Project Description

Peptidator P-800: Pathogens, you've been terminated!

Pathogens, you've been terminated!

This year, we chose to design a novel synthetic platform for high throughput isolation and characterization of peptides with antimicrobial properties (AMPs) to serve as The Terminator for multi-resistant bacterial pathogens. We used the available genomic sequencing data for Carcharodon carcharias (great white shark) as a source of novel peptide sequences that can be used instead of antibiotics. To identify such elements, we established a bioinformatic approach based on the tBLASTn algorithm and known antimicrobial peptides as quarries. We identified nine putative AMPs that successfully fulfilled our criteria for successful heterologous expression in E.coli - 10-30 amino acids in length, monomeric molecules, no unnatural amino acids, no C/N-terminus modifications. We designed expression constructs for them that consist of a T7 promotor, a strong RBS, an ATG start codon, the AMP-coding sequence, a TAA stop codon and a T7 terminator. These constructs were synthesised as gBlocks and cloned into pSB1C3 vector. We used KRX cells kindly provided by Promega, because they allow efficient cloning and T7-driven protein expression without the need for subcloning. Next, we tested the activity of these putative AMPs using different techniques and indicator strains. Meanwhile, we designed an overexpression cassette for AMP production that utilizes the DAMP4 protein fusion partner, masks toxicity and allows cheap and easy purification of the peptide of interest. In addition, we developed a design for a future second version of that cassette that has EDDIE instead of DAMP4 and allows the production of peptides with an intact N-terminus.