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

Sung Sun Yim, Nathan I. Johns, Jimin Park, Antonio L.C. Gomes, Ross M. McBee, Miles Richardson, Carlotta Ronda, Sway P. Chen, David Garenne, Vincent Noireaux, Harris H. Wang

Research output: Contribution to journalArticlepeer-review

12 Scopus citations

Abstract

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.

Original languageEnglish (US)
Article numbere8875
JournalMolecular Systems Biology
Volume15
Issue number8
DOIs
StatePublished - 2019

Bibliographical note

Funding 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.

Keywords

  • cell-free expression systems
  • gene expression
  • massively parallel reporter assay
  • synthetic biology
  • transcription

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