synthetic AHL fits Hill equation with R-squared value above 0.89.
We then characterized the sensitivity of the engineered biosensor by quantifying the β-galactosidase activity in response to a range of concentrations of C10-HSL. An empirical mathematical model, the Hill equation was used to model β-galactosidase activity as a function of the initial concentration of the synthetic AHLs.
As shown in Figure 11, the β-galactosidase activity of both engineered biosensor and the negative control was low at time 0 (Figure 11A). The β-galactosidase activity started to increase when the concentration of C10-HSL increased beyond 1.0 X 10-8 M and peaked at a steady rate when C10-HSL reached 1.0 X 10-6 M (Figure 11B). The Hill equation showed that the biosensor was highly sensitive to the C10-HSL with EC50 value as low as 1.0 X 10-8 M which lie within the average range of AHL concentrations in water environments indicating the feasibility of the engineered biosensor in detecting the C10-HSL.
Figure 12. Photographs of the biosensor pSB1C3-LasR-pLasR3 incubated with different concentrations of synthetic molecules 3OC12-HSL at a time point of 0 min (A) and 35 min (B) after incubation with X-gal solution. Genetically optimized biosensor pSB1C3-pLasR3 was used as the negative control spot. Biosensor incubated with water only labelled as -ve and it was used as the blank. The intensity of the blue colour was analysed using the ImageJ plugin and corrected by subtracting the blank value. The photographs are the typical results of triplicate quantitative X-gal assay.
Figure 12 showed no blue colour development was observed in both biosensor spots and negative control spots at a time point at 0 min of X-gal induction (Figure 12A). However, a high blue colour intensity was observed in the engineered biosensor incubated with 1.0 X 10-8 M to 1.0 X 10-5 M 3OC12-HSL. An increase in blue colour intensity was observed with increasing concentration of 3OC12-HSL in biosensor spots but not in negative control spots at 35 min of X-gal induction (Figure 12B). It was noted that the development of the blue colour intensity in biosensor was higher than that of C4-HSL and C10-HSL incubation. The visual results were then confirmed by quantifying the colour intensity using ImageJ software.
Figure 13. β-galactosidase activity in the biosensor pSB1C3-LasR-pLasR3 in response to different concentrations of synthetic molecules 3OC12-HSL at a time point of 0 min (A) and 35 min (B) after incubation with X-gal solution. Genetically optimized biosensor pSB1C3-pLasR3 was used as the negative control. The results are the means of six experiments; error bars indicating standard deviations.
The result showed that β-galactosidase activity of the biosensor for 3OC12-HSL increased from 0 arbitrary unit at 1.0 X 10-9 M to 0.17 arbitrary units at 1.0 X 10-5 M and peak at 1.0 X 10-6 M with 0.18 arbitrary unit after 35 min incubation with X-gal solution. There was very low background expression of β-galactosidase activity of the negative control from 1.0 X 10-11 M to 1.0 X 10-5 M (Figure 10). The development of the blue colour intensity in biosensor was higher than that of C4-HSL and C10-HSL incubation suggesting that the engineered biosensor was most responsive to 3OC12-HSL.
Figure 14. β-galactosidase activity in the biosensor pSB1C3-LasR-pLasR3 in response to different concentrations of synthetic molecules 3OC12-HSL at a time point of 0 min (A) and 35 min (B) after incubation with X-gal solution. Genetically optimized biosensor pSB1C3-pLasR3 was used as the negative control. The results are the means of six experiments; error bars indicating standard deviations. The relationship between β-galactosidase activity and the concentration of the synthetic AHL fits Hill equation with R-squared value above 0.97.
As shown in Figure 14, the β-galactosidase activity of both engineered biosensor and the negative control was low at time 0 (Figure 14A). The β-galactosidase activity started to increase when the concentration of 3OC12-HSL increased beyond 1.0 X 10-8 M and peaked at a steady rate when 3OC12-HSL reached 1.0 X 10-5 M (Figure 14B). The Hill equation showed that the biosensor was highly sensitive to the 3OC12-HSL with EC50 value as low as 3.2 X 10-8 M which lie within the average range of AHL concentrations in water environments indicating the feasibility of the engineered biosensor in detecting the 3OC12-HSL.
