Project inspiration

The use of plastic in modern society is inevitable, so are the consequences that come along. When larger pieces of plastics degrade in the ocean, they form smaller microplastics [1], and these little poisonous bits enter our food chain after being consumed by marine animals [2]. As we eat those affected marine organisms, we are indirectly consuming microplastics as well. Microplastics have been shown to cause harm to our body, including harm to gut microbiome and more [3]. The microplastics pollution problem is a pain in the neck for us humans. Our team noticed this problem and after numerous background research and literature reviews, we found that the engineering of whole-cell biosensors based on quorum sensing systems is mainly for biomedical application while the potential of biosensors for detection of microplastic level is unexplored. Thus, we decided to engineer a whole-cell biosensor based on LasI-LasR quorum sensing regulatory circuit in P. aeruginosa to quantitatively detect and monitor microplastics in samples of an area, therefore offers easier identification and tracking of microplastics. This approach is important for policy formulation and prioritizes the allocated resources to address the issue.

How is quorum sensing related to microplastic detection?

Microplastics are plastics that have been shattered by various weathering processes or fabricated into small sizes for specific purposes with diameters of 5mm or less.

Microplastics has been identified as hubs and carriers for microbial pathogens and their antimicrobial resistance (AMR) due to their hydrophobicity, greater surface area and pit forming nature. These pathogenic bacteria form biofilms on the microplastics known as “plastisphere” which in turn facilitates the transfer of AMR-genes (ARGs) via horizontal gene transfer and further escalates the occurrence and levels of AMR [4].

Biofilm formation is governed by the quorum sensing (QS) genes expressed as a result of the accumulation of a critical cell density and is involved in further cell to cell adhesion, maturation and dispersion of biofilm [5]. P. aeruginosa is a common Pseudomonas sps attach to the microplastics and contribute to microplastic-associated AMR. When a colony of P. aeruginosa finds a piece of microplastic in the ocean, they will live on it due to a smooth surface and numerous other factors such as high hydrophobicity [6]. They send out N-acyl-homoserine lactones (AHL) molecules, which are signaling molecules of P. aeruginosa>. These signaling molecules attract more P. aeruginosa> onto the piece of microplastic and forms a biofilm [5]. There is a complete quorum sensing regulatory circuit LasI-LasR in P. aeruginosa>. The LasI synthase constitutively produces the signal molecule 3OC₁₂-HSL, which binds the transcriptional regulator LasR and the resulting LasR-3OC₁₂-HSL complex activates gene transcription. Identification of microplastics in complex environmental matrices remains a challenge. Conventional detection methods requiring chemical or physical analytical techniques that are time-consuming and expensive. Engineered whole-cell biosensors with implemented quorum sensing circuit represent a good alternative for environmental monitoring of microplastics pollution. Therefore, we aimed to apply P. aeruginosa> LasI-LasR circuit to engineer biosensor for the detection of specific AHL molecules. The biosensor will ultimately use to quantitatively measure the presence of AHL molecules in water samples for monitoring microplastic pollution.

How do we design the whole-cell biosensor?

The whole-cell 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 molecule that are secreted from P. aeruginosa. Then the formation of LasR-AHL complex can bind to an inducible promoter, pLasRL or pLasR3 which lead to the activation of the reporting module. A green fluorescent protein-based system is used as the reporting module, in which the expression of GFP would be induced by the LasR-AHL complex. We integrate the LasR sensing module and the reporting module that expressed EGFP to demonstrate the functionality of the whole-cell biosensor in response to the quorum-sensing molecules of the P. aeruginosa>. This highly efficient expression and easy detection of the green fluorescence signal could facilitate the characterization and optimization of the biosensor system.

What is our ultimate goal?

In this study, the highly efficient expression and easy detection of the green fluorescence signal was used to facilitate the characterization and optimization of the biosensor system. Ultimately, we will develop a portable whole-cell paper strip biosensor for dose-dependent on-site monitoring of AHL molecules in a variety of environmental water samples for the detection of microplastic pollution levels. To this end, we will replace the EGFP expression system in the reporting module of our engineered biosensor with other reporter systems such as red lycopene pigment production or blue beta-galactosidase activity production to make the readout easily visible. The intensity of the developed outputs allows semi-quantitative measurements of the AHLs in a variety of environmental samples.

References

• [1] Zhang, K., Hamidian, A. H., Tubić, A., Zhang, Y., Fang, J. K. H., Wu, C., & Lam, P. K. S. (2021). Understanding plastic degradation and microplastic formation in the environment: A Review. Environmental Pollution, 274, 116554. https://doi.org/10.1016/j.envpol.2021.116554
• [2] Cverenkárová, K., Valachovičová, M., Mackuľak, T., Žemlička, L., & Bírošová, L. (2021, December 6). Microplastics in the food chain. MDPI. https://www.mdpi.com/2075-1729/11/12/1349
• [3] Ramasamy, E. V., & Harit, A. K. (2023). Impact of microplastics on human health. Microplastics in Human Consumption, 99–110. https://doi.org/10.1201/9781003201755-5
• [4] Lu, L., Luo, T., Zhao, Y., Cai, C., Fu, Z., & Jin, Y. (2019). Interaction between microplastics and microorganism as well as gut microbiota: A consideration on environmental animal and human health. Science of The Total Environment, 667, 94–100. https://doi.org/10.1016/j.scitotenv.2019.02.380
• [5] Ayush, P. T., Ko, J.-H., & Oh, H.-S. (2022). Characteristics of initial attachment and biofilm formation of pseudomonas aeruginosa on microplastic surfaces. Applied Sciences, 12(10), 5245. https://doi.org/10.3390/app12105245