The Experts below are selected from a list of 28935 Experts worldwide ranked by ideXlab platform
Sean F Brady - One of the best experts on this subject based on the ideXlab platform.
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Selective enrichment of environmental DNA Libraries for genes encoding nonribosomal peptides and polyketides by phosphopantetheine transferase-dependent complementation of siderophore biosynthesis.
ACS chemical biology, 2012Co-Authors: Zachary Charlop-powers, Jeffrey W Craig, Jacob J. Banik, Jeremy G. Owen, Sean F BradyAbstract:The cloning of DNA directly from environmental samples provides a means to functionally access biosynthetic gene clusters present in the genomes of the large fraction of bacteria that remains recalcitrant to growth in the laboratory. Herein, we demonstrate a method by which complementation of phosphopantetheine transferase deletion mutants can be used to restore siderophore biosynthesis and to therefore selectively enrich eDNA Libraries for nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) gene sequences to unprecedented levels. The common use of NRPS/PKS-derived siderophores across bacterial taxa makes this method generalizable and should allow for the facile selective enrichment of NRPS/PKS-containing biosynthetic gene clusters from large environmental DNA Libraries using a wide variety of phylogenetically diverse bacterial hosts.
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expanding small molecule functional metagenomics through parallel screening of broad host range cosmid environmental DNA Libraries in diverse proteobacteria
Applied and Environmental Microbiology, 2010Co-Authors: Jeffrey W Craig, Fangyuan Chang, Steven C Obiajulu, Sean F BradyAbstract:The small-molecule biosynthetic diversity encoded within the genomes of uncultured bacteria is an attractive target for the discovery of natural products using functional metagenomics. Phenotypes commonly associated with the production of small molecules, such as antibiosis, altered pigmentation, or altered colony morphology, are easily identified from screens of arrayed metagenomic library clones. However, functional metagenomic screening methods are limited by their intrinsic dependence on a heterologous expression host. Toward the goal of increasing the small-molecule biosynthetic diversity found in functional metagenomic studies, we report the phenotypic screening of broad-host-range environmental DNA Libraries in six different proteobacteria: Agrobacterium tumefaciens, Burkholderia graminis, Caulobacter vibrioides, Escherichia coli, Pseudomonas putida, and Ralstonia metallidurans. Clone-specific small molecules found in culture broth extracts from pigmented and antibacterially active clones, as well as the genetic elements responsible for the biosynthesis of these metabolites, are described. The host strains used in this investigation provided access to unique sets of clones showing minimal overlap, thus demonstrating the potential advantage conferred on functional metagenomics through the use of multiple diverse host species.
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Handbook of Molecular Microbial Ecology II: Metagenomics in Different Habitats - Expanding Small-Molecule Functional Metagenomics through Parallel Screening of Broad-Host-Range Cosmid Environmental DNA Libraries in Diverse Proteobacteria
Applied and Environmental Microbiology, 2010Co-Authors: Jeffrey W Craig, Fangyuan Chang, Steven C Obiajulu, Sean F BradyAbstract:The small-molecule biosynthetic diversity encoded within the genomes of uncultured bacteria is an attractive target for the discovery of natural products using functional metagenomics. Phenotypes commonly associated with the production of small molecules, such as antibiosis, altered pigmentation, or altered colony morphology, are easily identified from screens of arrayed metagenomic library clones. However, functional metagenomic screening methods are limited by their intrinsic dependence on a heterologous expression host. Toward the goal of increasing the small-molecule biosynthetic diversity found in functional metagenomic studies, we report the phenotypic screening of broad-host-range environmental DNA Libraries in six different proteobacteria: Agrobacterium tumefaciens, Burkholderia graminis, Caulobacter vibrioides, Escherichia coli, Pseudomonas putida, and Ralstonia metallidurans. Clone-specific small molecules found in culture broth extracts from pigmented and antibacterially active clones, as well as the genetic elements responsible for the biosynthesis of these metabolites, are described. The host strains used in this investigation provided access to unique sets of clones showing minimal overlap, thus demonstrating the potential advantage conferred on functional metagenomics through the use of multiple diverse host species.
Matthias Meyer - One of the best experts on this subject based on the ideXlab platform.
