Description

Missouri Miners 2024:

Tackling Antibiotic Resistance via Craspase Delivered by Lambda Phages

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Craspase is a RNA-activated protease, which is the combination of a Cas enzyme, gRAMP, and a protease, Csx29. When activated by the binding of an RNA molecule complementary to the guide RNA, the protease is transiently activated, cleaving a specific target protein. In the native host, this substrate is Csx30, which may lead to cell death or dormancy in response to viral infection.

At present, with the rising concern over the impacts of bacteria resistant to topical antibiotics, another method to control bacterial spread such as bacteriophage therapy needs development as an appropriate countermeasure. While antibiotics historically were favored over phage therapies due to their greater efficacy, we now benefit from understanding how bacteriophages function to inject/deliver genetic material and how phages can be altered to specifically target pathogenic bacteria. Furthermore, our understanding of phage reproductive cycles and how to harness their effects has increased. Phages follow a lytic, lysogenic, or temperate life cycle. Whereas lytic phages rapidly reproduce within a host often to lethal effect, lysogenic phages integrate within a host genome as a prophage, which retains the ability to enter a lytic state in the future. Temperate phages fall in between the two reproductive cycles, becoming either lytic or lysogenic when they infect a bacterium. Lytic phages are of greater interest for phage therapy due to their high lethality, although developments in synthetic biology have made it easier to convert a temperate phage into a strictly lytic one.

The Missouri Miners 2024 team aims to utilize a Craspase system delivered through lambda bacteriophages to target and neutralize antibiotic resistant genes in bacteria. Specifically, we plan to utilize a system akin to the modular oncogene targeting system, Proteus, developed collaboratively by McGill University. In lieu of using eukaryotic derived Gasdermins fused with our Craspase system that, upon proteolytic cleavage, create an immune response to lyse and ultimately kill the cell, we plan to deliver Craspase through bacteriophages to target antibiotic resistance coding RNAs in vivo. To both comply with iGEM safety policy and define the scope of our project, solely ampicillin resistance genes will be targeted with the Craspase system. As proof of concept, we plan to edit an E. Coli plasmid to code for our Craspase system, transform the plasmid into the ampicillin resistant bacteria, and monitor the response of the bacteria on agar plates containing ampicillin. Our hope is that this project provides understanding of the value of using phage-mediated delivery and Craspase as a tool to counteract multi-drug resistant bacteria, the proliferation of which can have disastrous implications for infectious disease control in both agricultural and healthcare settings. As a team, we decided on this project because it combines our previous experience in health diagnostics (Missouri Miners 2023) with our passion to safeguard public health. Many members of our team are pursuing careers in medicine and other health professions.