Using purified tyrosine site-specific recombinases in vitro to rapidly construct and diversify metabolic pathways

Wei Liu, Laura R. Tuck, Jon Marles Wright*, Yizhi Cai

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

The site-specific recombinase Cre was previously reported to have in vitro activity. Here, we describe the method of purifying two new tyrosine site-specific recombinases VCre and Dre along with Cre by nickel affinity chromatography. We proved the in vitro function of the VCre and Dre on their respective conditional recombination sites. We also developed a methodology to one-step construct and optimize the productivity of a biosynthetic pathway through the combinatorial integration of promoters into a plasmid-encoded pathway by simply incubating a DNA mixture with recombinase system at 37 °C in vitro.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press
Pages285-302
Number of pages18
ISBN (Electronic)978-1-4939-7169-5
ISBN (Print)978-1-4939-7167-1
DOIs
Publication statusPublished - 17 Aug 2017

Publication series

NameMethods in Molecular Biology
Volume1642
ISSN (Print)1064-3745

Keywords / Materials (for Non-textual outputs)

  • DNA recombination
  • metabolic engineering
  • protein purification
  • synthetic biology
  • tyrosine recombinase

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