CRAGE-Duet Facilitates Modular Assembly of Biological Systems for Studying Plant-Microbe Interactions

Bing Wang, Zhiying Zhao, Lauren K. Jabusch, Dawn M. Chiniquy, Koyo Ono, Jonathan M. Conway, Zheyun Zhang, Gaoyan Wang, David Robinson, Jan Fang Cheng, Jeffery L. Dangl, Trent R. Northen, Yasuo Yoshikuni

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

Abstract

Developing sustainable agricultural practices will require increasing our understanding of plant-microbe interactions. To study these interactions, new genetic tools for manipulating nonmodel microbes will be needed. To help meet this need, we recently reported development of chassis-independent recombinase-assisted genome engineering (CRAGE). CRAGE relies on cassette exchange between two pairs of mutually exclusive lox sites and allows direct, single-step chromosomal integration of large, complex gene constructs into diverse bacterial species. We then extended CRAGE by introducing a third mutually exclusive lox site, creating CRAGE-Duet, which allows modular integration of two constructs. CRAGE-Duet offers advantages over CRAGE, especially when a cumbersome recloning step is required to build single-integration constructs. To demonstrate the utility of CRAGE-Duet, we created a set of strains from the plant-growth-promoting rhizobacterium Pseudomonas simiae WCS417r that expressed various fluorescence marker genes. We visualized these strains simultaneously under a confocal microscope, demonstrating the usefulness of CRAGE-Duet for creating biological systems to study plant-microbe interactions.

Original languageEnglish (US)
Pages (from-to)2610-2615
Number of pages6
JournalACS Synthetic Biology
Volume9
Issue number9
DOIs
StatePublished - Sep 18 2020
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biomedical Engineering
  • Biochemistry, Genetics and Molecular Biology (miscellaneous)

Keywords

  • CRAGE
  • Cre-lox recombination
  • bacterial strain engineering
  • fluorescent protein
  • genome editing
  • genome engineering

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