iGEM 2025 — Team hkgtc

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## March 2024

* First round of iGEM team members recruitment, Project brainstorm, Literature review

## April 2024

* Project brainstorm, literature review, Environmental Justice Interactive video preparation and making, Education Booklet preparation, Board Game design

## May 2024

* Confirmed construct design - pSB1C3-LasR-pLasRL-LacZ (#1)

## July 2024

* Development of microplastic datasets for AI training
* 10/7: Transformation of PSB1C3-LasR-LasRL-LacZ into DH5α and BL21
* 11/7: Colony PCR to confirm positive clones
* 12/7: Pre-trials
* 16/7: Protocol development using different X-gal concentration
* 17/7, 23/7: Protocol development using different optical density (OD) of biosensor
* 24/7: Protocol development using different optical density (OD) of biosensor and the use of ImageJ
* 26/7: Protocol development using different AHL (3OC12-HSL) concentrations

## August 2024

* Comparison and evaluation of the performance of Mask-RCNN and YOLOv8
* 6/8: Protocol development of cell washing
* ⁠9-14/8: Sensitivity test of biosensor using a range of AHL (3OC12-HSL) concentrations (colour strip)

## February 2025

* Literature review

## March 2025

* Literature review

## April 2025

* Confirmed constructs design - pSB1C3-pLasRL-LacZ (#2), pSB1C3-LasR-pLasR3-LacZ (#3) and pSB1C3-pLasRL-LacZ (#4)
* 13/4: ZPL x iGEM Beach Cleanup Pre-fieldtrip
* 26/4: ZPL x iGEM Beach Cleanup

## May 2025

* 21/5: iGEM student advisors gave advice and experience to new research team students about experiments conduction
* 27/5: Youth Centre Sharing Session
* 28/5: Interview with Dr. David Jones from Just One Ocean

## July 2025

* 3/7: Sharing Session in G.T. (Ellen Yeung) College – Secondary School
* 8/7: Sharing Session in G.T. (Ellen Yeung) College – Primary School, Interview with Prof. Jose Jimenez from Imperial College London
* 9/7: Sharing Session in Queen Elizabeth School, Interview with Prof. Ville Santala from Tempere University
* 14/7: Interview with Environmental Protection Department (EPD), HKSAR
* 25/7: Trial experiments using pSB1C3-LasR-LasRL-LacZ (#1)

## August 2025

* Microplastic datasets making, AI training and Robotic car design and making
* 4/8: A visit to Green@SaiKung, Bacterial transformation of #2-4
* 5/8: Colony PCR to confirm positive clones of #2-4
* 7/8: AHLs (C10-HSL & 3OC12-HSL) induction with construct #3&4
* 12/8: AHLs (C4-HSL & C10-HSL) induction with construct #1&2
* 23/8: AHLs (C4-HSL, C10-HSL, 3OC12-HSL) induction with construct #3&4
* 26/8: AHLs (C4-HSL, C10-HSL, 3OC12-HSL) induction with construct #1&2
* 28/8: AHLs (C4-HSL, C10-HSL, 3OC12-HSL) induction with construct #3&4, Survey for exhibition in G.T. (Ellen Yeung) College
* 30/8: AHLs (C4-HSL, C10-HSL, 3OC12-HSL) induction with construct #1&2

## September 2025

* 3/9: Survey for Education booklet
* 4/9: AHLs (C4-HSL, C10-HSL, 3OC12-HSL) induction with construct #1&2
* 6/9: AHL (3OC12-HSL) induction with construct #3&4 with OD of 0.3, 0.6, 0.9
* 8, 16 & 30/9: Data Science Experience Course for G7 – G11 G.T. students
* 12/9: Board Game Trial Experience
* 13/9: AHL (3OC12-HSL) induction with construct #3&4 using tap water
* 16/9: AHL (3OC12-HSL) induction with construct #1-4
* 18/9: AHL (3OC12-HSL) induction with construct #3&4 using tap water using OD of 0.7
* 20/9: AHL (3OC12-HSL) induction with construct #3&4 using tap water
* 26/9: Project Exhibition in G.T. (Ellen Yeung) College, Green Event – Clean recycling
* 30/9: AHL (3OC12-HSL) induction with construct #3&4 using seawater and tap water

## October 2025

* 2/10: AHL (3OC12-HSL) induction with construct #3&4 with seawater and tap water
* 3/10: Green Event – Clean recycling
* 6/10: Sharing Session in Salesian Yip Hon Primary School

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#### 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.

![](https://static.igem.wiki/teams/5815/engineering-success/screenshot-2025-10-05-102754.webp)

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.

