Part:BBa_K4994002
Arabinose promoterr+SRRz cassette
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). 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. In addtion，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.
Usage and Biology
Figure 1: Gel electrophoresis images of AraBAD promoter and SRRz bacterial lysis cassete.
The Arabinose promoter and SRRz suicide gene (Azenta, USA) was synthesized，then we constructed the pSB1A3-AraBad vector (shown in Figure 1A), and then we cloned the SRRz gene downstream of the arabinose promoter (shown in Figure 2B). 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 2）
Figure 2 A: The working result of lysis system; B: Difference analysis of experimental results.
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.
We also designed a complex genetic circuit (shown in Figure 3). The gene circuit consists of two systems, system 1 is based on tyrosinase chromogenic system and system 2 is based on SRRz cleavage 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. This genetic circuit contains multiple elements, each of which plays a crucial role in a specific location.
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 synthesized the gene circuit and transformed the recombinant plasmid based pET23b into Escherichia coli Rosetta strain.
Figure 3: Design of gene circuit of chromogenic and lysis dual system.
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 4A). 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.
Figure 4: Changes in Bacterial Growth Curve; B: Colorimetric Assay of Bacterial Lysis Supernatant.
As shown in Figure 4B, 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.
Potential application directions
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.
References
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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1144
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 979
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 961
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