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.

We began by designing the gene circuit diagram for the first-generation p-cresol biosensor (as depicted in Figure 1). This circuit allowed for the emission of fluorescence, which served as a means to monitor the concentration of p-cresol in the environment. After the initial design, we had a discussion with an expert in the field, namely Calin.. These discussions were invaluable in providing insights into the biosensor's performance. During these conversations, it was brought to our attention that the cost of observation could be prohibitively high due to the requirement of a fluorescence microscope for monitoring. Calin recommended exploring alternatives that could reduce costs. To address the cost issue, we explored the concept of a visible color reaction as an alternative to fluorescence observation. Specifically, we looked into the blue-white version screening method. This method involves the introduction of X-Gal in the presence of galactosidase, resulting in a visible blue reaction that can be observed with the naked eye. This approach was considered a promising solution to mitigate the high observation costs associated with green fluorescent protein.

According to the suggestion of Calin, we designed the above gene circuit map（As shown in Figure 2）, and its working principle is similar to that of the first generation. When exogenous X-Gal is added to the sample, if there is p-cresol in the sample, β-galactosidase will be expressed. When β-galactosidase hydrolyzes X-Gal, it will produce blue hydrolysate. The higher the concentration of p-cresol, the darker the blue. This result does not need to be monitored under a fluorescence microscope, but can be monitored by a low-cost spectrophotometer. However, we consulted Professor Robert, who pointed out that the p-Cresol sensor system from Pseudomonas aeruginosa was not suitable for E. coli chassis microbes, and that pchR might be toxic to E. coli. Instead, we want to use Pseudomonas aeruginosa as the chassis microorganism to detect p-Cresol. Because pchR itself comes from Pseudomonas aeruginosa, it should be adaptable. But after we interviewed the doctor, the doctor told us that Pseudomonas aeruginosa is one of the more common clinical pathogens, and Pseudomonas aeruginosa is a class 3 microorganism. As a result, we gave up using p-Cresol sensing promoter as a part.

Considering possible problems during the experiment and in future applications, we abandoned the use of p-Cresol sensing system in Pseudomonas aeruginosa. So the third generation system was built. Tyrosinase can catalyze the conversion of p-cresol to 4-methylquinone, and further oxidation to 4-methyl-o-quinone. In the presence of MBTH(3-methyl-2-phenyl-2h-indazole-6-amine), the substance is rapidly converted into a pink complex that provides a visual indication of the presence or change of cresol concentration, providing an intuitive detection method. To create our biosensor using E. coli as a host, we synthesized the tyrosinase gene (Azenta, USA) and inserted it into the pET23b vector, ensuring the continuous expression of the target protein in E. coli (as depicted in Figure 1). The recombinant vector's fidelity was confirmed through DNA sequencing conducted by Generalbiol in China.

We then transformed the recombinant vector into E. coli Rosetta. The engineered E. coli were cultured overnight in LB medium, followed by centrifugation to collect cell pellets. These pellets were subsequently resuspended in Tris-HCl (pH 7.4). To extract the crude enzyme lysate, we employed ultrasonic lysis under an ice bath (150 W, 1s ultrasound followed by a 3s interval, for a total of 20 minutes).

In testing our biosensors, we prepared solutions of p-Cresol (Merck, Germany), tyrosinase, and MBTH (Merck, Germany) in phosphate buffer (pH 7.1), with extra precautions taken due to the toxicity and corrosiveness of p-Cresol. The reaction was initiated by adding 50 μL of 2 mg/mL crude enzyme solution to a pre-incubated mixture of 50 μL catechol solution and 50 μL of 24 mM MBTH solution at 37°C for 10 minutes. After 20 minutes, we measured the absorbance at 500 nm using an enzyme-linked immunosorbent assay (ELISA) system from Thermo Fisher, USA. The choice of absorbance at 500nm was due to the maximal absorbance of the pink reaction product formed by p-cresol and MBTH （Figure 4）.

To create a biosensor capable of precise environmental concentration monitoring, we conducted linear analysis (as illustrated in Figure 5). Using data collected from p-cresol concentrations ranging from 0 to 500 μM, we established a specific regression equation and performed linearization analysis. The regression equation exhibited a strong fit with the data, confirming an effective linear range for p-cresol concentration between 0 and 500 μM.

For concentrations exceeding 500 μM, while the experimental data remained significant, we postulated that non-linearity could be attributed to two factors: 1) High p-cresol concentrations potentially impact the growth and activity of engineered bacteria, and 2) Limited availability of engineered bacteria may restrict the treatment efficiency at higher p-cresol concentrations.

