Abstract
Escherichia coli cell-free transcription–translation (TXTL) systems offer versatile platforms for advanced biomanufacturing and for prototyping synthetic biological parts and devices. Production and testing could be accelerated with the use of linear DNA, which can be rapidly and cheaply synthesized. However, linear DNA is efficiently degraded in TXTL preparations from E. coli. Here, we show that double-stranded DNA encoding χ sites—eight base-pair sequences preferentially bound by the RecBCD recombination machinery—stabilizes linear DNA and greatly enhances the TXTL-based expression and activity of a fluorescent reporter gene, simple regulatory cascades, and T7 bacteriophage particles. The χ-site DNA and the DNA-binding λ protein Gam yielded similar enhancements, and DNA with as few as four χ sites was sufficient to ensure robust gene expression in TXTL. Given the affordability and scalability of producing the short χ-site DNA, this generalized strategy is expected to advance the broad use of TXTL systems across its many applications. Biotechnol. Bioeng. 2017;114: 2137–2141.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 2137-2141 |
| Number of pages | 5 |
| Journal | Biotechnology and bioengineering |
| Volume | 114 |
| Issue number | 9 |
| DOIs | |
| State | Published - Sep 23 2017 |
Bibliographical note
Funding Information:Correspondence to: V. Noireaux Contract grant sponsor: Office of Naval Research Contract grant number: N00014-13-1-0074 Contract grant sponsor: National Institutes of Health Contract grant number: 1R35GM119561-01 Contract grant sponsor: Defense Advanced Research Projects Agency Contract grant number: HR0011-16-C-01-34 Received 9 February 2017; Revision received 14 April 2017; Accepted 30 April 2017 Accepted manuscript online 5 May 2017; Article first published online 23 May 2017 in Wiley Online Library (http://onlinelibrary.wiley.com/doi/10.1002/bit.26333/abstract). DOI 10.1002/bit.26333
Funding Information:
V.N. thanks Meriem El Karoui (University of Edinburgh) for discussions about RecBCD and x sites. V.N. and C.L.B. thank Cold Spring Harbor Laboratory (CSHL). Preliminary experiments using x sites in TXTL were performed during the 2016 CSHL Synthetic Biology summer course. This material is based upon work supported by the Defense Advanced Research Projects Agency (contract HR0011-16-C-01-34, V.N. and C.L.B.), the Office of Naval Research (award N00014-13-1-0074, V.N.), and the National Institutes of Health (grant 1R35GM119561-01, C.L.B.).
Funding Information:
V.N. thanks Meriem El Karoui (University of Edinburgh) for discussions about RecBCD and ? sites. V.N. and C.L.B. thank Cold Spring Harbor Laboratory (CSHL). Preliminary experiments using ? sites in TXTL were performed during the 2016 CSHL Synthetic Biology summer course. This material is based upon work supported by the Defense Advanced Research Projects Agency (contract HR0011-16-C-01-34, V.N. and C.L.B.), the Office of Naval Research (award N00014-13-1-0074, V.N.), and the National Institutes of Health (grant 1R35GM119561-01, C.L.B.).
Publisher Copyright:
© 2017 Wiley Periodicals, Inc.
Keywords
- Escherichia coli
- RecBCD
- TXTL
- gene circuits
- prototyping
- synthetic biology