The Experts below are selected from a list of 3894 Experts worldwide ranked by ideXlab platform
Donald L Court - One of the best experts on this subject based on the ideXlab platform.
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Recombineering: genetic engineering in bacteria using homologous recombination.
Current protocols in molecular biology, 2020Co-Authors: Lynn Thomason, Donald L Court, Nina Costantino, Simanti Datta, Mikail Bubunenko, Helen Wilson, Amos OppenheimAbstract:The bacterial chromosome and plasmids can be engineered in vivo by homologous recombination using PCR products and synthetic oligonucleotides as substrates. This is possible because bacteriophage-encoded recombination functions efficiently to recombine sequences with homologies as short as 35 to 40 bases. This Recombineering allows DNA sequences to be inserted or deleted without regard to location of restriction sites. This unit first describes preparation of electrocompetent cells expressing the Recombineering functions and their transformation with dsDNA or ssDNA. Support protocols describe a two-step method of making genetic alterations without leaving any unwanted changes, and a method for retrieving a genetic marker (cloning) from the E. coli chromosome or a co-electroporated DNA fragment and moving it onto a plasmid. A method is also given to screen for unselected mutations. Additional protocols describe removal of defective prophage, methods for Recombineering.
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Recombineering: A Modern Approach to Genetic Engineering
Brenner's Encyclopedia of Genetics, 2020Co-Authors: James A. Sawitzke, Lynn C Thomason, Mikhail Bubunenko, X. Li, N. Costantino, Donald L CourtAbstract:Recombineering is a highly efficient and precise method for genetically engineering DNA in vivo. With Recombineering one can make gene replacements, deletions, insertions, inversions, and single and multiple point mutations. Gene cloning and gene/protein tagging is also possible. Recombineering is catalyzed by the bacteriophage λ Red or similar homologous recombination systems such as RecET from the Escherichia coli Rac prophage, which can utilize both double- and single-stranded DNAs as substrates. All genetic alterations are precise to the base pair as designed by the user and are mediated efficiently by DNA containing target homologies of ~50 bases. These homologies are short enough to be incorporated into commercially available oligonucleotides. Since Recombineering is directed by sequence homology, not convenient restriction sites, it can be used to create genetic alterations on large DNA molecules such as bacterial artificial chromosomes or bacterial genomes in a way not possible with classical in vitro genetic engineering techniques. Because of this, Recombineering has expanded the analytical genetic capability of many organisms. Recombineering with ssDNA can be over 100-fold more efficient than with dsDNA as a substrate. Using ssDNA it is possible to achieve recombination frequencies of over 50% of the cells that survive electroporation; thus, a selection is not necessary for finding the desired mutations. Key to these high frequencies is avoiding the methyl-directed mismatch repair system, which normally removes greater than 99% of the incorporated changes. Avoiding mismatch repair can be easily achieved even in wild-type cells with careful oligonucleotide design. Although the Red system is superior for most Recombineering reactions, the RecET proteins are more efficient at joining linear fragments containing terminal homologies. This is extremely useful for in vivo cloning.
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Recombineering: Genetic Engineering in Bacteria Using Homologous
2020Co-Authors: Recombination Lynn, James A. Sawitzke, Nina Costantino, Xintian Li, C. Thomason, Donald L CourtAbstract:The bacterial chromosome and bacterial plasmids can be engineered in vivo by homologous recombination using PCR products and synthetic oligonucleotides as substrates. This is possible because bacteriophage-encoded recombination proteins efficiently recombine sequences with homologies as short as 35 to 50 bases. Recombineering allows DNA sequences to be inserted or deleted without regard to location of restriction sites. This unit first describes preparation of electrocompetent cells expressing the Recombineering functions and their transformation with dsDNA or ssDNA. It then presents support protocols that describe several two-step selection/counter-selection methods of making genetic alterations without leaving any unwanted changes in the targeted DNA, and a method for retrieving onto a plasmid a genetic marker (cloning by retrieval) from the Escherichia coli chromosome or a co-electroporated DNA fragment. Additional protocols describe methods to screen for unselected mutations, removal of the defective prophage from Recombineering strains, and other useful techniques. Curr. Protoc. Mol. Biol. 106:1.16.1-1.16.39. C � 2014 by John Wiley & Sons, Inc.