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manual and automated preparation of single stranded DNA Libraries for the sequencing of DNA from ancient biological remains and other sources of highly degraded DNA
Nature Protocols, 2020Co-Authors: Marietheres Gansauge, Ayinuer Aximupetri, Sarah Nagel, Matthias MeyerAbstract:It has been shown that highly fragmented DNA is most efficiently converted into DNA Libraries for sequencing if both strands of the DNA fragments are processed independently. We present an updated protocol for library preparation from single-stranded DNA, which is based on the splinted ligation of an adapter oligonucleotide to the 3' ends of single DNA strands, the synthesis of a complementary strand using a DNA polymerase and the addition of a 5' adapter via blunt-end ligation. The efficiency of library preparation is determined individually for each sample using a spike-in oligonucleotide. The whole workflow, including library preparation, quantification and amplification, requires two work days for up to 16 Libraries. Alternatively, we provide documentation and electronic protocols enabling automated library preparation of 96 samples in parallel on a Bravo NGS Workstation (Agilent Technologies). After library preparation, molecules with uninformative short inserts (shorter than ~30-35 base pairs) can be removed by polyacrylamide gel electrophoresis if desired.
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Manual and automated preparation of single-stranded DNA Libraries for the sequencing of DNA from ancient biological remains and other sources of highly degraded DNA
Nature Protocols, 2020Co-Authors: Marietheres Gansauge, Sarah Nagel, Ayinuer Aximu-petri, Matthias MeyerAbstract:It has been shown that highly fragmented DNA is most efficiently converted into DNA Libraries for sequencing if both strands of the DNA fragments are processed independently. We present an updated protocol for library preparation from single-stranded DNA, which is based on the splinted ligation of an adapter oligonucleotide to the 3′ ends of single DNA strands, the synthesis of a complementary strand using a DNA polymerase and the addition of a 5′ adapter via blunt-end ligation. The efficiency of library preparation is determined individually for each sample using a spike-in oligonucleotide. The whole workflow, including library preparation, quantification and amplification, requires two work days for up to 16 Libraries. Alternatively, we provide documentation and electronic protocols enabling automated library preparation of 96 samples in parallel on a Bravo NGS Workstation (Agilent Technologies). After library preparation, molecules with uninformative short inserts (shorter than ~30−35 base pairs) can be removed by polyacrylamide gel electrophoresis if desired. Here the authors describe an updated protocol for single-stranded sequencing library preparation suitable for highly degraded DNA from ancient remains or other sources. The procedure can be performed manually or in an automated fashion.
Jeffrey W Craig - One of the best experts on this subject based on the ideXlab platform.
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Selective enrichment of environmental DNA Libraries for genes encoding nonribosomal peptides and polyketides by phosphopantetheine transferase-dependent complementation of siderophore biosynthesis.
ACS chemical biology, 2012Co-Authors: Zachary Charlop-powers, Jeffrey W Craig, Jacob J. Banik, Jeremy G. Owen, Sean F BradyAbstract:The cloning of DNA directly from environmental samples provides a means to functionally access biosynthetic gene clusters present in the genomes of the large fraction of bacteria that remains recalcitrant to growth in the laboratory. Herein, we demonstrate a method by which complementation of phosphopantetheine transferase deletion mutants can be used to restore siderophore biosynthesis and to therefore selectively enrich eDNA Libraries for nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) gene sequences to unprecedented levels. The common use of NRPS/PKS-derived siderophores across bacterial taxa makes this method generalizable and should allow for the facile selective enrichment of NRPS/PKS-containing biosynthetic gene clusters from large environmental DNA Libraries using a wide variety of phylogenetically diverse bacterial hosts.
