Summary
As industrialization gradually spreads worldwide, more and more plastic wastes are mass produced each year to fulfil the demands of the public. It is expected for global annual plastic production to reach a shocking 1.1 billion tonnes in 2050 [1], proving that the massive production and consumption of plastic in modern life is causing disastrous consequences. Within all negative impacts, the most striking problem is obviously marine pollution.
Identification of microplastics in complex environmental matrices remains a challenge. Fast and accurate detection of microplastics in environmental water samples is essential for understanding their sources, occurrences and ecological concern. Conventional detection methods require chemical or physical analytical techniques that are time-consuming and expensive. In contrast, whole cell biosensors, sensors that are formed from cells, can be modified by genetic engineering methods so that they can be used to detect a wider range of substances within a living cell. Due to their good sensitivity and high selectivity, they have been widely used in the field of environmental monitoring, food analysis, pharmacology and drug screening [2]. After numerous background research and literature reviews, we found that the engineering of whole-cell biosensors for detection of microplastic level is unexplored. Also, there is no test kit available for fast and accurate detection of microplastic. Thus, in this study, we engineered a whole-cell biosensor that expressed LacZ gene based on LasI-LasR quorum sensing regulatory circuit in P. aeruginosa for rapid and quantitative detection of microplastics in water samples of an area. The development of whole cell biosensors is essential for rapid high-throughput detection. It also offers easier identification and tracking of microplastics, policy formulation and prioritizes the allocated resources to address the issue.
During the process of research, we went through cycles of engineering (Research → Design → Build → Test → Learn → improve) to optimize a protocol of quantitative X-gal method to evaluate the biosensing system on the hydrogel-based assay. Besides, we have successfully engineered a functional biosensor which is able to detect AHL (3OC12-HSL) concentration as low as 1.0 X 10-8 M and a genetically optimized biosensor which act as a negative control to effectively reduced the background expression of β-galactosidase in the absence of AHL. This allows the use of the substrate X-Gal to quantify β-galactosidase levels produced intracellularly by the sensor cells.
Research
In this study, we engineered a whole cell biosensor that expressed LacZ gene, encoding -galactosidase, which is an enzyme extensively used as a reporter. The detection of blue β-galactosidase activity after incubation in an 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) solution act as a visible readout.
We attempt to develop a whole cell biosensor which is physically immobilized in the alginate hydrogel for the development of rapid, sensitive, cost effective and quantitative detection of microplastic.
The performance of the hydrogel-based biosensor can be affected by many factors, including the amount of the LasR for AHL induction, optical density (OD) at 600 nm, the concentration of X-gal and the time duration for incubation after AHL induction. Thus we first investigated the best combination of these factors for the functionality of the biosensor.
Design
The engineered biosensor consists of two modules, the sensing module and the reporting module. The sensing module expresses the transcription factor LasR which can bind to the AHL molecules that are secreted from P. aeruginosa that attach on microplastics. The formation of the LasR-AHL complex can then bind to an inducible promoter, pLasRL which activates the transcription of the reporting module. A β-galactosidase, or LacZ system is used as the reporting module, in which the expression of bioluminescence would be induced by the LasR-AHL complex after the addition of 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) to show a blue color. The intensity of blue color emitted by the biosensor is directly proportional to the amount of AHL molecules present on the microplastics. Thus, we can quantify and compare the extent of microplastic pollution in certain samples of water.
Build
The whole-cell biosensor 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. It was cloned into EcoRI and PstI sites of pSB1C3 vector. The resulting plasmid was named pSB1C3-LasR-pLasRL. A synthetic AHL molecule, N-3-oxo-dodecanoyl L-homoserine lactone (3OC12-HSL) was used to determine the specificity and sensitivity of the engineered biosensor.
Test, learn and improve
After the confirmation of the presence of LasR and pLasRL in the engineered biosensor, we preformed different tests to optimize the protocol for a more sensitive quantitative X-gal assay.
1. Hydrogel percentage
The whole-cell biosensor was immobilized in 2% hydrogel and added in a 96-well plate. The viscosity of hydrogel affects the performance of the biosensor, for example, if the hydrogel percentage is too high, the pellet would not be able to resuspend. On the other hand, if the hydrogel is too watery, it will easily evaporate.
Figure 1. Images of sample using 3% hydrogel to resuspend (left) and 2% hydrogel to resuspend (right).
The above result demonstrates that when the hydrogel percentage is too high (3%), suspension remains in the well which affects the performance and visibility of the biosensor. Hence, 2% hydrogel is chosen for the following investigation.
2. Bacterial Optical Density
We studied the effect of induction cell density at induction time by comparing the β- galactosidase activity on two bacterial densities y at OD600 of 0.3 and OD600 of 0.7 for 3OC12-HSL induction.
Figure 2. β-galactosidase activity of biosensor on two different bacterial concentrations (OD600) for AHL induction. The biosensor was induced by 3OC12-HSL at 1.0 X 10-6 M for 0h and 2h, and the blue luminescence production rate is quantified as β-galactosidase activity. The biosensor without AHL induction was used as the negative control. The result are the means of triplicate experiments; error bars indicate the standard deviations.
