High-throughput synthesis and measurement of horizontally transferred regulatory elementsView all posters
Columbia University Medical Center, United States
Horizontal gene transfer (HGT) is known to play a fundamental role in the evolution of microorganisms. HGT likely plays a large role in shaping microbial communities, including those that make up the human microbiome, which has great influence on health and disease. While a great deal of research has been done on the transfer of genes, little research has been done on the transfer of regulatory elements and how they function after acquisition. Here we develop a system for high-throughput measurement of promoter activities after laboratory transformation. A large library (>50,000) of chip-synthesized promoters with barcodes were cloned into species-specific vectors upstream of GFP. The library consists of regulatory sequences from known horizontally transferred genes as well as a comprehensive set selected to cover various COGs, antibiotic resistance genes, virulence factors, and phylogenetic groups. The forced horizontal transfer of the promoter library through transformation into diverse microbial species allows for multiplexed measurement of transcription levels under various growth conditions by RNA-seq. Cells can also be sequenced after FACS to measure translation levels. This approach will allow us to determine how phylogeny, ecology, and other factors affect fate of horizontally transferred regulatory elements. It may also shed light on microbial acquisition of antibiotic resistance and pathogenicity. Additionally, the results also provide us with a large set of promoters characterized in multiple species that should prove useful for synthetic biologists aiming to engineer diverse species and synthetic ecosystems.