title: Pseudomonas putida KT2440 markerless gene deletion using a combination of λ Red recombineering and Cre/loxP site-specific recombination.
abstract: Pseudomonas putida KT2440 is a saprophytic, environmental microorganism that plays important roles in the biodegradation of environmental toxic compounds and production of polymers, chemicals and secondary metabolites. Gene deletion of KT2440 usually involves cloning of the flanking homologous fragments of the gene of interest into a suicide vector followed by transferring into KT2440 via triparental conjugation. Selection and counterselection steps are then employed to generate gene deletion mutant. However, these methods are tedious and are not suitable for the manipulation of multiple genes simultaneously. Herein, a two-step, markerless gene deletion method is presented. First, homologous armsflanked loxP-neo-loxP was knocked-in to replace the gene of interest, then the kanamycin resistance marker is removed by Cre recombinase catalyzed site-specific recombination. Both two-plasmid and one-plasmid gene systems were established. MekR/PmekA regulated gene expression system was found to be suitable for tight Cre expression in one-plasmid deletion system. The straightforward, time saving and highly efficient markerless gene deletion strategy has the potential to facilitate the genetics and functional genomics study of P. putida KT2440.
authors.affiliation: Jiangsu Key Laboratory for Microbes and Functional Genomics, College of Life Sciences, Nanjing Normal University, Nanjing, Jiangsu Province 210023, China.
authors.forename: Xi
authors.initials: X
authors.lastname: Luo
authors.affiliation: Jiangsu Key Laboratory for Microbes and Functional Genomics, College of Life Sciences, Nanjing Normal University, Nanjing, Jiangsu Province 210023, China.
authors.forename: Yunwen
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Jiangsu Key Laboratory for Microbes and Functional Genomics, College of Life Sciences, Nanjing Normal University, Nanjing, Jiangsu Province 210023, China.
authors.forename: Wen
authors.initials: W
authors.lastname: Ling
authors.affiliation: Jiangsu Key Laboratory for Microbes and Functional Genomics, College of Life Sciences, Nanjing Normal University, Nanjing, Jiangsu Province 210023, China.
authors.forename: Hao
authors.initials: H
authors.lastname: Zhuang
authors.affiliation: Jiangsu Key Laboratory for Microbes and Functional Genomics, College of Life Sciences, Nanjing Normal University, Nanjing, Jiangsu Province 210023, China.
authors.forename: Qin
authors.initials: Q
authors.lastname: Li
authors.affiliation: Jiangsu Key Laboratory for Microbes and Functional Genomics, College of Life Sciences, Nanjing Normal University, Nanjing, Jiangsu Province 210023, China shanggd@hotmail.com.
authors.forename: Guangdong
authors.initials: G
authors.lastname: Shang
mesh_terms: D001709:Biotechnology / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D015870:Gene Expression; D019426:Integrases; D010957:Plasmids; D016958:Pseudomonas putida / Q000235:genetics*; D011995:Recombination, Genetic
chemical_list: C045073:Cre recombinase; D019426:Integrases
keywords: Cre/loxP; MekR/PmekA; Pseudomonas putida KT2440; markerless deletion; λ Red recombineering