The result shows that biosensor cells with OD600 of 0.3 has a higher β-galactosidase activity with more obvious difference between samples with 3OC12-HSL and without 3OC12-HSL induction. While the OD600 0.77 sample have less difference with the control samples due to the rapid background expression of β-galactosidase in the absence of inducer, X-gal caused by the large cell number in the sample. Thus, OD600 0.3 is chosen for the following investigation to minimize the effect of background expression.
3. Concentration of X-gal
X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside), the cell-permeable chromogenic β-galactosidase substrate, is the key factor for blue-coloured product on hydrogel-based assay. After resuspension using hydrogel, X-gal substrate solution is carefully added into the 96-well plate with sensing cells. The X-gal substrate will bind with the β-galactosidase enzyme formed from the reporting module of the biosensor, showing a blue colour in X-gal assay.
The concentration of X-gal would affect the visibility of the blue colour formed. The intensity of the blue color with different concentrations of X-gal (5mg/mL and 10mg/mL) were compared to determine the most suitable X-gal concentration for the blue product observation and quantification.
Figure 3. Mean Auncorr (an “uncorrected value” as an output value from the Readplate 3.0 analysis which indicate the intensity of blue colour formed after X-gal induction) on different X-gal concentration. The biosensor was induced by 3OC12-HSL at 1.0 X 10-6 M for 2hr. Then, 5mg/mL and 10mg/mL of X-gal were added to the samples. Colour development was carried out for 15 minutes at room temperature. Samples without X-gal induction (0h) were used as negative controls. The blue colour of samples was compared to samples at 0h without AHL induction. The result are the means of triplicate experiments; error bars indicate the standard deviations.
The result shows that the X-gal with 10mg/mL concentration has the highest mean auncorr value which indicates the highest intensity of blue luminescence emission. Comparing with 5mg/mL samples, X-gal with 10mg/mL is more favourable to observe the β-galactosidase activity between samples with AHL and without AHL as it has a more obvious colour difference than that of 5mg/mL X-gal without saturation. Therefore, 10mg/mL X-gal is chosen for the subsequent investigation for a better visualization of the blue luminescence.
4. AHL Induction Time
To evaluate the ability of the engineered biosensor to detect synthetic AHL compound 3OC12-HSL, the biosensor was induced by 1.0 X 10-6 M 3OC12-HSL for 0h, 2h, 4h and 6h. Meanwhile, bacterial optical density was recorded at every time point to study the growth of biosensor cells.
Figure 4. Optical density at 600 nm (OD600) at every two hours after AHL induction. The biosensor was induced in AHL at 1.0 X 10-6 M and incubated in shaker at 33°C, 220 rpm. The biosensor without AHL induction was used as the negative control. The result are the means of triplicate experiments.
A visible blue colour was developed in the biosensor spot after 2h, 4h and 6h of incubation with 1.0 X 10-6 M 3OC12-HSL using 5mg/mL and 10mg/mL X-gal. The negative control spot showed a yellow colour representing the uninduced biosensor cells.
Figure 5. 96-well plate photo of biosensor with different X-gal concentration every two hours after AHL induction. The biosensor was induced in AHL at 1.0 X 10-6 M (+ve) and incubated in shaker at 33°C, 220 rpm. The biosensor without AHL induction (-ve) was used as the negative control. Three X-gal concentrations, 0mg/µL, 5mg/µL and 10mg/µL were added in samples. The result is a triplicate experiment. Image is taken 15 minutes after X-gal induction.
The result shows the obvious colour difference between 0h and 2h AHL incubation time, where 2h samples have relatively higher colour intensity than that of 0h. Although 4h and 6h samples have high colour intensity like the 2h samples, the colour difference between samples with and without AHL induction are smaller than that of 2h samples. Meanwhile, there were a gradual increase of OD600 from 0h to 4h, while the OD600 has a slight decline after 4h (Figure 4). It indicated that the bacterial growth was inhibited as the incubation time increased.
Therefore, we chose to incubate the samples 2 hours after AHL induction to minimize the effect caused by fluctuated bacterial density for a better visualization of the result.
Taken together, we have developed a protocol which allows for the fast and convenient detection of 3OC12-HSL after 2 hour AHL induction time for visualization of the blue color development.
5. Sensitivity of the hydrogel-based Assay to detect AHL molecules
For detection of microplastic in water samples, the hydrogel-based biosensor is able to distinguish between different concentrations of AHL by comparing the colour intensity of the blue end product.
The sensitivity of the biosensor is characterized by quantifying the β-galactosidase activity in response to a range of concentrations of 3OC12-HSL.
Figure 6. β-galactosidase activity of biosensor towards eight concentrations of AHL. The biosensor was induced in AHL at 1.0 X 10-5 M to 1.0 X 10-11 M for 2hr, and the blue luminescence production rate is quantified as β-galactosidase activity. The biosensor without AHL induction was used as the negative control. The result are the means of triplicate experiments; error bars indicate the standard deviations. Result is taken 30 minutes after X-gal induction.
