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Guangdong Shang - One of the best experts on this subject based on the ideXlab platform.
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recombineering and i scei mediated pseudomonas putida kt2440 scarless Gene Deletion
Fems Microbiology Letters, 2016Co-Authors: Zhongqiu Chen, Wen Ling, Guangdong ShangAbstract:: Pseudomonas putida KT2440 is a saprophytic and Generally recognized as safe microorganism that plays important roles in the biodegradation and production of value-added chemicals. Chromosomal Gene Deletion of P. putida KT2440 usually involves time-consuming Gene cloning, conjugal transfer and counterselection. Recently, we developed a P. putida KT2440 markerless Gene Deletion method based on recombineering and Cre/loxP site-specific recombination. PCR-based λ Red recombineering circumvents the tedious cloning steps and is more amenable to high-throughput manipulation. Here we report an improved scarless Gene Deletion strategy based on recombineering and intron-encoded homing endonuclease I-SceI-mediated double-strand break repair. Sixteen drug exporter Gene(s) were deleted and the minimal inhibition concentrations of the mutants to a variety of antibiotics were determined. The robustness of the procedure was also demonstrated by sequential Deletion of five large genomic regions. Up to 59% recombination efficiency was achieved for a 54.8 kb Deletion, and the efficiency of RecA-mediated double-strand break repair, which was boosted by λ Red recombinase, was nearly 100%. The strain with a 3.76% genome reduction showed an improved growth rate and transformation efficiency. The straightforward, time-saving and highly efficient scarless Deletion approach has the potential to facilitate the Genetic study, and biotechnological and environmental applications of P. putida KT2440.
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pseudomonas putida kt2440 markerless Gene Deletion using a combination of λ red recombineering and cre loxp site specific recombination
Fems Microbiology Letters, 2016Co-Authors: Yunwen Yang, Wen Ling, Hao Zhuang, Qin Li, Guangdong ShangAbstract: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 tri-parental 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. Firstly, homologous arms flanked 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.
Wen Ling - One of the best experts on this subject based on the ideXlab platform.
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recombineering and i scei mediated pseudomonas putida kt2440 scarless Gene Deletion
Fems Microbiology Letters, 2016Co-Authors: Zhongqiu Chen, Wen Ling, Guangdong ShangAbstract:: Pseudomonas putida KT2440 is a saprophytic and Generally recognized as safe microorganism that plays important roles in the biodegradation and production of value-added chemicals. Chromosomal Gene Deletion of P. putida KT2440 usually involves time-consuming Gene cloning, conjugal transfer and counterselection. Recently, we developed a P. putida KT2440 markerless Gene Deletion method based on recombineering and Cre/loxP site-specific recombination. PCR-based λ Red recombineering circumvents the tedious cloning steps and is more amenable to high-throughput manipulation. Here we report an improved scarless Gene Deletion strategy based on recombineering and intron-encoded homing endonuclease I-SceI-mediated double-strand break repair. Sixteen drug exporter Gene(s) were deleted and the minimal inhibition concentrations of the mutants to a variety of antibiotics were determined. The robustness of the procedure was also demonstrated by sequential Deletion of five large genomic regions. Up to 59% recombination efficiency was achieved for a 54.8 kb Deletion, and the efficiency of RecA-mediated double-strand break repair, which was boosted by λ Red recombinase, was nearly 100%. The strain with a 3.76% genome reduction showed an improved growth rate and transformation efficiency. The straightforward, time-saving and highly efficient scarless Deletion approach has the potential to facilitate the Genetic study, and biotechnological and environmental applications of P. putida KT2440.
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pseudomonas putida kt2440 markerless Gene Deletion using a combination of λ red recombineering and cre loxp site specific recombination
Fems Microbiology Letters, 2016Co-Authors: Yunwen Yang, Wen Ling, Hao Zhuang, Qin Li, Guangdong ShangAbstract: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 tri-parental 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. Firstly, homologous arms flanked 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.
Ursula Fleig - One of the best experts on this subject based on the ideXlab platform.
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Targeted Gene Deletion in Saccharomyces cerevisiae and Schizosaccharomyces pombe.
Methods in molecular biology (Clifton N.J.), 2014Co-Authors: Johannes H. Hegemann, Sven Boris Heick, Jennifer Pöhlmann, Marcus M. Langen, Ursula FleigAbstract:Gene Deletion is an important element in the functional characterization of Gene and protein function. Efficient tools for Gene Deletion have been developed in the model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, all of which rely on the replacement of the endogenous Gene of interest with a selectable marker Gene by homologous recombination. In order to minimize incidental recombination events between DNA sequences within the marker Gene and a chromosomal sequence, Gene Deletion cassettes consisting entirely of heterologous DNA sequences are preferred. The Gene Deletion cassettes, which are composed of the marker Gene flanked by short DNA segments homologous to the chromosomal sequences lying to the left and right of the Gene to be deleted, are Generated by PCR and mediate highly efficient one-step Gene Deletion events. Incorporation of loxP sites flanking the marker Gene allows Cre recombinase-mediated rescue, so that the marker can be reused for the next Gene Deletion. This is particularly useful for the characterization of Gene families in S. cerevisiae. The one-step Gene Deletion method is not limited to the elimination of individual Genes, but can also be used for the removal of chromosomal segments exceeding 100 kbp in length. Here we describe a comprehensive set of Gene Deletion cassettes and outline their use in S. cerevisiae and S. pombe.
Zhongqiu Chen - One of the best experts on this subject based on the ideXlab platform.
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recombineering and i scei mediated pseudomonas putida kt2440 scarless Gene Deletion
Fems Microbiology Letters, 2016Co-Authors: Zhongqiu Chen, Wen Ling, Guangdong ShangAbstract:: Pseudomonas putida KT2440 is a saprophytic and Generally recognized as safe microorganism that plays important roles in the biodegradation and production of value-added chemicals. Chromosomal Gene Deletion of P. putida KT2440 usually involves time-consuming Gene cloning, conjugal transfer and counterselection. Recently, we developed a P. putida KT2440 markerless Gene Deletion method based on recombineering and Cre/loxP site-specific recombination. PCR-based λ Red recombineering circumvents the tedious cloning steps and is more amenable to high-throughput manipulation. Here we report an improved scarless Gene Deletion strategy based on recombineering and intron-encoded homing endonuclease I-SceI-mediated double-strand break repair. Sixteen drug exporter Gene(s) were deleted and the minimal inhibition concentrations of the mutants to a variety of antibiotics were determined. The robustness of the procedure was also demonstrated by sequential Deletion of five large genomic regions. Up to 59% recombination efficiency was achieved for a 54.8 kb Deletion, and the efficiency of RecA-mediated double-strand break repair, which was boosted by λ Red recombinase, was nearly 100%. The strain with a 3.76% genome reduction showed an improved growth rate and transformation efficiency. The straightforward, time-saving and highly efficient scarless Deletion approach has the potential to facilitate the Genetic study, and biotechnological and environmental applications of P. putida KT2440.
Yunwen Yang - One of the best experts on this subject based on the ideXlab platform.
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pseudomonas putida kt2440 markerless Gene Deletion using a combination of λ red recombineering and cre loxp site specific recombination
Fems Microbiology Letters, 2016Co-Authors: Yunwen Yang, Wen Ling, Hao Zhuang, Qin Li, Guangdong ShangAbstract: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 tri-parental 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. Firstly, homologous arms flanked 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.