Short DNA containing χ sites enhances DNA stability and gene expression in E. coli cell‐free transcription–translation systems

• DOI: 10.1002/bit.26333
• Publication Date: 2017-05-05T13:43:03Z
• Authors: Ryan Marshall; Colin S. Maxwell; Scott P. Collins; Chase L. Beisel; Vincent Noireaux
• Keywords: RecBCD; Synthetic Biology; Transcription; In vitro recombination; Cell-free protein synthesis

Escherichia coli cell-free transcription-translation (TXTL) systems offer versatile platforms for advanced biomanufacturing and prototyping synthetic biological parts devices. Production testing could be accelerated with the use of linear DNA, which can rapidly cheaply synthesized. However, DNA is efficiently degraded in TXTL preparations from E. coli. Here, we show that double-stranded encoding χ sites-eight base-pair sequences preferentially bound by RecBCD recombination machinery-stabilizes greatly enhances TXTL-based expression activity a fluorescent reporter gene, simple regulatory cascades, T7 bacteriophage particles. The χ-site DNA-binding λ protein Gam yielded similar enhancements, as few four sites was sufficient to ensure robust gene TXTL. Given affordability scalability producing short this generalized strategy expected advance broad across its many applications. Biotechnol. Bioeng. 2017;114: 2137-2141. © 2017 Wiley Periodicals, Inc.