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Recombineering highly efficient in vivo genetic engineering using single strand oligos
Methods in Enzymology, 2013Co-Authors: James A. Sawitzke, Lynn C Thomason, Nina Costantino, Mikhail Bubunenko, Xintian Li, Donald L CourtAbstract:Abstract Recombineering provides the ability to make rapid, precise, and inexpensive genetic alterations to any DNA sequence, either in the chromosome or cloned onto a vector that replicates in E. coli (or other Recombineering-proficient bacteria), and to do so in a highly efficient manner. Complicated genetic constructs that are impossible to make with in vitro genetic engineering can be created in days with Recombineering. Recombineering with single-strand DNA (ssDNA) can be used to create single or multiple clustered point mutations, small or large (up to 10 kb) deletions, and small (10–20 base) insertions such as sequence tags. Using optimized conditions, point mutations can be made with such high frequencies that they can be found without selection. This technology excels at creating both directed and random mutations.
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Recombineering: a homologous recombination-based method of genetic engineering
Nature Protocols, 2009Co-Authors: Shyam K Sharan, Lynn C Thomason, Sergey G Kuznetsov, Donald L CourtAbstract:Recombineering is an efficient method of in vivo genetic engineering applicable to chromosomal as well as episomal replicons in E. coli. This method circumvents the need for most standard in vitro cloning techniques. Recombineering allows construction of DNA molecules with precise junctions without constraints being imposed by restriction enzyme site location. Bacteriophage homologous recombination proteins catalyze these Recombineering reactions using double- and single-strand linear DNA substrates, so-called targeting constructs, introduced by electroporation. Gene knockouts, deletions and point mutations are readily made, gene tags can be inserted, and regions of bacterial artificial chromosomes (BACs) or the E. coli genome can be subcloned by gene retrieval using Recombineering. Most of these constructs can be made within about a week's time.
Francis A Stewart - One of the best experts on this subject based on the ideXlab platform.
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single stranded dna binding protein and exogenous recbcd inhibitors enhance phage derived homologous recombination in pseudomonas
iScience, 2019Co-Authors: Wentao Zheng, Chanjuan Jiang, Xiaotong Diao, Hanna Chen, Ruijuan Li, Aiying Li, Francis A Stewart, Shanshan Li, Hailong Wang, Youming ZhangAbstract:Summary The limited efficiency of the available tools for genetic manipulation of Pseudomonas limits fundamental research and utilization of this genus. We explored the properties of a lambda Red-like operon (BAS) from Pseudomonas aeruginosa phage Ab31 and a Rac bacteriophage RecET-like operon (RecTEPsy) from Pseudomonas syringae pv. syringae B728a. Compared with RecTEPsy, the BAS operon was functional at a higher temperature indicating potential to be a generic system for Pseudomonas. Owing to the lack of RecBCD inhibitor in the BAS operon, we added Redγ or Pluγ and found increased Recombineering efficiencies in P. aeruginosa and Pseudomonas fluorescens but not in Pseudomonas putida and P. syringae. Overexpression of single-stranded DNA-binding protein enhanced Recombineering in several contexts including RecET recombination in E. coli. The utility of these systems was demonstrated by engineering P. aeruginosa genomes to create an attenuated rhamnolipid producer. Our work enhances the potential for functional genomics in Pseudomonas.
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recet direct cloning and red alpha beta Recombineering of biosynthetic gene clusters large operons or single genes for heterologous expression
Nature Protocols, 2016Co-Authors: Hailong Wang, Aiying Li, Francis A Stewart, Jun Fu, Xiaoying Bian, Rolf Muller, Zhen Li, Youming ZhangAbstract:Wang et al. present a protocol for direct cloning and engineering of biosynthetic gene clusters, large operons or single genes using combined RecET and Redαβ Recombineering systems present within a single E. coli host.
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rac tagging Recombineering and cas9 assisted targeting for protein tagging and conditional analyses
Scientific Reports, 2016Co-Authors: Oliver Baker, Youming Zhang, Ashish Gupta, Mandy Obst, Konstantinos Anastassiadis, Jun Fu, Francis A StewartAbstract:A fluent method for gene targeting to establish protein tagged and ligand inducible conditional loss-of-function alleles is described. We couple new Recombineering applications for one-step cloning of gRNA oligonucleotides and rapid generation of short-arm (~1 kb) targeting constructs with the power of Cas9-assisted targeting to establish protein tagged alleles in embryonic stem cells at high efficiency. RAC (Recombineering And Cas9)-tagging with Venus, BirM, APEX2 and the auxin degron is facilitated by a Recombineering-ready plasmid series that permits the reuse of gene-specific reagents to insert different tags. Here we focus on protein tagging with the auxin degron because it is a ligand-regulated loss-of-function strategy that is rapid and reversible. Furthermore it includes the additional challenge of biallelic targeting. Despite high frequencies of monoallelic RAC-targeting, we found that simultaneous biallelic targeting benefits from long-arm (>4 kb) targeting constructs. Consequently an updated Recombineering pipeline for fluent generation of long arm targeting constructs is also presented.
