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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
Part of the Methods in Molecular Biology book series (MIMB, volume 1642)

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.

Key words

Protein purification Tyrosine recombinase DNA recombination Metabolic engineering Synthetic biology 

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.School of Biological Sciences, The University of EdinburghEdinburghUK
  2. 2.School of Biology, Newcastle UniversityNewcastle upon TyneUK

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