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.
Bibliographical noteFunding Information:
We thank members of the Wang Lab for advice and comments on the manuscript. H.H.W. acknowledges funding support from NSF (MCB-1453219), NIH/NIGMS (U01GM110714-01A1), NIH/NIAID (1R01AI132403-01), DARPA (HR0011-17-C-0068), DoD ONR (N00014-15-1-2704), and the Sloan Foundation (FR-2015-65795). V.N. acknowledges funding support from DoD ONR (N00014-13-1-0074) and Human Frontier Science Program (RGP0037/2015). S.S.Y. thanks support from Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education (NRF-2017R1A6A3A03003401). N.I.J. is supported by an NSF Graduate Research Fellowship (DGE-1644869). We also thank K.J. Jeong (KAIST, Daejeon, Korea) for providing pCES208 plasmid.
- cell-free expression systems
- gene expression
- massively parallel reporter assay
- synthetic biology