Taken together, the engineered biosensors pSB1C3-LasR-pLasRL and pSB1C3-LasR-pLasR3 are not responsive to C4-HSL. Besides, pSB1C3-LasR-pLasR3 is more sensitive than pSB1C3-LasR-pLasRL in the detection of AHLs, specifically C10-HSL and 3OC12-HSL. EC50 values represent the concentration of AHL compounds required for half-maximal activation of the biosensor. The Hill equation showed that the biosensor pSB1C3-LasR-pLasR3 was highly sensitive to the AHLs with EC50 values as low as 1.0 X 10-8 M for C10-HSL and 3.2 X 10-8 M for 3OC12-HSL. This biosensor shows similar level of sensitivity towards C10-HSL and 3OC12-HSL.
3. Real sample analysis of the Engineered Biosensor pSB1C3-LasR-pLasR3
To evaluate the potential matrix effect of tap water and seawater due to interfering substances such as chlorine, microorganisms and various chemical contaminants on the performance of the engineered biosensor pSB1C3-LasR-pLasR3, we further characterized the dose-response curve of the biosensor in real water samples of tap water and seawater spiked with 3OC12-HSL, which is the cognate AHL produced by P. aeruginosa at different concentrations.
Figure 15. Photographs of the biosensor pSB1C3-LasR-pLasR3 incubated with different concentrations of synthetic molecules 3OC12-HSL at a time point of 0 min (A) 50 min (B) after incubation with X-gal solution and in tap water samples spiked with different concentrations of synthetic molecules 3OC12-HSL at a time point of 50 min (C) after incubation with X-gal solution. Genetically optimized biosensor pSB1C3-pLasR3 was used as the negative control spot. Biosensor incubated with water only labelled as -ve and it was used as the blank. The intensity of the blue colour was analysed using the ImageJ plugin and corrected by subtracting the blank value. The photographs are the typical results of triplicate quantitative X-gal assay.
The engineered biosensor pSB1C3-LasR-pLasR3 was incubated with different concentrations of synthetic molecules 3OC12-HSL for 2 h and allow for blue colour development after X-gal addition. Figure 15 showed that no blue colour development was observed in both biosensor spots and negative control spots at a time point of 0 min of X-gal induction (Figure 15A). However, a high blue colour intensity was observed in the engineered biosensor incubated with 1.0 X 10-8 M to 1.0 X 10-5 M 3OC12-HSL at 50 min of X-gal induction (Figure 15B). The result was consistent with our previous findings (Figure 12B).
To analyze the matrix effect, 0.2 mL of real tap water samples spiked with different concentration of 3OC12-HSL was added in triplicate to culture tube containing 1.8 mL of biosensor cells. The biosensor with tap water samples was then incubated with different concentrations of synthetic molecules 3OC12-HSL for 2 h and allow for blue colour development after X-gal addition. An increase in blue colour intensity was observed with increasing concentration of 3OC12-HSL in biosensor spots but not in negative control spots at 50 min of X-gal induction (Figure 15C). It was noted that the development of the blue colour intensity in biosensor with actual tap water samples was higher than that of biosensor cells only at 1 X 10-8 M 3OC12-HSL (Figure 15B and 15C). The visual results were then confirmed by quantifying the colour intensity using ImageJ software.
Figure 16. β-galactosidase activity in the biosensor pSB1C3-LasR-pLasR3 in response to different concentrations of synthetic molecules 3OC12-HSL at a time point of 0 min (A) and 50 min (B) after incubation with X-gal solution. β-galactosidase activity in the biosensor pSB1C3-LasR-pLasR3 in tap water samples spiked with different concentrations of synthetic molecules 3OC12-HSL at a time point of 0 min (C) and 50 min (D). Genetically optimized biosensor pSB1C3-pLasR3 was used as the negative control. The results are the means of four experiments; error bars indicating standard deviations.
Figure 16 showed that β-galactosidase activity of the biosensor for 3OC12-HSL increased from 0 arbitrary unit at 1.0 X 10-8 M to 0.17 arbitrary units at 1.0 X 10-5 M after 50 min incubation with X-gal solution (Figure 16B). There were very low background expression of β-galactosidase activity of the negative control from 1.0 X 10-11 M to 1.0 X 10-5 M (Figure 16A and 16C). The β-galactosidase activity of the biosensor with actual tap water samples increased from 0 arbitrary unit at 1.0 X 10-8 M to 0.27 arbitrary units at 1.0 X 10-5 M after 50 min incubation with X-gal solution (Figure 16D). It suggested that the performance of engineered biosensor was functional in the real tap water environment.
