Multiplex transcriptional characterizations across diverse bacterial species using cell‐free systems

Cell‐free expression systems enable rapid prototyping of genetic programs in vitro . However, current throughput of cell‐free measurements is limited by the use of channel‐limited fluorescent readouts. 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 promoter activity that can be quantitatively modeled, providing a rich resource for tuning gene expression in diverse bacterial species. Finally, we examine the transcriptional capacities of dual‐species hybrid lysates that can simultaneously harness gene expression properties of multiple organisms. We expect that this cell‐free multiplex transcriptional measurement approach will improve genetic part prototyping in new bacterial chassis for synthetic biology. Synopsis A cell‐free framework (DRAFTS) is developed to quantitatively characterize transcriptional activities of thousands of bacterial regulatory sequences. DRAFTS shows high and robust correlations between in vitro and in vivo transcriptional measurements. Cell‐free systems of 10 diverse bacterial species from three phyla (Proteobacteria, Firmicutes, and Actinobacteria) are generated. Transcriptional capacities of the 10 bacterial species assessed by DRAFTS revealed a divergence of regulatory function as phylogenetic distance increases. Transcription models trained on large‐scale dataset generated by DRAFTS enable assessment of different features for transcriptional activation from regulatory sequences in diverse bacterial species. Graphical Abstract A cell‐free framework (DRAFTS) is developed to quantitatively characterize transcriptional activities of thousands of bacterial regulatory sequences.