As shown in Figure 6,the β-galactosidase activity is at a steady rate from 0 to 10-8 M 3OC12-HSL, and the rate started to increase when the concentration of 3OC12-HSL increased beyond 1.0 X 10-8 M and peaked at 1.0 X 10-5 M, the highest concentration of 3OC12-HSL used. The result suggested that the hydrogel biosensor is able to detect AHL concentration as low as 1.0 X 10-8 M upon colour development for 20 minutes. It is more sensitive when higher concentration of 3OC12-HSL is induced.
Figure 7. Image of biosensor activity towards eight AHL concentration. The biosensor was induced in AHL at 1.0 X 10-5 M(left) to 1.0 X 10-11 M(right) for 2hr. The biosensor without AHL induction(-ve) was used as the negative control. Image is taken 20 minutes after X-gal induction.
As shown in Figure 7, a visible blue colour was developed in the biosensor spot after 2 h of incubation with 1.0 X 10-11 M to 1.0 X 10-5 M respectively while the negative control spot showed a yellow colour representing the biosensor without AHL induction after 20 minutes of X-gal induction. The result suggested that the engineered biosensor is able to detect AHL concentration as low as 1.0 X 10-8 M upon color development for 20 minutes. It was more sensitive when higher concentration of AHL was induced.
Figure 8. Image of biosensor activity towards eight AHL concentration after X-gal induction of 0, 10, 20, 30 and 40 minutes. The biosensor was induced in AHL at 1.0 X 10-5 M(left) to 1.0 X 10-11 M(right) for 2hr. The biosensor without AHL induction(-ve) was used as the negative control.
However, it was noted that the uninduced biosensor showed a blue colour after 20 minutes of X-gal induction. The blue colour development in the control biosensor was obvious after 30 min and 40 min X-gal induction (Figure 8). The colour development time in the negative control limit the detection time, level of gene expression and thus the sensitivity of engineered biosensor. It might be due to the background expression of β-galactosidase in the uninduced biosensor.
Research
From literature review and professors’ interview, we learned that the sensitivity of biosensor can be improved by testing the strength of promoters in order to improve the binding of transcription factor LasR to AHL molecules. Besides, increasing affinity of transcription factor LasR to AHL molecules can also increase the biosensor sensitivity. Inspired by the suggestion, we decided to take an approach to reduce the leaky expression of β-galactosidase in the uninduced biosensor by removing the transcription factor LasR in the sensing module so that no LasR-AHL complex will be formed and no complex will bind on the reporting module and thus no β-galactosidase expression.
Design and Build
The biosensor that was genetically optimized to act as a negative control of the engineered biosensor is composed of reporting module only. It is consisting of the pLasRL inducible promoter upstream of a LacZ coding region. The sensing module is removed to prevent leaky expression of LacZ gene. It was cloned into EcoRI and PstI sites of pSB1C3 vector. The resulting plasmid was named pSB1C3-pLasRL. A synthetic AHL molecule, N-3-oxo-dodecanoyl L-homoserine lactone (3OC12-HSL) was used to determine the specificity and sensitivity of the engineered biosensor.
Test, learn and improve
Figure 9. Image of biosensor activity of pSB1C3-LasR-pLasRL and pSB1C3-pLasRL towards eight AHL concentration. The biosensor was induced in AHL at 1.0 X 10-5 M(left) to 1.0 X 10-11 M(right) for 2hr. The biosensor without AHL induction(-ve) was used as the blank. Image is taken after 40 minutes after X-gal induction.
As shown in Figure 9, increasing shades of blue arise in biosensor cells showed that the β-galactosidase activity of the biosensor increases as the concentration of AHL increase from 1.0 X 10-11 M to 1.0 X 10-5 M. There was no blue colour development in the genetically optimized biosensor which acts as a negative control of the biosensor cells after 40 minutes with AHL added. Thus, we have successfully engineered a control of biosensor which reduce background expression of β-galactosidase. With the genetically optimized biosensor as a control, the blue colour development time will not be restricted by the negative control which allow a higher level of gene expression and thus the sensitivity of the engineered biosensor.
Future prospective
This engineering cycle has led us to engineer a whole-cell biosensor where detection is based on the use of β-galactosidase as the reporter protein for microplastic detection. We also develop an optimized protocol of quantitative X-gal assay to evaluate the biosensing system on the hydrogel-based system. Moreover, we have successfully engineered an optimized biosensor which act as a control of the whole-cell biosensor to reduce background expression of β-galactosidase in the absence of AHL molecule. These data allow us to further characterize the engineered biosensor towards the three AHL molecules and development of a new biosensor with higher sensitivity for microplastic detection.
References
[1] Geyer, R. (2020). Production, use, and fate of synthetic polymers. In Plastic waste and recycling (pp. 13-32). Academic Press.
[2] Gui, Q., et al. The Application of Whole Cell-Based Biosensors for Use in Environmental Analysis and in Medical Diagnostics. Sensors, 2017. 17, DOI: 10.3390/s17071623.