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 employ this method in active cell lysates developed from ten diverse bacterial species. Interspecies analysis of transcriptional profiles from >1,000 diverse regulatory sequences reveals functional differences in gene expression that can be predictively modeled. Finally, we explore the potential 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.