p-Cresol is an organic compound with a chemical formula of C7H8O. Its molecular structure includes a hydroxyl group (OH) and a methyl group (CH3). Despite its various industrial and laboratory applications, it is considered a hazardous substance that can have adverse effects on human health. Prolonged exposure to and inhalation of p-Cresol can lead to various adverse reactions, including skin irritation and respiratory irritation. In more severe cases, it may even have detrimental effects on vital organs such as the central nervous system and the liver.

In industrial production and laboratory settings, p-Cresol is typically used as a solvent, raw material, and disinfectant, among other purposes. Therefore, we have decided to develop a biocensor for monitoring p-cresol based on Escherichia coli (E. coli). This biocensor consists of two key components: a colorimetric system and a lysis system.

The colorimetric system involves the catalytic conversion of p-Cresol into 4-methylquinone by tyrosinase, which is further oxidized to 4-methyl-o-quinone. In the presence of MBTH (3-methyl-2-phenyl-2H-indazol-6-amine), this substance rapidly transforms into a pink complex, providing a visual indication of the presence or changes in p-cresol concentration, offering an intuitive detection method.

On the other hand, the lysis system is controlled by the L-arabinose promoter. When L-arabinose is present, the SRRz gene is expressed, causing the engineered strain to lyse, thereby releasing tyrosinase. Besides, the addition of the lysis system also ensures the biosafety of the engineered strains

This E. coli-based biocensor holds promise for the rapid, sensitive, and visual monitoring of p-Cresol and is expected to find widespread applications in industrial and laboratory environments. This technology development will help enhance the safety monitoring of p-Cresol in workplaces and laboratories, reducing the associated health risks associated with prolonged exposure.

To construct a biosensor based on E. coli, we synthesized the tyrosinase gene (Azenta, USA) and cloned it into the pET23b vector, allowing the target protein to be constitutively expressed in E. coli（As shown in Figure 1）. The recombinant vector was verified by DNA sequencing (Generalbiol, China) and then transformed into E. coli Rosetta. The recombinant E. coli were cultured overnight in LB medium, centrifuged to collect cell pellets, and resuspended in Tris-HCl (pH 7.4). Under ice bath, the cells were then ultrasonically lysed (150 W, ultrasound for 1s, interval 3s, for 20 minutes) to obtain crude enzyme lysate. The total protein concentration in the lysate was measured using Bradford assasy kit (Beyotime, China).

To test the capabilities of the biosensors, p-Cresol (Merck, Germany), tyrosinase, and MBTH (Merck, Germany) were dissolved in phosphate buffer (pH 7.1). Due to the toxicity and corrosiveness of p-Cresol, it was prepared in a fume hood. 50 μL of catechol solution and 50 μL of 24 mM MBTH solution were pre-incubated at 37°C for 10 min, then 50 μL of 2 mg/mL crude enzyme solution was added to start the reaction. After 20 minutes, the absorbance at 500 nm was analyzed using an enzyme-linked immunosorbent assay (Thermo Fisher, USA). The absorbance here is chosen because the pink product of the reaction of p-cresol with MBTH has the maximum absorbance at 500nm wavelength. As shown in the Figure 2 above, the absorbance of 500nm increases with the increase of p-cresol concentration, that is, the higher the p-cresol concentration, the thicker the pink of the reaction solution.

Obviously, the curve growth shown in Figure 2 cannot accurately represent the numerical value. In order to design a p-cresol biosensor which can accurately monitor the environmental concentration, we need to carry out linear analysis（As shown in Figure 3). We took the results of p-cresol in the concentration of 0 ~ 500 μ M, obtained the specific regression equation, and carried on the linearization analysis. The regression equation has a high degree of fit to the results, which proves that there is a good linearization range when the concentration of p-cresol is 0 ~ 500 μ M.

For the part with p-cresol concentration above 500 μ M, the experimental data are also considerable, and it is speculated that the reasons for its non-linearity are: (1) high concentration of p-cresol has an effect on the growth and working ability of engineering bacteria; (2) the number of engineering bacteria is limited, and the treatment efficiency of high concentration of p-cresol is limited.

In the reaction system of 50 μ L 100 mM p-cresol solution, 50 μ L 24 mM MBTH solution and 50 μ L 2 mg/mL crude enzyme solution, in order to study the effect of pH on biosensor, we adjusted the pH of phosphate buffer to 5.5,6.2,7.1,8.05. To determine the working range of p-cresol biosensor and the best working pH.

As shown in Figure 4，the results show that the p-cresol biosensor can work normally in the range of pH 5.5-8.1. When pH is 6.2, the efficiency of p-cresol biosensor is the highest.

