Bacteriocin Production and Screening Using Cell-Free Protein Synthesis

Extensive use of antibiotics in the community and hospitals has resulted in the occurrence of multidrug-resistant bacteria that have been significant threats. In the United States, at least 2 million people are suffered from the infection from bacteria which have resistance to one or more antibiotics, and 23,000 people die by the results of direct infection of these pathogens in each year. As antibiotics are treated, bacteria rapidly develop the ways to protect themselves by regulating intrinsic antibiotic resistance genes or acquiring new genes from other bacteria that are already equipped the antibiotic resistance mechanisms. Therefore, novel strategies are required to effectively eradicate antibiotic resistant bacteria. Bacteriocins are antimicrobial peptides produced by bacteria and exhibit narrow or broad spectrum of killing other bacteria. These peptides have great potential as alternatives of traditional antibiotics, since the killing mechanisms of bacteriocins are different from those of antibiotics: bacteriocins kill cells via pore-forming on the membrane, nuclease activity, and inhibiting peptidoglycan production, while antibiotics usually kill cells via inhibiting transcription and translation. In this study, we utilize cell-free protein synthesis (CFPS) platforms to produce colicin, a type of bacteriosin, which is toxic to Escherichia coli strains. CFPS systems are not constrained by cell-viability requirements and are therefore impervious to the cytotoxic nature of bacteriocins. We generated colicin variant library via error-prone PCR and synthesized using a CFPS platform. We will assess their efficacy of killing E. coli strains including pathogenic E. coli. We will discuss developing cell-free high-throughput screening methods for peptide-based drugs. The rapid design-build-test cycle of CFPS platforms provides a time and cost advantage in screening antimicrobial peptides and have phenomenal potential to open up a new area of pharmaceutical biotechnology.