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Short DNA containing χ sites enhances DNA stability and gene expression in E. coli cell‐free transcription–translation systems
Author(s) -
Marshall Ryan,
Maxwell Colin S.,
Collins Scott P.,
Beisel Chase L.,
Noireaux Vincent
Publication year - 2017
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.26333
Subject(s) - dna , recbcd , biology , synthetic biology , escherichia coli , gene , transcription (linguistics) , in vitro recombination , cell free protein synthesis , base pair , reporter gene , computational biology , microbiology and biotechnology , genetics , gene expression , protein biosynthesis , molecular cloning , linguistics , philosophy
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. © 2017 Wiley Periodicals, Inc.