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a new Recombineering system for photorhabdus and xenorhabdus
Nucleic Acids Research, 2015Co-Authors: Xuezhi Ding, Francis A Stewart, Jun Fu, Thomas Hoffmann, Michael B Hoffmann, Xiaoying Bian, Rolf Muller, Youming ZhangAbstract:Precise and fluent genetic manipulation is still limited to only a few prokaryotes. Ideally the highly advanced technologies available in Escherichia coli could be broadly applied. Our efforts to apply lambda Red technology, widely termed ‘Recombineering’, in Photorhabdus and Xenorhabdus yielded only limited success. Consequently we explored the properties of an endogenous Photorhabdus luminescens lambda Red-like operon, Plu2934/Plu2935/Plu2936. Bioinformatic and functional tests indicate that Plu2936 is a 5’-3’ exonuclease equivalent to Redα and Plu2935 is a single strand annealing protein equivalent to Redβ. Plu2934 dramatically enhanced Recombineering efficiency. Results from bioinformatic analysis and Recombineering assays suggest that Plu2934 may be functionally equivalent to Redγ, which inhibits the major endogenous E. coli nuclease, RecBCD. The Recombineering utility of Plu2934/Plu2935/Plu2936 was demonstrated by engineering Photorhabdus and Xenorhabdus genomes, including the activation of the 49-kb non-ribosomal peptide synthase (NRPS) gene cluster plu2670 by insertion of a tetracycline inducible promoter. After tetracycline induction, novel secondary metabolites were identified. Our work unlocks the potential for bioprospecting and functional genomics in the Photorhabdus, Xenorhabdus and related genomes.
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high efficiency counterselection Recombineering for site directed mutagenesis in bacterial artificial chromosomes
Nature Methods, 2012Co-Authors: Alexander W Bird, Youming Zhang, Jun Fu, Marcello Maresca, Axel Erler, Jeankarim Heriche, Anthony A Hyman, Francis A StewartAbstract:Site-directed seamless modification of bacterial artificial chromosomes is enhanced more than tenfold in efficiency by improving the counterselection step. A set of plasmids and oligonucleotide design software also make this E. coli Recombineering approach markedly faster and easier.
Youming Zhang - One of the best experts on this subject based on the ideXlab platform.
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Recombineering for Genetic Engineering of Natural Product Biosynthetic Pathways.
Trends in Biotechnology, 2020Co-Authors: Muhammad Nazeer Abbasi, Youming Zhang, Hailong Wang, Jun Fu, Xiaoying Bian, Aiying LiAbstract:Microbial genomes encode many cryptic and uncharacterized biosynthetic gene clusters (BGCs). Exploiting this unexplored genetic wealth to discover microbial novel natural products (NPs) remains a challenging issue. We review homologous recombination (HR)-based Recombineering, mediated by the recombinases RecE/RecT from Rac prophage and Redα/Redβ from lambda phage, which has developed into a highly inclusive tool for direct cloning of large DNA up to 100 kb, seamless mutation, multifragment assembly, and heterologous expression of microbial NP BGCs. Its utilization in the refactoring, engineering, and functional expression of long BGCs for NP biosynthesis makes it easy to elucidate NP-producing potential in microbes. This review also highlights various applications of Recombineering in NP-derived drug discovery.
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single stranded dna binding protein and exogenous recbcd inhibitors enhance phage derived homologous recombination in pseudomonas
iScience, 2019Co-Authors: Wentao Zheng, Chanjuan Jiang, Xiaotong Diao, Hanna Chen, Ruijuan Li, Aiying Li, Francis A Stewart, Shanshan Li, Hailong Wang, Youming ZhangAbstract:Summary The limited efficiency of the available tools for genetic manipulation of Pseudomonas limits fundamental research and utilization of this genus. We explored the properties of a lambda Red-like operon (BAS) from Pseudomonas aeruginosa phage Ab31 and a Rac bacteriophage RecET-like operon (RecTEPsy) from Pseudomonas syringae pv. syringae B728a. Compared with RecTEPsy, the BAS operon was functional at a higher temperature indicating potential to be a generic system for Pseudomonas. Owing to the lack of RecBCD inhibitor in the BAS operon, we added Redγ or Pluγ and found increased Recombineering efficiencies in P. aeruginosa and Pseudomonas fluorescens but not in Pseudomonas putida and P. syringae. Overexpression of single-stranded DNA-binding protein enhanced Recombineering in several contexts including RecET recombination in E. coli. The utility of these systems was demonstrated by engineering P. aeruginosa genomes to create an attenuated rhamnolipid producer. Our work enhances the potential for functional genomics in Pseudomonas.
