US Department of Energy Joint Genome Institute, Lawrence Berkeley National Laboratory, Berkeley, California 94720, United States.
Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, United States.
ACS Synth Biol. 2020 Sep 18;9(9):2610-2615. doi: 10.1021/acssynbio.0c00280. Epub 2020 Aug 13.
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 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 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 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.
发展可持续农业实践需要增加我们对植物-微生物相互作用的理解。为了研究这些相互作用，需要新的遗传工具来操纵非模式微生物。为了满足这一需求，我们最近报道了底盘独立重组酶辅助基因组工程（CRAGE）的发展。CRAGE 依赖于两对互斥 位点之间的盒式交换，允许将大型复杂基因构建体直接、一步到位地整合到多种细菌物种中。然后，我们通过引入第三个互斥 位点扩展了 CRAGE，创建了 CRAGE-Duet，它允许两个构建体的模块化整合。CRAGE-Duet 比 CRAGE 具有优势，特别是在构建单整合构建体时需要繁琐的重新克隆步骤时。为了证明 CRAGE-Duet 的实用性，我们从植物促生根际细菌 WCS417r 中创建了一组表达各种荧光标记基因的菌株。我们在共聚焦显微镜下同时观察这些菌株，证明了 CRAGE-Duet 用于创建用于研究植物-微生物相互作用的生物系统的有用性。