In the reaction system of 50 μ L 100 mM p-cresol solution, 50 μ L 24 mm MMBTH solution and 50 μ L 2 mg/mL crude enzyme solution, in order to study the effect of temperature on the biosensor, we controlled and maintained the temperature at 25 °C, 37 °C, 45 °C and 55 °C to determine the operating range and optimum operating temperature of p-cresol biosensor.

As shown in figure 5，the results show that the p-cresol biosensor can work normally in the temperature range of 25-55 ℃. When the temperature is 37 ℃, the working efficiency of p-cresol biosensor is the highest, and when the temperature is 25 ℃, the working efficiency is considerable, so there is no need for heating, so the sensor can be used at room temperature.

The SRRz gene cluster is composed of a linked set of genes, including the S gene, the R gene (encoding a soluble transglycosylase enzyme that degrades peptidoglycan in the cell wall), and the RZ gene (encoding an endopeptidase enzyme that cleaves between oligosaccharides in peptidoglycan and crosslinks between peptidoglycan and the outer membrane of the cell). The product of the S gene functions to alter the permeability of the cell membrane, forming a porous structure on the membrane, allowing the enzymes produced by the R and RZ genes to pass through the membrane and reach the cell wall. As a result, the cell wall is acted upon, leading to its rupture and the release of cellular contents. Therefore, the SRRz gene cluster facilitates cell wall disruption.

The Arabinose promoter and SRRz suicide gene (Azenta, USA) was synthesized，then we constructed the pSB1A3-AraBad vector (shown in Figure 6A), and then we cloned the SRRz gene downstream of the arabinose promoter (shown in Figure 6B). The recombinant plasmid was transformed into E. coli Rosetta competent cells.

The recombinant strains were inoculated into LB medium and induced with different concentrations of arabinose to express SRRz at 37°C and 180 rpm. After 12 hours, 1 mL of bacterial culture was collected and the OD600 was measured with a UV spectrophotometer to assess the lysis effect of SRRz on bacterial growth（shown in Figure 7）

The results showed that when there was no arabinose in the environment, the solution OD600 value was slightly more than 1.0; when the arabinose concentration in the environment was 1mM, the solution OD600 value was less than 0.5, the bacterial growth was significantly inhibited, which proved that the SRRz gene was expressed and inhibited the bacterial growth.

In addition to separately validating the coloration and lysis systems, we also undertook the integration of these two components, synthesizing and incorporating them into the pET23b vector to create a complex genetic circuit（As shown in figure 8）. This genetic circuit encompasses multiple elements, each playing a crucial role at specific positions.

The T7 promoter is a robust promoter sequence capable of driving high-level expression of the target gene. It is typically used in conjunction with T7 RNA polymerase for efficient gene transcription. Tyrosinase is used as reporter gene. This allows us to quantitatively evaluate the p-Cresol concentration. The B0015 terminator plays a crucial role in ensuring the proper termination of transcription for the target gene in this genetic circuit. The AraC promoter is another promoter sequence, allows us to modulate the expression of the target gene by adding or removing arabinose. The SRRz gene cassette enables bacteria to undergo cell wall disruption when induced by arabinose.

Following this, we transformed the recombinant plasmid into Escherichia coli Rosetta strain with ampicillin selection. Escherichia coli Rosetta is a specialized strain resistant to multiple codons, making it highly valuable for heterologous protein expression.

We inoculated the engineered bacterial strain into 5 mL of M9 minimal medium and cultured it for 12 hours. Afterward, we added 1 mM arabinose to induce the expression of the lysis genes. It has been demonstrated that SRRz induced by 1 mM arabinose leads to bacterial lysis (Figure 9A). Following overnight cultivation, we collected the bacterial lysate induced with 1 mM arabinose for use as a colorimetric reagent. To assess the colorimetric effect of the bacterial lysate, we pre-incubated 50 μL of various concentrations of catechol solution and 50 μL of 24 mM MBTH solution at 37°C for 10 minutes. Then, we added 50 μL of bacterial lysate to initiate the reaction. After 20 minutes, we observed the color reaction.

As shown in Figure 9B, when the sample does not contain catechol, the solution remains colorless. With an increasing concentration of catechol in the sample, the reaction between catechol and MBTH intensifies, resulting in a deeper color, indicating the activity of the lysis system. As the cell membrane and cell wall rupture, the pink reaction product from within the cells is released into the solution, presenting a visual color that allows experimenters and users to easily observe the experimental phenomenon. This enables the monitoring of catechol concentrations in the environmental surroundings.

Min, Kyoungseon, et al. "A perspective on the biotechnological applications of the versatile tyrosinase." Bioresource technology 289 (2019): 121730.

Leuzzi, Adriano, et al. "Role of the SRRz/Rz1 lambdoid lysis cassette in the pathoadaptive evolution of Shigella." International Journal of Medical Microbiology 307.4-5 (2017): 268-275.