Figure 17. β-galactosidase activity in the biosensor pSB1C3-LasR-pLasR3 in response to different concentrations of synthetic molecules 3OC12-HSL at a time point of 0 min (A) and 50 min (B) after incubation with X-gal solution.
β-galactosidase activity in the biosensor pSB1C3-LasR-pLasR3 in tap water samples spiked with different concentrations of synthetic molecules 3OC12-HSL at a time point of 0 min (C) and 50 min (D) after incubation with X-gal solution.
Genetically optimized biosensor pSB1C3-pLasR3 was used as the negative control. The results are the means of four experiments; error bars indicating standard deviations. The relationship between β-galactosidase activity and the concentration of the synthetic AHL fits Hill equation with R-squared value above 0.92.
As shown in Figure 17, the β-galactosidase activity of engineered biosensor without and with real tap water samples and the negative control was low at time 0 (Figure 17A and 17C). The β-galactosidase activity started to increase when the concentration of 3OC12-HSL increased beyond 1.0 X 10-8 M and peaked at a steady rate when 3OC12-HSL reached 1.0 X 10-5 M in biosensor without and with real tap water samples (Figure 17B and 17D). The Hill equation showed that the biosensor with and without real tap water samples were highly sensitive to the 3OC12-HSL with EC50 value as low as 1.8 X 10-8 M for biosensor only and 1.6 X 10-8 M for biosensor with real tap water samples respectively. Both EC50 values lie within the average range of AHL concentrations in water environments indicating the feasibility of the engineered biosensor in detecting the 3OC12-HSL in real tap water environment.
Figure 18. Photographs of the biosensor pSB1C3-LasR-pLasR3 incubated with different concentrations of synthetic molecules 3OC12-HSL at a time point of 0 min (A) 50 min (B) after incubation with X-gal solution.
Photographs of the biosensor pSB1C3-LasR-pLasR3 in tap water samples spiked with different concentrations of synthetic molecules 3OC12-HSL at a time point of 50 min (C) after incubation with X-gal solution.
Photographs of the biosensor pSB1C3-LasR-pLasR3 in sea water samples spiked with different concentrations of synthetic molecules 3OC12-HSL at a time point of 50 min (D) after incubation with X-gal solution. Genetically optimized biosensor pSB1C3-pLasR3 was used as the negative control spot. Biosensor incubated with water only labelled as -ve and it was used as the blank. The intensity of the blue colour was analysed using the ImageJ plugin and corrected by subtracting the blank value. The photographs are the typical results of triplicate quantitative X-gal assay.
To compare the potential matrix effect of tap water and seawater on the performance of the engineered biosensor, the engineered biosensor pSB1C3-LasR-pLasR3 only, biosensor with real tap water samples and biosensor with sea water samples were incubated with different concentrations of synthetic molecules 3OC12-HSL for 2 h and allow for blue colour development after X-gal addition. Figure 18 showed that no blue colour development was observed in biosensor spots and negative control spots at a time point of 0 min of X-gal induction (Figure 18A). However, a high blue colour intensity was observed in the engineered biosensor incubated with 1.0 X 10-8 M to 1.0 X 10-5 M 3OC12-HSL at 50 min of X-gal induction (Figure 18B). The result was consistent with our previous findings (Figure 12B and 15B).
For biosensor with real tap water samples, an increase in blue colour intensity was observed with increasing concentration of 3OC12-HSL from 1.0 X 10-8 M to 1.0 X 10-5 M in biosensor spots but not in negative control spots at 50 min of X-gal induction (Figure 18C). The result was consistent to previous findings (Figure 15C).
For biosensor with seawater samples, an increase in blue colour intensity was also observed with increasing concentration of 3OC12-HSL from 1.0 X 10-8 M to 1.0 X 10-5 M in biosensor spots but not in negative control spots at 50 min of X-gal induction (Figure 18D).
The results demonstrated that our engineered biosensor is functional in the real tap water environment and seawater without significant matrix effect which affects the real sample analysis.
Figure 19. β-galactosidase activity in the biosensor pSB1C3-LasR-pLasR3 in response to different concentrations of synthetic molecules 3OC12-HSL at a time point of 0 min (A) and 50 min (B) after incubation with X-gal solution.