![](https://static.igem.wiki/teams/5815/engineering-success/screenshot-2025-10-05-102834.webp)

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.

![](https://static.igem.wiki/teams/5815/engineering-success/screenshot-2025-10-05-102844.webp)

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.

![](https://static.igem.wiki/teams/5815/engineering-success/screenshot-2025-10-05-102855.webp)

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.

![](https://static.igem.wiki/teams/5815/engineering-success/screenshot-2025-10-05-102903.webp)

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.

![](https://static.igem.wiki/teams/5815/engineering-success/screenshot-2025-10-05-102911.webp)

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.

![](https://static.igem.wiki/teams/5815/engineering-success/screenshot-2025-10-05-102921.webp)

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.

![](https://static.igem.wiki/teams/5815/engineering-success/screenshot-2025-10-05-102931.webp)

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

![](https://static.igem.wiki/teams/5815/engineering-success/screenshot-2025-10-05-102948.webp)

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.

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### Transformation

After receiving the gene synthesized by Synbio Technologies, 2 μg of the powder in the vial was
dissolved with 20 μL of MilliQ water, forming a solution with a concentration of 100 μg/μL.
It is kept at -20°C. Bacterial transformation was performed in order to produce multiple copies
of DNA for experiments.

1. Mix 1 μL of the plasmid DNA with 200 μL of *E. coli* BL21 cells.
2. Put the bacterial mixture in ice for 30 minutes.
3. Incubate the bacterial mixture at 42°C for 90 seconds.
4. Put in the ice again for 1-2 minutes.
5. Add 1mL of LB medium to the bacterial mixture.
6. Incubate the mixture at 37°C at 200 rpm for 1 hour.
7. Plate 200 μL and all of the bacterial mixture onto a LB agar plate containing 34 mg/mL chloramphenicol.
8. Incubate the plates at 37°C overnight.

### Colony PCR

1. Set up the following reaction mixture:

   | Mastermix Components (for 11 reactions) | μL |
   | --- | --- |
   | 2× EasyTaq | 5.5 |
   | Forward primer | 2.2 |
   | Reverse primer | 2.2 |
   | Distilled water | 39.6 (variable) |
   | DNA template | variable |
    Total volume | 110 |

2. Aliquot 9 μL into each tube. Pick bacterial colonies with a sterile tip and add them to each
   reaction mix.

3. PCR programme was set as follow:
   1. DNA samples were first heated at 95°C for 5 minutes, and then the cycle began.
   2. Reaction mixture was heated to a temperature of 95°C for 30 seconds to denature.
   3. The samples were annealed at a temperature of 55°C for 30 seconds.
   4. The temperature was then increased to 72°C for 1 minute for DNA extension and complete one cycle.
   5. The cycle was repeated 30 times.
   6. After the cycle, the DNA was incubated at 72°C for 5 minutes.
   7. The DNA was stored at 4°C.

### AHL preparation

We have ordered 3 synthetic AHL molecules from commercial companies, named N-butyryl L-
homoserine lactone (C4HSL), N-3-oxo-decanoyl L-homoserine lactone (C10HSL) and N-3-oxo-
dodecanoyl L-homoserine lactone (3OC12-HSL). The AHL is prepared by dissolving it in DMSO to
result in a 10mM stock solution and the Eppendorf is labelled as tube 1.

|  | Molecular Mass (g/mol) | Volume of DMSO used to make a 10 mM stock solution (μL) |
| --- | --- | --- |
| 30C4-HSL | 171.2 | 584.1 |
| 3OC10-HSL | 269.3 | 371.3 |
| 3OC12-HSL | 297.3 | 336.3 |

The preparation of AHLs in seven concentrations is done by serial dilution. 9 eppendorfs are prepared
with 450 μl milliQ water each, each is diluted with 50 μl of the previous eppendorf.

| Eppendorf | AHL Concentration |  |
| --- | --- | --- |
| 2 | 10-5 | 10 mM |
| 3 | 10-6 | 1.0 mM |
| 4 | 10-7 | 0.1 mM |
| 5 | 10-8 | 0.01 mM |
| 6 | 10-9 | 0.001 mM |
| 7 | 10-10 | 0.0001 mM |
| 8 | 10-11 | 0.00001 mM |
| 9 | 10-12 | 0.000001 mM |

### Bacterial Subculture

1. Inoculate a single colony of the biosensor into 5 mL of LB broth with 34 mg/mL chloramphenicol and incubate overnight in a shaking incubator at 37°C and 210 rpm.
2. Transfer 4 mL overnight culture into 100 mL LB broth with 34 mg/mL chloramphenicol and incubate in a shaking incubator at 37°C and 220 rpm until OD600 = 0.3.
3. 2 mL of cell culture was then transferred into a 15 mL culture tubes for AHL induction with three synthetic AHLs (3OC12-HSL, C10-HSL and C4-HSL respectively) at seven concentrations (1.0 × 10-5, 1.0 × 10-6, 1.0 × 10-7, 1.0 × 10-8, 1.0 × 10-9, 1.0 × 10-10, 1.0 × 10-11M). The culture tube with MilliQ water added was used as a negative control.

