Protecting Linear DNA Templates in Cell-Free Expression Systems from Diverse Bacteria

Sung Sun Yim, Nathan I. Johns, Vincent Noireaux, Harris H. Wang

Research output: Contribution to journalArticlepeer-review

13 Scopus citations

Abstract

Recent advances in cell-free systems have opened up new capabilities in synthetic biology from rapid prototyping of genetic circuits and metabolic pathways to portable diagnostics and biomanufacturing. A current bottleneck in cell-free systems, especially those employing non-E. coli bacterial species, is the required use of plasmid DNA, which can be laborious to construct, clone, and verify. Linear DNA templates offer a faster and more direct route for many cell-free applications, but they are often rapidly degraded in cell-free reactions. In this study, we evaluated GamS from λ-phage, DNA fragments containing Chi-sites, and Ku from Mycobacterium tuberculosis for their ability to protect linear DNA templates in diverse bacterial cell-free systems. We show that these nuclease inhibitors exhibit differential protective activities against endogenous exonucleases in five different cell-free lysates, highlighting their utility for diverse bacterial species. We expect these linear DNA protection strategies will accelerate high-throughput approaches in cell-free synthetic biology.

Original languageEnglish (US)
Pages (from-to)2851-2855
Number of pages5
JournalACS Synthetic Biology
Volume9
Issue number10
DOIs
StatePublished - Oct 16 2020

Keywords

  • Chi
  • GamS
  • Ku
  • RecBCD
  • cell-free expression systems
  • linear DNA

PubMed: MeSH publication types

  • Journal Article
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, Non-U.S. Gov't
  • Research Support, N.I.H., Extramural

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