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Biomolecules 2018, 8(4), 137; https://doi.org/10.3390/biom8040137

Genome Editing in Model Strain Myxococcus xanthus DK1622 by a Site-Specific Cre/loxP Recombination System

1
State Key Laboratory of Microbial Technology, Microbiology Technology Institute, Shandong University, Qingdao 266237, China
2
Marine Agriculture Research Center, Tobacco Research Institute of Chinese Academy of Agricultural Sciences, Qingdao 266101, China
3
Research and Development Department, Biotechnology, Uttaranchal University, Dehradun 248007, India
4
Department of Bio-Chemistry, Qingdao Technical College, Qingdao 266555, China
*
Authors to whom correspondence should be addressed.
Received: 31 August 2018 / Revised: 24 October 2018 / Accepted: 25 October 2018 / Published: 6 November 2018
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Abstract

Myxococcus xanthus DK1622 is a rich source of novel secondary metabolites, and it is often used as an expression host of exogenous biosynthetic gene clusters. However, the frequency of obtaining large genome-deletion variants by using traditional strategies is low, and progenies generated by homologous recombination contain irregular deletions. The present study aims to develop an efficient genome-engineering system for this bacterium based on the Cre/loxP system. We first verified the functionality of the native cre system that was integrated into the chromosome with an inducible promoter PcuoA. Then we assayed the deletion frequency of 8-bp-spacer-sequence mutants in loxP by Cre recombinase which was expressed by suicide vector pBJ113 or self-replicative vector pZJY41. It was found that higher guanine content in a spacer sequence had higher deletion frequency, and the self-replicative vector was more suitable for the Cre/loxP system, probably due to the leaky expression of inducible promoter PcuoA. We also inspected the effects of different antibiotics and the native or synthetic cre gene. Polymerase chain reaction (PCR) and sequencing of new genome joints confirmed that the Cre/loxP system was able to delete a 466 kb fragment in M. xanthus. This Cre/loxP-mediated recombination could serve as an alternative genetic manipulation method. View Full-Text
Keywords: Cre/loxP; large fragment deletion; Myxococcus xanthus; loxP spacer mutant; copper inducible promoter; Escherichia coli-Myxococcus shuttle plasmid; genome editing Cre/loxP; large fragment deletion; Myxococcus xanthus; loxP spacer mutant; copper inducible promoter; Escherichia coli-Myxococcus shuttle plasmid; genome editing
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Yang, Y.-J.; Singh, R.P.; Lan, X.; Zhang, C.-S.; Li, Y.-Z.; Li, Y.-Q.; Sheng, D.-H. Genome Editing in Model Strain Myxococcus xanthus DK1622 by a Site-Specific Cre/loxP Recombination System. Biomolecules 2018, 8, 137.

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