### AHL Induction

1. Add 2 mL of cell cultures with 20 μL of three synthetic AHLs (3OC12-HSL, C10-HSL and C4-HSL respectively) at seven concentrations (1.0 × 10-5, 1.0 × 10-6, 1.0 × 10-7, 1.0 × 10-8, 1.0 × 10-9, 1.0 × 10-10, 1.0 × 10-11M). The culture tube with 20 μL of MilliQ water added was used as a negative control.
2. To test the effect of tap water and sea water on the performance of the engineered biosensors, 0.2 mL of tap water and sea water spiked with 20 μL 3OC12-HSL, C10-HSL and C4-HSL were added to culture tubes containing 1.8 mL of cell cultures.
3. Incubate culture tubes in a shaking incubator at 37°C and 220 rpm for 2 h.

### Washing Cells with Phosphate Buffered Saline (PBS)

1. Add three aliquots of the 300 μL culture into the Eppendorf.
2. Centrifuge the cells at 13,500 rpm for 2 minutes to collect the cell pellets.
3. Discard the supernatant and wash the cells with 500 μL 1× PBS.
4. Vortex and centrifuge for 2 minutes.
5. Repeat steps 2-4 once.
6. Discard all the supernatant to collect the cell pellets.

### Hydrogel Preparation and Resuspension

1. Add 98 mL milliQ water to 2 g of sodium alginate powder.
2. Heat the mixture until the powder is completely dissolved.
3. Let the hydrogel cool down at room temperature.
4. Add 200 μL hydrogel to each pellet.
5. Pipette up and down to mix well.
6. Transfer the hydrogel with biosensor cells to a transparent 96-well plate.

### X-gal Induction

1. Add 10 μL of 10 mg/mL X-gal solution into each well and mix well.
2. Incubate the plate at room temperature to allow blue color development.
3. Take photos of the 96-well plate in five minute intervals.

### Obtaining Data Using ImageJ

Download ImageJ from <https://imagej.net/ij/download.html> and Readplate 3.0 from <https://forum.image.sc/t/readplate-3-0-release/42008>.

1. Set the contrast and saturation of the image to 100 to articulate subtle differences in color intensity.
2. Open the ImageJ software. Go to "Analyze" → "Set Measurements". Select "Area", "Mean gray value", "standard deviation", "modal gray value" and "Min & max gray value". Select the results to be 5 decimal places. These are selected to improve the accuracy of quantifying the color intensity even if the color is unevenly distributed in a well.
3. "File" → "Open" and choose the image from your computer.
4. Drag the rectangle such that the vertices of the rectangle are on the center of the wells A1, A12, H1 and H12.
5. "Plugins" → "Macros" → "Run"
6. From "Readplate\_wr", select "source code" → "Readplate 3.0". Select 96 for the number of wells.
7. Select "Red" for "Channel"
8. For the "parameters", use the default settings.
9. Check whether the grid generated by the software fits each well. If the grid is at the center of each well, and the color intensity is evenly distributed in each well, click "OK". Else, click "cancel" and increase the "Diameter of main circle (in pixels)" in Step 8.
10. Click "OK" in "Checking Results", then, select "Yes" in save results.
11. Name your file with the type of document as .csv or .xlsx to compile a spreadsheet.

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In this study, we engineer a set of whole-cell biosensors for rapid and quantitative detection of microplastics in water samples of an area based on the quorum sensing regulatory circuit LasI-LasR in *P. aeruginosa*, a common *Pseudomonas* sps attach to the microplastics and contribute to microplastic-associated antimicrobial resistance [1]. The LasI synthase constitutively produces signal molecule, N-acylhomoserine lactone (AHL) which binds to the transcriptional regulator LasR and the resultant LasR-AHL complex activates gene transcription. AHLs are quorum sensing signal molecules involved in cell-to-cell communication that help in the formation of biofilm on microplastics [1]. This whole cell biosensor contains a *lacZ* gene as the reporter gene, encoding β-galactosidase. The detection and quantification of blue β-galactosidase activity after incubation in an 5-bromo-4-chloro-3-indolyl-b-galactoside (X-gal) solution act as a visible readout for the amount of quorum sensing signalling molecules, AHLs which ultimately can be used for rapid, sensitive and quantitative detection of microplastics. The important findings of our study are as below.

