Part:BBa_K5304004
pnirB::hlyA
The pnirB::hlyA composite part drives the hypoxia-inducible expression of the hlyA gene, encoding listeriolysin O, which is critical for the release of short hairpin RNA (shRNA) into the host cell cytoplasm in a transkingdom RNA interference (tkRNAi) system.
Background
TkRNAi is a bacterial-mediated RNA interference delivery system that enables efficient gene silencing in vitro and in vivo. The tkRNAi system uses genetically modified bacteria to produce and deliver shRNA, which is then processed by the host cell to initiate the RNA interference pathway. The hlyA gene encodes listeriolysin O, a pore-forming toxin derived from Listeria monocytogenes, which facilitates the release of shRNA from the endosome into the cytoplasm by rupturing the endosomal membrane. The pnirB promoter, which is hypoxia-inducible, is used to ensure that hlyA is expressed in low-oxygen environments, such as the tumor microenvironment. This selective expression reduces the likelihood of off-target effects, making this part particularly useful for targeting hypoxic tumors.
Usage and Biology
The pnirB::hlyA part is designed for use in bacterial systems that deliver shRNA for tkRNAi. In this system, genetically engineered bacteria invade target cells and release shRNA into the host cell cytoplasm after bacterial lysis. The hlyA gene product, listeriolysin O, is critical in this process. After the bacteria invade host cells and are enclosed within endosomes, listeriolysin O creates pores in the endosomal membrane, allowing the shRNA to escape into the cytoplasm, where it is processed by the host's RNA interference machinery. The pnirB promoter ensures that hlyA is expressed under hypoxic conditions, such as those found in solid tumors, enhancing the specificity of tkRNAi for cancer therapy. This mechanism minimizes systemic side effects by confining bacterial lysis and shRNA release to the hypoxic tumor environment.
Design
The pnirB promoter was combined with the hlyA gene, encoding listeriolysin O, to create a system where gene expression is triggered under hypoxic conditions, such as in tumor tissues. This composite sequence was then incorporated into the plasmid pSilencer-CLDN6 designed for tkRNAi, enabling efficient delivery and release of shRNA into target cells. This design ensures that bacterial lysis and shRNA release occur specifically in hypoxic environments, optimizing therapeutic targeting and reducing off-target effects.
Figure 1. plasmid pSilencer-CLDN6.
Validation of Listeriolysin O Expression under Hypoxic Conditions
To validate the hypoxia-inducible expression of listeriolysin O driven by the pnirB promoter, Salmonella was cultured under low-oxygen conditions, followed by Western blot analysis to detect listeriolysin O expression. A negative control group, which was not subjected to hypoxic conditions, was included for comparison. For anaerobic treatment, Salmonella was first grown in LB medium, then transferred to a low-oxygen environment prepared using Na₂SO₃ and pre-reduced LB medium. After overnight incubation in an anaerobic tank, bacterial lysates were collected, and the expression levels of listeriolysin O were analyzed by Western blot. The Western blot results showed a significant increase in listeriolysin O expression in the hypoxic group compared to the negative control group, where no or minimal expression was detected. This validates the functionality of the pnirB::hlyA composite part, demonstrating that it effectively induces listeriolysin O expression in low-oxygen environments, such as those found in tumor tissues.
Figure 2. Listeriolysin O Expression under Hypoxic Conditions
Conclusion and Outlook
In conclusion, the validation of listeriolysin O expression under hypoxic conditions confirms the functionality of the pnirB::hlyA composite part. The pnirB promoter reliably induced the expression of listeriolysin O in low-oxygen environments, with minimal expression in the control group, demonstrating the part's responsiveness to hypoxia. Given its specific role in the tkRNAi system, where listeriolysin O is essential for shRNA release into host cells, this part is well-suited for controlled gene silencing applications within hypoxic tumor microenvironments. While its use may be limited to such contexts, this composite part provides an effective solution for targeted shRNA delivery in bacteria-based therapeutic systems.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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