Chloe S.Y. Yeung, Elly Y. Luo, Adrian Y.L. Chu, Christine K.Y. Yu*
G.T. (Ellen Yeung) College
*Email:
Microplastic pollution
As industrialization spreads worldwide, more and more plastic wastes are produced to fulfill the demand. They are commonly found accumulated in the ocean due to the degradation of plastics into smaller fragments, insufficient sewage treatment, microplastic particles in cosmetic products and fibers like polyester and polyamide. After being consumed by marine animals, the plastic debris enters the food chain which shows dangers to both humans and marine creatures, for example it may cause health impacts like oxidative stress, alteration of metabolism, and pose harm to gut microbiome.
Quorum Sensing System in P. aeruginosa
Pseudomonas aeruginosa (or P. aeruginosa), is a type of gram-negative bacterium which contains a quorum sensing system LuxI/LuxR circuit. The Luxl homolog LasI synthesizes the signal molecule Acyl-homoserine lactones (AHL). The signaling molecule AHL is detected by the cytoplasmic LuxR homolog LasR. The LasR-AHL complex formed activates the transcription of target genes.
P. aeruginosa is a common Pseudomonas species that can 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 occupy it and send out their signaling molecules, AHL, through quorum sensing. These signaling molecules attract more P. aeruginosa onto the microplastic and form a biofilm.
In this study, we engineered a biosensor that expressed lacZ gene, encoding β-galactosidase, which is an enzyme used as a reporter. The detection of blue β-galactosidase activity after incubation with 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) solution acts as a visible readout for the development of biosensors for rapid, sensitive and quantitative detection of microplastic.
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, pLasR 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 3-indolyl-β-D-galactoside (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.
Materials and methods
Preparation of the Whole-cell Biosensor
After receiving the gene synthesized by Synbio Technologies, it is dissolved in a solution with a concentration of 100 μg/μL, kept at -20 °C. Bacterial transformation was performed for DNA cloning, to produce multiple copies of DNA for experiments. The resulting transformed biosensor cells were selected in LB medium with 34 μg/ml chloramphenicol. The presence of the LasR gene was confirmed by Polymerase Chain Reaction (PCR).
Preparation of hydrogel
The 2% hydrogel was made from sodium alginate powder and Milli-Q water. The mixture is then microwaved until the powder is fully dissolved and cooled down to room temperature.
AHL induction
For starter culture, a bacterial colony is picked from the bacterial plate and transferred into 1 mL LB medium with 34 μg/mL chloramphenicol (LCM). It was incubated at 37 °C, 230 rpm overnight.
For bacterial subculture, the starter culture is added to LCM in the ratio of 1:99 starter culture to LCM in 50 mL falcons. The falcon is put to incubate at 33 °C, 220 rpm. It is allowed to grow until the bacterial concentration reaches 0.3 OD600 for AHL induction. At each time point, the bacterial concentration OD600 was measured using a NanoDrop™ One/OneC Microvolume UV-Vis Spectrophotometer. 300 μL samples were collected at each time point.
X-gal induction
After resuspension using hydrogel, 10 μg/μL X-gal substrate solution is carefully added into the 96-well plate. For every 100 μL of X-gal solution, 20 μL of X-gal is added into 80 μL of Milli-Q water and mixed well with pipette up and down. X-gal is highly sensitive to moisture and light. Such that it is wrapped in aluminum foil and stored in -20 °C. It is prepared only before X-gal induction.
Bioimaging – ImageJ
After X-gal induction, pictures of the result need to be taken every 5 minutes to record the colour development. The 96-well plate containing the result was scanned using a smartphone and a CanoScan LiDE 120 scanner. Then the colour intensity is quantified using the software, ImageJ.
Results and discussion
We developed a hydrogel-based assay with the whole-cell biosensor immobilized in the alginate hydrogel. To test the sensitivity of the hydrogel-based assay to detect AHL molecule, the biosensor was induced in different concentration of AHL.
The biosensor was induced in AHL at 1.0 x 10-5 M to 1.0 x 10-11 M for 2 hr, and the blue luminescence production rate is quantified as β-galactosidase activity. The biosensor without AHL induction was used as the negative control. The results are the means of triplicate experiments; error bars indicate the standard deviations. Results were taken 30 minutes after X-gal induction.
As shown in Fig 2, the β-galactosidase activity is at a steady rate from 0 to 10-8 M AHL, and the rate started to increase when the concentration of AHL increased beyond 1.0 x 10-8 and peaked at 1.0 x 10-5, the highest concentration of AHL used. The result suggested that the hydrogel biosensor is able to detect AHL concentration as low as 1.0 x 10-7 M upon color development for 20 minutes. It is more sensitive when higher concentration of AHL is induced.
We also developed a colour strip using a range of AHL standards for semiquantitative detection of AHL in water samples as shown in Fig. 3.
Conclusion
In this study, we have developed a hydrogel-based assay utilizing whole-cell biosensor for microplastic quantification. The biosensor immobilized in hydrogel provides a convenient method for AHL detection where the sensitivity of the biosensor is able to detect AHL concentration as low as 1.0 x 10-7 M. Therefore, it could be a simple and cheap test kit for microplastic in water bodies in the future.
References
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