Latest revision as of 11:45, 12 October 2023

lambda lysis cassette[SRRz/Rz1]

This part causes cell death. It contains holin/antiholin, endolysin and Rz/Rz1 genes.

XHD-WuHan-China 2023

Description

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.

Potential application directions

The part of arabinose-controlled SRRz can be used to artificially control bacterial lysis, thereby releasing cell contents. In our study, we used it to release tyrosinase, thus laying the foundation for the application of p-cresol biosensors.

References

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