We also explored the impact of pH on the biosensor by adjusting the pH of the phosphate buffer to 5.5, 6.2, 7.1, and 8.05 (as shown in Figure 6). This analysis aimed to ascertain the operational range and optimal pH for the p-cresol biosensor. The results indicated that the biosensor effectively operated within a pH range of 5.5 to 8.1. At pH 6.2, the biosensor demonstrated the highest efficiency.

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 7，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.

Through experiments, we have learned that the p-cresol biosensor operates most efficiently at a pH of 6.2 and a temperature of 37°C. This information is invaluable as it provides us with the optimal conditions for practical applications, ensuring accurate and reliable results. We have established a well-defined linear range for our biosensor, extending from 0 to 500 μM of p-cresol concentration. Beyond this range, we observed non-linearity, which we attribute to factors such as the potential inhibitory effect of high p-cresol concentrations on engineered bacteria. This insight informs the practical limitations of our biosensor's performance and guides its application in different scenarios.

We encountered challenges related to the compatibility of Escherichia coli as the host organism for our biosensor. We learned from Professor Robert that the p-cresol sensing system from Pseudomonas putida might not be suitable for Escherichia coli due to potential toxicity and compatibility issues. This discovery led us to reconsider our choice of host organism and promoter components.

Consultations with medical professionals emphasized the clinical relevance and potential safety concerns associated with Pseudomonas putida, a pathogenic bacterium. We recognized the importance of responsible biosensor design and decided to abandon the use of the p-cresol sensing promoter from Pseudomonas putida due to these ethical and safety considerations.

By adopting a visible color reaction instead of relying solely on fluorescence, we have learned how to significantly reduce the cost of monitoring. This practical insight allows us to make our biosensor more accessible and economically viable for widespread use.

The gene circuit diagram of the fourth generation is shown in the figure above. The first module is the chromogenic module, and tyrosine will be continuously expressed in the interior of the engineered bacteria. The second module is the lysis module, when arabinose is present in the environment, the permeability of the cell membrane of the engineered bacteria is changed, the cell wall is degraded, and the cell contents are released into the solution. When we culture the engineered bacteria for a period of time, add MBTH and arabinose to the system, it will produce a color reaction, and judge the concentration of p-cresol in the environment by the presence and depth of color.

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.

We first synthesized arabinose promoter and SRRz suicide gene (Azenta, USA), constructed pSB1A3-AraBad vector (Figure 9A), and cloned SRRz gene downstream of arabinose promoter (Figure 9B). The recombinant plasmid was transfected into Escherichia coli Rosetta receptor cells. The engineered Escherichia coli was named after BAD-SRRZ.

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 genetic circuit（As shown in Figure 8). Following this, we transformed the recombinant plasmid into Escherichia coli Rosetta strain with ampicillin selection.

The recombinant strains pBAD-SRRz 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 10）

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.

Subsequently, we inoculated the engineered strains containing chromogenic and lysis dual systems into 5 ml M9 micromedium and cultured them for 12h. Then 1 mM arabinose was added to induce the expression of lytic gene. Studies have shown that SRRz induced by 1 mM Arabinose leads to bacterial lysis (Figure 11A). After overnight culture, we collected 1mm arabinose induced bacterial lysate as a colorimetric reagent. In order to evaluate the colorimetric effect of the bacterial lysate, 50 μL catechol solution with different concentrations and 50 μL 24 mM MBTH solution were pre-incubated at 37℃ for 10 minutes. Then 50 μL bacterial lysate was added to start the reaction. After 20 minutes, we observe the color response.

As shown in Figure 11B, 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.

The Arabitol-controlled SRRz segment represents a powerful tool for selectively controlling bacterial lysis, thereby enabling the release of cellular contents. In our research, we successfully utilized this mechanism to achieve the controlled release of tyrosine enzymes, laying a solid foundation for the application of our cresol biosensor.

At the core of this technology is Arabitol serving as an inducer capable of activating the SRRz protein. Once activated, SRRz acts on the bacterial cell wall, leading to cell lysis and the release of contents. We carefully designed this process to ensure the ordered release of tyrosine enzymes. The release of the tyrosine enzyme not only exhibits precise timing but is also controllable, allowing us to initiate the operation of the cresol biosensor when needed.

Through this approach, we are able to deliver the tyrosine enzyme into a specific reaction environment, facilitating efficient catalysis of cresol. This not only enhances the sensor's performance but also ensures the accuracy and repeatability of the reaction. This strategy not only provides ideal reaction conditions for the cresol biosensor but also opens up new possibilities for future research and applications.

In conclusion, the Arabitol-controlled SRRz segment played a crucial role in our research, offering robust support and innovative methods for the controlled bacterial lysis, ordered release of cellular contents, and enhancement of the performance of the cresol biosensor. The successful implementation of this strategy provides promising directions for future biotechnological research and applications.

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.