Difference between revisions of "Part:BBa K5001005"

Latest revision as of 02:58, 12 October 2023

Temperature-controlled promoter + cleavage gene

The basal transcription activity of the gene can be regulated within a specific temperature range by using a temperature-controlled promoter. We chose the pTcl42 promoter because its ideal activation temperature threshold is 42°C, and it has low expression levels at 37°C. MazF is a widely studied toxin-antitoxin (TA) system in Escherichia coli, and its mechanism of action is well defined. MazF can recognize ACA sequences and hydrolyze the phosphodiester bond at the first A position at either the 5' or 3' end, causing ribosome release from cleaved mRNA and preventing protein synthesis. Subsequently, improperly encoded polypeptides are released and degraded by intracellular proteases, leading to cell death. Therefore, we plan to use the temperature-controlled promoter TC1-42 and mazF to induce bacterial lysis.

Usage and Biology

In order to achieve temperature-induced self-lysis in the engineered strain, the pTcl42 promoter was placed upstream of the bacterial lysis gene mazF. Subsequently, the Tcl42 promoter and the mazF gene were cloned together into the pSB1A3 plasmid. The successfully constructed plasmid was transformed into E. coli DH5a bacteria using heat shock method, and recombinant bacteria were screened on LB agar plates containing 100 μg/mL ampicillin.

Characterization

To evaluate the expression of the mazF gene at different temperatures, the transformed bacteria were cultured for 12 hours at 37°C and 42°C, and their OD600 values were measured using a spectrophotometer. Wild-type DH5a and DH5a carrying only the pTcl42 of pSB1A3 plasmid were used as controls. All experiments were performed in triplicate to ensure the reliability of the results. The results are shown in Figure 2A.

To evaluate the time dependence of the bacterial lysis system driven by the pTcl42 promoter at 42°C, a time-course test was performed. The engineered bacteria were added to a 96-well plate and cultured in a shaking incubator at 42°C. Every 2 hours, the OD600 values were measured using a microplate reader to assess bacterial growth. The results are shown in Figure 2B. All experiments were performed in triplicate to ensure the reliability of the results.

Figure 1 Gel electrophoresis of Tcl42-pR-pL and mazF.

Figure 2 The experimental results related to pTcl42-mazF.

The experimental results showed that the temperature-inducible promoter was induced at 42°C, causing almost complete lysis of the bacteria within 12 hours. In the future, we plan to modify common suppositories to have a heating function. When it is necessary to completely kill the engineered bacteria, the heating function of the suppository will be activated to initiate the bacterial suicide system.

Potential application directions

Engineered bacteria with this system could be used as targeted therapeutic agents. They could be administered to patients, and when necessary, an external heating source (e.g., localized heating device or suppository with a heating function) could activate the temperature-controlled promoter to induce bacterial lysis within the body, effectively eliminating the engineered bacteria and release therapautic agent. This could be particularly useful in the treatment of localized infections or tumors. The technology could also be utilized in biocontainment and safety measures for genetically modified organisms (GMOs). By incorporating the temperature-controlled promoter and mazF system, GMOs could be designed to self-destruct when exposed to specific temperatures, providing a fail-safe mechanism in case of accidental release into the environment.

Sequence and Features