β-galactosidase activity in the biosensor pSB1C3-LasR-pLasR3 in tap water samples spiked with different concentrations of synthetic molecules 3OC12-HSL at a time point of 0 min (C) and 50 min (D) after incubation with X-gal solution.
β-galactosidase activity in the biosensor pSB1C3-LasR-pLasR3 in sea water samples spiked with different concentrations of synthetic molecules 3OC12-HSL at a time point of 0 min (E) and 50 min (F) after incubation with X-gal solution. Genetically optimized biosensor pSB1C3-pLasR3 was used as the negative control. The results of (A) to (D) are the means of four experiments; The results of (E) and (F) are the means of duplicate experiments; error bars indicating standard deviations.
The β-galactosidase activity of the engineered biosensor pSB1C3-LasR-pLasR3 only, biosensor with real tap water samples and biosensor with sea water samples were compared. There were very low background expression of β-galactosidase activity of the negative control from 1.0 X 10-11 M to 1.0 X 10-5 M in all three biosensors (Figure 19A, 19C and 19E). The β-galactosidase activity of the engineered biosensor for 3OC12-HSL increased from 0 arbitrary unit at 1.0 X 10-8 M to 0.16 arbitrary units at 1.0 X 10-5 M after 50 min incubation with X-gal solution (Figure 19B). The β-galactosidase activity of the engineered biosensor with real tap water samples for 3OC12-HSL increased from 0 arbitrary unit at 1.0 X 10-8 M to 0.27 arbitrary units at 1.0 X 10-5 M after 50 min incubation with X-gal solution (Figure 19D).
The β-galactosidase activity of the biosensor with seawater samples increased from 0 arbitrary unit at 1.0 X 10-8 M to 0.30 arbitrary units at 1.0 X 10-5 M after 50 min incubation with X-gal solution. The results demonstrated that the performance of engineered biosensor was functional in both real tap water environment and seawater.
Figure 20. β-galactosidase activity in the biosensor pSB1C3-LasR-pLasR3 in sea water samples spiked with different concentrations of synthetic molecules 3OC12-HSL at a time point of 0 min (A) and 50 min (B) after incubation with X-gal solution. Genetically optimized biosensor pSB1C3-pLasR3 was used as the negative control. The results are the means of duplicate experiments; error bars indicating standard deviations. The relationship between β-galactosidase activity and the concentration of the synthetic AHL fits Hill equation with R-squared value above 0.95.
As shown in Figure 20, the β-galactosidase activity of engineered biosensor with seawater samples and the negative control was low at time 0 (Figure 20A). The β-galactosidase activity started to increase when the concentration of 3OC12-HSL increased beyond 1.0 X 10-8 M and peaked at a steady rate when 3OC12-HSL reached 1.0 X 10-5 M in biosensor with seawater samples (Figure 20B). The Hill equation showed that the biosensor with seawater samples was highly sensitive to the 3OC12-HSL with EC50 value as low as 2.5 X 10-8 M which lie within the average range of AHL concentrations in water environments indicating the feasibility of the engineered biosensor in detecting the 3OC12-HSL in seawater environment.
Taken together, we have engineered a set of biosensors including two whole-cell biosensors, pSB1C3-LasR-pLasRL and pSB1C3-LasR-pLasR3 and their negative control biosensors, pSB1C3-pLasRL and pSB1C3-pLasR3 respectively. The whole-cell biosensors are based on the quorum sensing regulatory circuit LasI-LasR in P. aeruginosa and the lacZ gene was used in the reporting module. To reduce background expression of β-galactosidase activity in the absence of AHLs, we modified the engineered biosensors by removing the sensing module which acts as a negative control of the engineered biosensors. The negative control biosensors, pSB1C3-pLasRL and pSB1C3-pLasR3 are consisting of the pLasRL and pLasR3 inducible promoter upstream of a lacZ coding region. Both negative control biosensors were proved to effectively reduce background expression of β-galactosidase activity so that the blue colour development time will not be limited by the rapid blue colour development in the negative control which allow a higher level of gene expression and thus the sensitivity of the engineered biosensor.