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simple and rapid direct cloning and heterologous expression of natural product biosynthetic gene cluster in bacillus subtilis via red et Recombineering
Scientific Reports, 2016Co-Authors: Qiyao Shen, Hanna Chen, Hailong Wang, Jun Fu, Xiaoying Bian, Wu Chen, Dingjun Li, Youming ZhangAbstract:Simple and rapid direct cloning and heterologous expression of natural product biosynthetic gene cluster in Bacillus subtilis via Red/ET Recombineering
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recet direct cloning and red alpha beta Recombineering of biosynthetic gene clusters large operons or single genes for heterologous expression
Nature Protocols, 2016Co-Authors: Hailong Wang, Aiying Li, Francis A Stewart, Jun Fu, Xiaoying Bian, Rolf Muller, Zhen Li, Youming ZhangAbstract:Wang et al. present a protocol for direct cloning and engineering of biosynthetic gene clusters, large operons or single genes using combined RecET and Redαβ Recombineering systems present within a single E. coli host.
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rac tagging Recombineering and cas9 assisted targeting for protein tagging and conditional analyses
Scientific Reports, 2016Co-Authors: Oliver Baker, Youming Zhang, Ashish Gupta, Mandy Obst, Konstantinos Anastassiadis, Jun Fu, Francis A StewartAbstract:A fluent method for gene targeting to establish protein tagged and ligand inducible conditional loss-of-function alleles is described. We couple new Recombineering applications for one-step cloning of gRNA oligonucleotides and rapid generation of short-arm (~1 kb) targeting constructs with the power of Cas9-assisted targeting to establish protein tagged alleles in embryonic stem cells at high efficiency. RAC (Recombineering And Cas9)-tagging with Venus, BirM, APEX2 and the auxin degron is facilitated by a Recombineering-ready plasmid series that permits the reuse of gene-specific reagents to insert different tags. Here we focus on protein tagging with the auxin degron because it is a ligand-regulated loss-of-function strategy that is rapid and reversible. Furthermore it includes the additional challenge of biallelic targeting. Despite high frequencies of monoallelic RAC-targeting, we found that simultaneous biallelic targeting benefits from long-arm (>4 kb) targeting constructs. Consequently an updated Recombineering pipeline for fluent generation of long arm targeting constructs is also presented.
Lynn C Thomason - One of the best experts on this subject based on the ideXlab platform.
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Recombineering: A Modern Approach to Genetic Engineering
Brenner's Encyclopedia of Genetics, 2020Co-Authors: James A. Sawitzke, Lynn C Thomason, Mikhail Bubunenko, X. Li, N. Costantino, Donald L CourtAbstract:Recombineering is a highly efficient and precise method for genetically engineering DNA in vivo. With Recombineering one can make gene replacements, deletions, insertions, inversions, and single and multiple point mutations. Gene cloning and gene/protein tagging is also possible. Recombineering is catalyzed by the bacteriophage λ Red or similar homologous recombination systems such as RecET from the Escherichia coli Rac prophage, which can utilize both double- and single-stranded DNAs as substrates. All genetic alterations are precise to the base pair as designed by the user and are mediated efficiently by DNA containing target homologies of ~50 bases. These homologies are short enough to be incorporated into commercially available oligonucleotides. Since Recombineering is directed by sequence homology, not convenient restriction sites, it can be used to create genetic alterations on large DNA molecules such as bacterial artificial chromosomes or bacterial genomes in a way not possible with classical in vitro genetic engineering techniques. Because of this, Recombineering has expanded the analytical genetic capability of many organisms. Recombineering with ssDNA can be over 100-fold more efficient than with dsDNA as a substrate. Using ssDNA it is possible to achieve recombination frequencies of over 50% of the cells that survive electroporation; thus, a selection is not necessary for finding the desired mutations. Key to these high frequencies is avoiding the methyl-directed mismatch repair system, which normally removes greater than 99% of the incorporated changes. Avoiding mismatch repair can be easily achieved even in wild-type cells with careful oligonucleotide design. Although the Red system is superior for most Recombineering reactions, the RecET proteins are more efficient at joining linear fragments containing terminal homologies. This is extremely useful for in vivo cloning.
