Difference between revisions of "Part:BBa K4986001"

Latest revision as of 11:48, 12 October 2023

Suicide system: temperature-controlled promoter +SRRZ cutting gene

Considering the need to achieve sufficient efficacy without causing harm to Escherichia coli (E. coli), we have decided to design a targeted suicide gene strategy focused on the bacterial cell wall. The suicide gene system we are utilizing comprises a series of linked genes known as SRRz. We have focused our attention on a specific lambdoid lysis cassette (LC) which typically consist of four genes: S, R, Rz and Rz1. the S gene encodes holin and its inhibitor, the R gene encodes the endolysin, Rz and Rz1 are nested genes that encode proteins involved in resolving the oligopeptide linkages between peptidoglycan (PG) and the Lpp protein .Furthermore, since this gene chain exclusively targets cell wall degradation, it poses no harm to human cells, thereby aligning with biological safety requirements. Temperature-responsive promoter is a special promoter sequence that can regulate gene expression through temperature changes. Temperature-responsive promoters usually contain responsive elements associated with heat shock response. When the cellular environment temperature increases, the heat shock response is activated, thereby activating the function of temperature-responsive promoters and initiating gene expression in their vicinity.

Usage and Biology

We used the brick SRRz as the basis(Figure 1). Use promoter Tcl42 and terminator B0015 to express SRRz gene chain and transformed the plasmids into E. coli BL21 Strain.

Figure1 The design of Tcl42-pR-pL and SRRz.

Characterization

Figure 2 (A) Gel image of Tcl42-pR-pL and SRRz. (B) Map of recombinant plasmid.

Figure3 The result of expression of Tcl42 and SRRz.

To validate the functionality of the Tcl42 temperature-sensitive promoter, we first coupled it upstream of the mRFP reporter gene and cloned it into the pSB1A3 plasmid. Subsequently, we transformed the recombinant plasmid into E. coli DH5α. The transformed strains were cultured at 37°C and 42°C for 12 hours. We measured the initial mRFP fluorescence intensity at these temperatures and normalized it based on OD600 to evaluate the activity of the Tcl42 promoter.As shown in Figure 3A~D, the Tcl42 temperature-sensitive promoter exhibits the best expression at 42°C.

We placed Tcl42 upstream of the bacterial lysis gene SRRz. Firstly, we cloned the Tcl42 promoter together with the SRRz gene into the pSB1A3 plasmid. Subsequently, we transformed this recombinant plasmid into E. coli DH5α bacteria using a heat shock method and selected it on LB agar plates containing 100 µg/mL Ampicillin.To assess the expression of the SRRz gene at different temperatures, we cultured the transformed bacteria at 25°C, 37°C, and 42°C for 12 hours, measuring their OD600 values. Additionally, we included wild-type DH5α and DH5α carrying only the pTcl42 pSB1A3 plasmid as controls. We observed in the results from Figure 3E that the bacterial survival rate under the Tcl42-SRRz condition was the lowest,approximately five times lower than the other two conditions. Furthermore，to assess the effectiveness of the Tcl42 promoter in driving the SRRz gene at 42°C, we cultured the samples in a 42°C shaking incubator for 12 hours. Every 2 hours, we took out 500µL samples and measured their OD600 values to evaluate bacterial growth. Under the same conditions, we also cultured DH5α strains carrying only the pTcl42 pSB1A3 plasmid and wild-type DH5α as controls.（figure3F）The results indicate that under the influence of the pTcl42 pSB1A3 plasmid-carrying DH5α and wild-type DH5α, bacterial survival rates range from 0.4 to 0.5 . However, under the Tcl42-SRRz condition, the survival rate drops significantly to 0.1 which indicate Tcl42-SRRz is the most efficient choice.

Potential application directions

The strain suicide system has extensive application potential. It can be used in areas such as biosafety control, gene editing, drug production, and environmental management, providing important support for biotechnology research and applications.

Reference

[1]Wu M,Nissim L,Stupp D, et al. A high-throughput screening and computation platform for identifying synthetic promoters with enhanced cell-state specificity (SPECS)[J]. Nature Communications,2019,10(1). [2]Leuzzi A,Grossi M,Martino D L M, et al. Role of the SRRz/Rz1 lambdoid lysis cassette in the pathoadaptive evolution of Shigella[J]. International Journal of Medical Microbiology,2017,307(4-5).

Sequence and Features