Our results demonstrated that the engineered biosensor pSB1C3-LasR-pLasRL was responsive to 3OC12-HSL and C10-HSL but not C4-HSL. However, it exhibited sensitivity at the lowest concentrations of 1.0 X 10-7 M which was not sensitive enough to detect the presence of AHL molecules under the average range of AHL concentration in water environments. Thus we engineered a new biosensor, pSB1C3-LasR-pLasRL by replacing pLasR3 as the inducible promoter in the reporting module. It was found that pSB1C3-LasR-pLasR3 was responsive to 3OC12-HSL and C10-HSL but not C4-HSL. The β-galactosidase activity of the engineered biosensor in response to 3OC12-HSL was 0.17 arbitrary unit (Figure 13B) double the level of C10-HSL which was 0.075 arbitrary unit (Figure 10B). Thus, the new biosensor was most responsive to 3OC12-HSL. The biosensor pSB1C3-LasR-pLasR3 was highly sensitive to the AHLs with EC50 values as low as 1.0 X 10-8 M for C10-HSL and 3.2 X 10-8 M for 3OC12-HSL. This biosensor shows similar level of sensitivity towards C10-HSL and 3OC12-HSL.
Real sample analysis demonstrated that the biosensor pSB1C3-LasR-pLasR3 is functional in both real tap water and seawater environment. Matrix effect of tap water such as chlorine, microorganisms and other chemical contaminants have no interference effect to our engineered biosensor. Similarly, matrix effect of seawater such as salt concentration, organic macromolecules such as proteins, polysaccharides and humic substances have no interference effect to our engineered biosensor. It was noted that the β-galactosidase activity was the highest in biosensor with seawater samples followed by biosensor with tap water samples (Figure 19). The major difference in the samples lies in the salinity, thus we will further investigate the effect of salinity on the sensitivity of the engineered biosensor.
Future Perspectives
Among the common microplastics, polyethylene (PE) and polypropylene (PP), polytetrafluoroethylene (PTFE) and polystyrene (PS), it was found that biofilms formed by P. aeruginosa were better on polyethylene (PE) and polypropylene (PP) microplastic surface which have similar densities to water and float on water surface. Besides, PE which exhibited a relatively high hydrophobicity was observed to have the best bacterial attachment at the initial stage of biofilm formation [4]. PE and PP are microplastics commonly found in freshwater ecosystem, marine water ecosystem, urban water ecosystem and wastewater ecosystem [1]. The design of our engineered biosensor is based on LasI-LasR regulatory circuit in P. aeruginosa. With high detection sensitivity and selectivity, our engineered whole-cell biosensor, pSB1C3-LasR-pLasR3 may potentially be used for rapid detection and quantification of PE and PP in environmental water samples.
It was found that the common microplastics in Hong Kong marine waters are PE and PP which contributed average proportions of 61.6% and 19.8% respectively [5]. Our engineered biosensor may have potential to serve as an alternative approach to the sampling and detection method of microplastics used in Hong Kong.
Current detection method of microplastics detection and quantification are time-consuming, labour intensive, require specialized equipment such as Fourier transform infrared spectroscopy (FTIR), expensive reagents or procedural charge and trained technicians which prevent wide implementation and regular monitoring of microplastics. Besides, quantification methods varied from different countries making inconsistency in the global comparison. Therefore, our engineered biosensor has potentials to allow microplastic detection in a rapid, high throughput, low cost, regular basis and consistent comparison. Besides, no specialized training and equipment is required.
We have developed a standardized protocol in a testing kit as attached. It can be used in the lab to ensure data consistency for global comparison about microplastic pollution in marine waters. In our developed protocol of quantitative X-gal assay, a visible blue colour will be developed in 1 h and the total experiment time is less than 6 h after preparation of overnight culture of biosensor cells. Our biosensor is feasible to deploy in EPD’s Water Management Division for routine and high-throughput microplastics monitoring and assessment in marine waters, rivers, beaches, stormwater drains and effluent from wastewater treatment plants; Drainage Service Department laboratory to manage load of microplastics entering and leaving wastewater treatment plants; Water Supplies Department laboratory to monitor reservoir water and treated drinking water for microplastic contamination to ensure public health; University research for research discovery and commercial environmental testing laboratories for providing analytical testing services to clients such as NGOs on a commercial basis.
Whole-cell biosensors are the ideal solution for continuous microplastics monitoring which is important to assess the effects of microplastics on our environment and ecosystem for developing strategies for sustainability.