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efficient and precise genome editing in shewanella with Recombineering and crispr cas9 mediated counter selection
ACS Synthetic Biology, 2019Co-Authors: Anna D Corts, Lynn C Thomason, Ryan T Gill, Jeffrey A GralnickAbstract:Dissimilatory metal-reducing bacteria, particularly those from the genus Shewanella, are of importance for bioremediation of metal contaminated sites and sustainable energy production. However, studies on this species have suffered from a lack of effective genetic tools for precise and high throughput genome manipulation. Here we report the development of a highly efficient system based on single-stranded DNA oligonucleotide Recombineering coupled with CRISPR/Cas9-mediated counter-selection. Our system uses two plasmids: a sgRNA targeting vector and an editing vector, the latter harboring both Cas9 and the phage recombinase W3 Beta. Following the experimental analysis of Cas9 activity, we demonstrate the ability of this system to efficiently and precisely engineer different Shewanella strains with an average efficiency of >90% among total transformed cells, compared to ≃5% by Recombineering alone, and regardless of the gene modified. We also show that different genetic changes can be introduced: mismatche...
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Recombineering highly efficient in vivo genetic engineering using single strand oligos
Methods in Enzymology, 2013Co-Authors: James A. Sawitzke, Lynn C Thomason, Nina Costantino, Mikhail Bubunenko, Xintian Li, Donald L CourtAbstract:Abstract Recombineering provides the ability to make rapid, precise, and inexpensive genetic alterations to any DNA sequence, either in the chromosome or cloned onto a vector that replicates in E. coli (or other Recombineering-proficient bacteria), and to do so in a highly efficient manner. Complicated genetic constructs that are impossible to make with in vitro genetic engineering can be created in days with Recombineering. Recombineering with single-strand DNA (ssDNA) can be used to create single or multiple clustered point mutations, small or large (up to 10 kb) deletions, and small (10–20 base) insertions such as sequence tags. Using optimized conditions, point mutations can be made with such high frequencies that they can be found without selection. This technology excels at creating both directed and random mutations.
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Recombineering: a homologous recombination-based method of genetic engineering
Nature Protocols, 2009Co-Authors: Shyam K Sharan, Lynn C Thomason, Sergey G Kuznetsov, Donald L CourtAbstract:Recombineering is an efficient method of in vivo genetic engineering applicable to chromosomal as well as episomal replicons in E. coli. This method circumvents the need for most standard in vitro cloning techniques. Recombineering allows construction of DNA molecules with precise junctions without constraints being imposed by restriction enzyme site location. Bacteriophage homologous recombination proteins catalyze these Recombineering reactions using double- and single-strand linear DNA substrates, so-called targeting constructs, introduced by electroporation. Gene knockouts, deletions and point mutations are readily made, gene tags can be inserted, and regions of bacterial artificial chromosomes (BACs) or the E. coli genome can be subcloned by gene retrieval using Recombineering. Most of these constructs can be made within about a week's time.
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Modifying Bacteriophage $\lambda$ with Recombineering
2009Co-Authors: Lynn C Thomason, Amos B. Oppenheim, Donald L CourtAbstract:Recombineering is a recently developed method of in vivo genetic engineering used in Escherichia coli and other Gram-negative bacteria. Recombineering can be used to create single-base changes, small and large deletions, and small insertions in phage lambda as well as in bacterial chromosomes, plasmids, and bacterial artificial chromosomes (BACS). This technique uses the bacteriophage lambda generalized recombination system, Red, to catalyze homologous recombination between linear DNA and a replicon using short homologies of 50 base pairs. With Recombineering, single-stranded oligonucleotides or double-stranded PCR products can be used to directly modify the phage lambda genome in vivo. It may also be possible to modify the genomes of other bacteriophages with Recombineering.
Jun Fu - One of the best experts on this subject based on the ideXlab platform.
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Recombineering for Genetic Engineering of Natural Product Biosynthetic Pathways.
