(650c) Lego-like Micropillar/Microwell Chip for High-Throughput Functional Analysis of Genes Encoding Pathogen-Specific Antimicrobial Enzymes

The overuse and misuse of antibiotics have resulted in the emergence of antibiotic-resistant pathogens along with the perturbation of healthy human microbiota via killing putatively beneficial commensal bacteria. Hence, targeting infectious bacterial pathogens is important for reducing the evolution of antibiotic-resistant bacteria and preserving the endogenous human microbiome. Cell lytic enzymes including bacteriophage endolysins, autolysins, and peptidoglycan targeting enzymes are useful antibiotic alternatives, and genetic information of numerous cell lytic enzymes is currently available. However, the identification of their antimicrobial function and specificity has been limited due to time-intensive protocols to identify their specific targets. Here, we developed Lego-like sandwich microchip for rapidly accessing the function of diverse genes that are suggestive of encoding cell lytic enzymes. This approach can be used to quantify antimicrobial activity in a high-throughput manner by detecting bacterial cell growth in 40 nL alginate gel spots on micropillar supports. In addition, cell-free protein synthesis could be achieved in a complementary microwell chip. The combination of the chip-based antimicrobial assay with parallel cell-free protein synthesis enabled the rapid identification and evaluation of species-specific antimicrobial function of 11 genes encoding known and putative cell lytic enzymes.