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expanding small molecule functional metagenomics through parallel screening of broad host range cosmid environmental DNA Libraries in diverse proteobacteria
Applied and Environmental Microbiology, 2010Co-Authors: Jeffrey W Craig, Fangyuan Chang, Steven C Obiajulu, Sean F BradyAbstract:The small-molecule biosynthetic diversity encoded within the genomes of uncultured bacteria is an attractive target for the discovery of natural products using functional metagenomics. Phenotypes commonly associated with the production of small molecules, such as antibiosis, altered pigmentation, or altered colony morphology, are easily identified from screens of arrayed metagenomic library clones. However, functional metagenomic screening methods are limited by their intrinsic dependence on a heterologous expression host. Toward the goal of increasing the small-molecule biosynthetic diversity found in functional metagenomic studies, we report the phenotypic screening of broad-host-range environmental DNA Libraries in six different proteobacteria: Agrobacterium tumefaciens, Burkholderia graminis, Caulobacter vibrioides, Escherichia coli, Pseudomonas putida, and Ralstonia metallidurans. Clone-specific small molecules found in culture broth extracts from pigmented and antibacterially active clones, as well as the genetic elements responsible for the biosynthesis of these metabolites, are described. The host strains used in this investigation provided access to unique sets of clones showing minimal overlap, thus demonstrating the potential advantage conferred on functional metagenomics through the use of multiple diverse host species.
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Handbook of Molecular Microbial Ecology II: Metagenomics in Different Habitats - Expanding Small-Molecule Functional Metagenomics through Parallel Screening of Broad-Host-Range Cosmid Environmental DNA Libraries in Diverse Proteobacteria
Applied and Environmental Microbiology, 2010Co-Authors: Jeffrey W Craig, Fangyuan Chang, Steven C Obiajulu, Sean F BradyAbstract:The small-molecule biosynthetic diversity encoded within the genomes of uncultured bacteria is an attractive target for the discovery of natural products using functional metagenomics. Phenotypes commonly associated with the production of small molecules, such as antibiosis, altered pigmentation, or altered colony morphology, are easily identified from screens of arrayed metagenomic library clones. However, functional metagenomic screening methods are limited by their intrinsic dependence on a heterologous expression host. Toward the goal of increasing the small-molecule biosynthetic diversity found in functional metagenomic studies, we report the phenotypic screening of broad-host-range environmental DNA Libraries in six different proteobacteria: Agrobacterium tumefaciens, Burkholderia graminis, Caulobacter vibrioides, Escherichia coli, Pseudomonas putida, and Ralstonia metallidurans. Clone-specific small molecules found in culture broth extracts from pigmented and antibacterially active clones, as well as the genetic elements responsible for the biosynthesis of these metabolites, are described. The host strains used in this investigation provided access to unique sets of clones showing minimal overlap, thus demonstrating the potential advantage conferred on functional metagenomics through the use of multiple diverse host species.
Florian Hollfelder - One of the best experts on this subject based on the ideXlab platform.
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split mix assembly of DNA Libraries for ultrahigh throughput on bead screening of functional proteins
Nucleic Acids Research, 2020Co-Authors: Laurens Lindenburg, Tuomas Huovinen, Kayleigh Van De Wiel, Michael Herger, Michael Snaith, Florian HollfelderAbstract:Site-saturation Libraries reduce protein screening effort in directed evolution campaigns by focusing on a limited number of rationally chosen residues. However, uneven library synthesis efficiency leads to amino acid bias, remedied at high cost by expensive custom synthesis of oligonucleotides, or through use of proprietary library synthesis platforms. To address these shortcomings, we have devised a method where DNA Libraries are constructed on the surface of microbeads by ligating dsDNA fragments onto growing, surface-immobilised DNA, in iterative split-and-mix cycles. This method-termed SpliMLiB for Split-and-Mix Library on Beads-was applied towards the directed evolution of an anti-IgE Affibody (ZIgE), generating a 160,000-membered, 4-site, saturation library on the surface of 8 million monoclonal beads. Deep sequencing confirmed excellent library balance (5.1% ± 0.77 per amino acid) and coverage (99.3%). As SpliMLiB beads are monoclonal, they were amenable to direct functional screening in water-in-oil emulsion droplets with cell-free expression. A FACS-based sorting of the library beads allowed recovery of hits improved in Kd over wild-type ZIgE by up to 3.5-fold, while a consensus mutant of the best hits provided a 10-fold improvement. With SpliMLiB, directed evolution workflows are accelerated by integrating high-quality DNA library generation with an ultra-high throughput protein screening platform.