Trends in Biotechnology, 2020Co-Authors: Muhammad Nazeer Abbasi, Youming Zhang, Hailong Wang, Jun Fu, Xiaoying Bian, Aiying LiAbstract:Microbial genomes encode many cryptic and uncharacterized biosynthetic gene clusters (BGCs). Exploiting this unexplored genetic wealth to discover microbial novel natural products (NPs) remains a challenging issue. We review homologous recombination (HR)-based Recombineering, mediated by the recombinases RecE/RecT from Rac prophage and Redα/Redβ from lambda phage, which has developed into a highly inclusive tool for direct cloning of large DNA up to 100 kb, seamless mutation, multifragment assembly, and heterologous expression of microbial NP BGCs. Its utilization in the refactoring, engineering, and functional expression of long BGCs for NP biosynthesis makes it easy to elucidate NP-producing potential in microbes. This review also highlights various applications of Recombineering in NP-derived drug discovery.
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simple and rapid direct cloning and heterologous expression of natural product biosynthetic gene cluster in bacillus subtilis via red et Recombineering
Scientific Reports, 2016Co-Authors: Qiyao Shen, Hanna Chen, Hailong Wang, Jun Fu, Xiaoying Bian, Wu Chen, Dingjun Li, Youming ZhangAbstract:Simple and rapid direct cloning and heterologous expression of natural product biosynthetic gene cluster in Bacillus subtilis via Red/ET Recombineering
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recet direct cloning and red alpha beta Recombineering of biosynthetic gene clusters large operons or single genes for heterologous expression
Nature Protocols, 2016Co-Authors: Hailong Wang, Aiying Li, Francis A Stewart, Jun Fu, Xiaoying Bian, Rolf Muller, Zhen Li, Youming ZhangAbstract:Wang et al. present a protocol for direct cloning and engineering of biosynthetic gene clusters, large operons or single genes using combined RecET and Redαβ Recombineering systems present within a single E. coli host.
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rac tagging Recombineering and cas9 assisted targeting for protein tagging and conditional analyses
Scientific Reports, 2016Co-Authors: Oliver Baker, Youming Zhang, Ashish Gupta, Mandy Obst, Konstantinos Anastassiadis, Jun Fu, Francis A StewartAbstract:A fluent method for gene targeting to establish protein tagged and ligand inducible conditional loss-of-function alleles is described. We couple new Recombineering applications for one-step cloning of gRNA oligonucleotides and rapid generation of short-arm (~1 kb) targeting constructs with the power of Cas9-assisted targeting to establish protein tagged alleles in embryonic stem cells at high efficiency. RAC (Recombineering And Cas9)-tagging with Venus, BirM, APEX2 and the auxin degron is facilitated by a Recombineering-ready plasmid series that permits the reuse of gene-specific reagents to insert different tags. Here we focus on protein tagging with the auxin degron because it is a ligand-regulated loss-of-function strategy that is rapid and reversible. Furthermore it includes the additional challenge of biallelic targeting. Despite high frequencies of monoallelic RAC-targeting, we found that simultaneous biallelic targeting benefits from long-arm (>4 kb) targeting constructs. Consequently an updated Recombineering pipeline for fluent generation of long arm targeting constructs is also presented.
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a new Recombineering system for photorhabdus and xenorhabdus
Nucleic Acids Research, 2015Co-Authors: Xuezhi Ding, Francis A Stewart, Jun Fu, Thomas Hoffmann, Michael B Hoffmann, Xiaoying Bian, Rolf Muller, Youming ZhangAbstract:Precise and fluent genetic manipulation is still limited to only a few prokaryotes. Ideally the highly advanced technologies available in Escherichia coli could be broadly applied. Our efforts to apply lambda Red technology, widely termed ‘Recombineering’, in Photorhabdus and Xenorhabdus yielded only limited success. Consequently we explored the properties of an endogenous Photorhabdus luminescens lambda Red-like operon, Plu2934/Plu2935/Plu2936. Bioinformatic and functional tests indicate that Plu2936 is a 5’-3’ exonuclease equivalent to Redα and Plu2935 is a single strand annealing protein equivalent to Redβ. Plu2934 dramatically enhanced Recombineering efficiency. Results from bioinformatic analysis and Recombineering assays suggest that Plu2934 may be functionally equivalent to Redγ, which inhibits the major endogenous E. coli nuclease, RecBCD. The Recombineering utility of Plu2934/Plu2935/Plu2936 was demonstrated by engineering Photorhabdus and Xenorhabdus genomes, including the activation of the 49-kb non-ribosomal peptide synthase (NRPS) gene cluster plu2670 by insertion of a tetracycline inducible promoter. After tetracycline induction, novel secondary metabolites were identified. Our work unlocks the potential for bioprospecting and functional genomics in the Photorhabdus, Xenorhabdus and related genomes.