#### **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.

![](https://static.igem.wiki/teams/5815/results/fig-1.webp)

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.

![](https://static.igem.wiki/teams/5815/results/fig-2.webp)

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.

![](https://static.igem.wiki/teams/5815/results/fig-3.webp)

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.

![](https://static.igem.wiki/teams/5815/results/fig-4.webp)

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.

![](https://static.igem.wiki/teams/5815/results/fig-5.webp)

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.

![](https://static.igem.wiki/teams/5815/results/fig-6.webp)

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**

![](https://static.igem.wiki/teams/5815/results/fig-7.webp)

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.

![](https://static.igem.wiki/teams/5815/results/fig-8.webp)

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.

![](https://static.igem.wiki/teams/5815/results/fig-9.webp)

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.

![](https://static.igem.wiki/teams/5815/results/fig-10.webp)

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).

![](https://static.igem.wiki/teams/5815/resultsnew/screenshot-2025-10-09-160835.avif)
![](https://static.igem.wiki/teams/5815/resultsnew/screenshot-2025-10-09-160902.avif)

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.

![](https://static.igem.wiki/teams/5815/results/fig-12.webp)

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.

![](https://static.igem.wiki/teams/5815/results/fig-13.webp)

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.

![](https://static.igem.wiki/teams/5815/resultsnew/fig-14-a.avif)
![](https://static.igem.wiki/teams/5815/resultsnew/fig-14-b.avif)

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.

![](https://static.igem.wiki/teams/5815/results/fig-15.webp)

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.

![](https://static.igem.wiki/teams/5815/resultsnew/fig-16-a.avif)
![](https://static.igem.wiki/teams/5815/resultsnew/fig-16-b.avif)
![](https://static.igem.wiki/teams/5815/resultsnew/fig-16-c.avif)
![](https://static.igem.wiki/teams/5815/resultsnew/fig-16-d.avif)

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.

![](https://static.igem.wiki/teams/5815/resultsnew/fig-17-a.avif)
![](https://static.igem.wiki/teams/5815/resultsnew/fig-17-b.avif)
![](https://static.igem.wiki/teams/5815/resultsnew/fig-17-c.avif)
![](https://static.igem.wiki/teams/5815/resultsnew/fig-17-d.avif)

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.

![](https://static.igem.wiki/teams/5815/results/fig-18-part-1.webp)
![](https://static.igem.wiki/teams/5815/results/fig-18-part-2.webp)

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.

![](https://static.igem.wiki/teams/5815/resultsnew/fig-19-a.avif)
![](https://static.igem.wiki/teams/5815/resultsnew/fig-19-b.avif)
![](https://static.igem.wiki/teams/5815/resultsnew/fig-19-c.avif)
![](https://static.igem.wiki/teams/5815/resultsnew/fig-19-d.avif)
![](https://static.igem.wiki/teams/5815/resultsnew/fig-19-e.avif)
![](https://static.igem.wiki/teams/5815/resultsnew/fig-19-f.avif)

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.

![](https://static.igem.wiki/teams/5815/resultsnew/fig-20-a.avif)
![](https://static.igem.wiki/teams/5815/resultsnew/fig-20-b.avif)

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.

#### References

[1]. K. Kaur, S. Reddy, P. Barathe, U. Oak, V. Shriram, S. S. Kharat, et al. (2022)
Microplastic-associated pathogens and antimicrobial resistance in environment. Chemosphere Vol. 291 Pages 133005

[2]. Y. Wu, C.-W. Wang, D. Wang and N. Wei. (2021) A Whole-Cell Biosensor for Point-of-Care Detection of Waterborne Bacterial Pathogens. *ACS Synthetic Biology* Vol. 10 Issue 2 Pages 333-344

[3]. J. Chappell, K. Jensen and P. S. Freemont. (2013) Validation of an entirely in vitro approach for rapid prototyping of DNA regulatory elements for synthetic biology. Nucleic Acids Research 2013 Vol. 41 Issue 5 Pages 3471-3481

[4]. P. T. Ayush, J.-H. Ko and H.-S. Oh. (2022) Characteristics of Initial Attachment and Biofilm
Formation of *Pseudomonas aeruginosa* on Microplastic Surfaces. *Applied Sciences* Vol. 12
Issue 10 Pages 5245

[5]. K. Zhang, M. C. Y. Cheng, M. Liu, S. Xu, Y. Ma, H. S. Chau, et al. (2024). Microplastics in Hong Kong's marine waters: Impact of rainfall and Pearl River discharge. Marine Pollution Bulletin 2024 Vol. 205 Pages 116635

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