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Split & mix assembly of DNA Libraries for ultrahigh throughput on-bead screening of functional proteins
Nucleic acids research, 2020Co-Authors: Laurens Lindenburg, Tuomas Huovinen, Kayleigh Van De Wiel, Michael Herger, Michael Snaith, Florian HollfelderAbstract:Site-saturation Libraries reduce protein screening effort in directed evolution campaigns by focusing on a limited number of rationally chosen residues. However, uneven library synthesis efficiency leads to amino acid bias, remedied at high cost by expensive custom synthesis of oligonucleotides, or through use of proprietary library synthesis platforms. To address these shortcomings, we have devised a method where DNA Libraries are constructed on the surface of microbeads by ligating dsDNA fragments onto growing, surface-immobilised DNA, in iterative split-and-mix cycles. This method-termed SpliMLiB for Split-and-Mix Library on Beads-was applied towards the directed evolution of an anti-IgE Affibody (ZIgE), generating a 160,000-membered, 4-site, saturation library on the surface of 8 million monoclonal beads. Deep sequencing confirmed excellent library balance (5.1% ± 0.77 per amino acid) and coverage (99.3%). As SpliMLiB beads are monoclonal, they were amenable to direct functional screening in water-in-oil emulsion droplets with cell-free expression. A FACS-based sorting of the library beads allowed recovery of hits improved in Kd over wild-type ZIgE by up to 3.5-fold, while a consensus mutant of the best hits provided a 10-fold improvement. With SpliMLiB, directed evolution workflows are accelerated by integrating high-quality DNA library generation with an ultra-high throughput protein screening platform.
Marietheres Gansauge - One of the best experts on this subject based on the ideXlab platform.
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manual and automated preparation of single stranded DNA Libraries for the sequencing of DNA from ancient biological remains and other sources of highly degraded DNA
Nature Protocols, 2020Co-Authors: Marietheres Gansauge, Ayinuer Aximupetri, Sarah Nagel, Matthias MeyerAbstract:It has been shown that highly fragmented DNA is most efficiently converted into DNA Libraries for sequencing if both strands of the DNA fragments are processed independently. We present an updated protocol for library preparation from single-stranded DNA, which is based on the splinted ligation of an adapter oligonucleotide to the 3' ends of single DNA strands, the synthesis of a complementary strand using a DNA polymerase and the addition of a 5' adapter via blunt-end ligation. The efficiency of library preparation is determined individually for each sample using a spike-in oligonucleotide. The whole workflow, including library preparation, quantification and amplification, requires two work days for up to 16 Libraries. Alternatively, we provide documentation and electronic protocols enabling automated library preparation of 96 samples in parallel on a Bravo NGS Workstation (Agilent Technologies). After library preparation, molecules with uninformative short inserts (shorter than ~30-35 base pairs) can be removed by polyacrylamide gel electrophoresis if desired.
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Manual and automated preparation of single-stranded DNA Libraries for the sequencing of DNA from ancient biological remains and other sources of highly degraded DNA
Nature Protocols, 2020Co-Authors: Marietheres Gansauge, Sarah Nagel, Ayinuer Aximu-petri, Matthias MeyerAbstract:It has been shown that highly fragmented DNA is most efficiently converted into DNA Libraries for sequencing if both strands of the DNA fragments are processed independently. We present an updated protocol for library preparation from single-stranded DNA, which is based on the splinted ligation of an adapter oligonucleotide to the 3′ ends of single DNA strands, the synthesis of a complementary strand using a DNA polymerase and the addition of a 5′ adapter via blunt-end ligation. The efficiency of library preparation is determined individually for each sample using a spike-in oligonucleotide. The whole workflow, including library preparation, quantification and amplification, requires two work days for up to 16 Libraries. Alternatively, we provide documentation and electronic protocols enabling automated library preparation of 96 samples in parallel on a Bravo NGS Workstation (Agilent Technologies). After library preparation, molecules with uninformative short inserts (shorter than ~30−35 base pairs) can be removed by polyacrylamide gel electrophoresis if desired. Here the authors describe an updated protocol for single-stranded sequencing library preparation suitable for highly degraded DNA from ancient remains or other sources. The procedure can be performed manually or in an automated fashion.