Multiplex transcriptional characterizations across diverse and hybrid bacterial cell-free expression systems

Cell-free expression systems enable rapid prototyping of genetic programs in vitro. However, current throughput of cell-free measurements is often limited by the use of single-channel reporter assays. Here, we describe DNA Regulatory element Analysis by cell-Free Transcription and Sequencing (DRAFTS), a rapid and robust in vitro approach for multiplexed measurement of transcriptional activities from thousands of regulatory sequences in a single reaction. We employed this method in active cell lysates developed from ten diverse bacterial species. Interspecies analysis of transcriptional profiles from u003e1,000 diverse regulatory sequences revealed functional differences in gene expression that could be predictively modeled. Finally, we constructed and examined the transcriptional capacities of dual-species hybrid cell lysates that can simultaneously harness gene expression properties of multiple organisms. We expect that this cell-free multiplex transcriptional measurement approach will improve genetic circuit prototyping in new bacterial chassis for synthetic biology.