The Experts below are selected from a list of 9 Experts worldwide ranked by ideXlab platform
Banaszak Holl, Mark M. - One of the best experts on this subject based on the ideXlab platform.
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Quantification of cytosolic plasmid DNA degradation using high‐throughput Sequencing: implications for gene delivery
'Wiley', 2014Co-Authors: Rattan Rahul, Bielinska, Anna U., Banaszak Holl, Mark M.Abstract:Background Although cytosolic DNA degradation plays an important role in decreasing transgene expression, the plasmid degradation pattern remains largely unexplored. Methods Illumina Dye Sequencing was employed to provide degradation site information for S1 and cytosolic nucleases. S1 nuclease provided a positive control for a comparison between the agarose gel method and Sequencing approaches. Results The poly(A) region between the β‐lactamase gene and the cytomegalovirus (CMV) promoter was identified as the most likely cut site for polyplex‐treated cytosol. The second most likely site, at the 5' end of the β‐lactamase gene, was identified by gel electrophoresis and Sequencing. Additional sites were detected in the OriC region, the SV40/poly(A) region, the luciferase gene and the CMV promoter. Sequence analysis of plasmid treated with cytosol from control cells showed the greatest cut activity in the OriC region, the β‐lactamase gene and the poly(A) region following the luciferase gene. Additional regions of cut activity include the SV40 promoter and the β‐lactamase poly(A) termination sequence. Both cytosolic nucleases and the S1 nuclease showed substantial activity at the bacterial origin of replication ( OriC ). Conclusions High‐throughput plasmid Sequencing revealed regions of the luciferase plasmid DNA sequence that are sensitive to cytosolic nuclease degradation. This provides new targets for improving plasmid and/or polymer design to optimize the likelihood of protein expression. Copyright © 2014 John Wiley & Sons, Ltd.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/106947/1/jgm2761.pd
Pyne, Michael E - One of the best experts on this subject based on the ideXlab platform.
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Development of genetic tools for metabolic engineering of Clostridium pasteurianum
'University of Waterloo', 2014Co-Authors: Pyne, Michael EAbstract:Reducing the production cost of industrial biofuels will greatly facilitate their proliferation and co-integration with fossil fuels. The cost of feedstock is the largest cost in most fermentation bioprocesses and therefore represents an important target for cost reduction. Meanwhile, the biorefinery concept advocates revenue growth through complete utilization of by-products generated during biofuel production. Taken together, the production of biofuels from low-cost crude glycerol, available in oversupply as a by-product of bioethanol production, in the form of thin stillage, and biodiesel production, embodies a remarkable opportunity to advance affordable biofuel development. However, few bacterial species possess the natural capacity to convert glycerol as a sole source of carbon and energy into value-added bioproducts. Of particular interest is the anaerobe Clostridium pasteurianum, the only microorganism known to convert glycerol alone directly into butanol, which currently holds immense promise as a high-energy biofuel and bulk chemical. Unfortunately, genetic and metabolic engineering of C. pasteurianum has been fundamentally impeded due to a complete lack of genetic tools and techniques available for the manipulation of this promising bacterium. This thesis encompasses the development of fundamental genetic tools and techniques that will permit extensive genetic and metabolic engineering of C. pasteurianum. We initiated our genetic work with the development of an electrotransformation protocol permitting high-level DNA transfer to C. pasteurianum together with accompanying selection markers and vector components. The CpaAI restriction-modification system was found to be a major barrier to DNA delivery into C. pasteurianum which we overcame by in vivo methylation of the recognition site (5’-CGCG-3’) using the M.FnuDII methyltransferase. Systematic investigation of various parameters involved in the cell growth, washing and pulse delivery, and outgrowth phases of the electrotransformation procedure significantly elevated the electrotransformation efficiency up to 7.5 × 104 transformants µg-1 DNA, an increase of approximately three orders of magnitude. Key factors affecting the electrotransformation efficiency include cell-wall-weakening using glycine, ethanol-mediated membrane solubilization, field strength of the electric pulse, and sucrose osmoprotection. Following development of a gene transfer methodology, we next aimed to sequence the entire genome of C. pasteurianum. Using a hybrid approach involving 454 pyroSequencing, Illumina Dye Sequencing, and single molecule real-time Sequencing platforms, we obtained a near-complete genome sequence comprised of 12 contigs, 4,420,100 bp, and 4,056 candidate protein-coding genes with a GC content of 30.0%. No extrachromosomal elements were detected. We provide an overview of the genes and pathways involved in the organism’s central fermentative metabolism. We used our developed electrotransformation procedure to investigate the use of established clostridial group II intron biology for constructing chromosomal gene knockout mutants of C. pasteurianum. Through methylome analysis of C. pasteurianum genome Sequencing data and transformation assays of various vector deletion constructs, we identified a new Type I restriction-modification system that inhibits transfer of vectors harboring group II intron gene knockout machinery. We designated the new restriction system CpaAII and proposed a recognition sequence of 5’-AAGNNNNNCTCC-3’. Overcoming restriction by CpaAII, in addition to low intron retrohoming efficiency, allowed the isolation of a gene knockout mutant of C. pasteurianum with a disrupted CpaAI Type II restriction system. The resulting mutant strain should be efficienty transformed with plasmid DNA lacking M.FnuDII methylation. Lastly, we investigated the use of plasmid-based gene overexpression and chromosomal gene downregulation to alter gene expression in C. pasteurianum. Using a β-galactosidase reporter gene, we characterized promoters corresponding to the ferredoxin and thiolase genes of C. pasteurianum and show that both promoters permitted high-level, constitutive gene expression. The thiolase promoter was then utilized to drive transcription of an antisense RNA molecule possessing complementarity to mRNA of our β-galactosidase reporter gene. Our antisense RNA system demonstrated 52-58% downregulation of plasmid encoded β-galactosidase activity throughout the duration of growth. In an attempt to perturb the central fermentative metabolism of C. pasteurianum and enhance butanol titers, we prepared several antisense RNA constructs for downregulation of 1,3-propanediol, butyrate, and hydrogen production pathways. The resulting downregulation strains are expected to exhibit drastically altered central fermentative metabolism and product distribution. Taken together, we have demonstrated that C. pasteurianum is amendable to genetic manipulation through the development of methods for plasmid DNA transfer and gene overexpression, knockdown, and knockout. Further, our genome sequence should provide valuable nucleotide sequence information for the application of our genetic tools. Thus, the genome sequence, electrotransformation method, and associated genetic tools and techniques reported here should promote extensive genetic manipulation and metabolic engineering of this biotechnologically important bacterium.1 yea
Rattan Rahul - One of the best experts on this subject based on the ideXlab platform.
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Quantification of cytosolic plasmid DNA degradation using high‐throughput Sequencing: implications for gene delivery
'Wiley', 2014Co-Authors: Rattan Rahul, Bielinska, Anna U., Banaszak Holl, Mark M.Abstract:Background Although cytosolic DNA degradation plays an important role in decreasing transgene expression, the plasmid degradation pattern remains largely unexplored. Methods Illumina Dye Sequencing was employed to provide degradation site information for S1 and cytosolic nucleases. S1 nuclease provided a positive control for a comparison between the agarose gel method and Sequencing approaches. Results The poly(A) region between the β‐lactamase gene and the cytomegalovirus (CMV) promoter was identified as the most likely cut site for polyplex‐treated cytosol. The second most likely site, at the 5' end of the β‐lactamase gene, was identified by gel electrophoresis and Sequencing. Additional sites were detected in the OriC region, the SV40/poly(A) region, the luciferase gene and the CMV promoter. Sequence analysis of plasmid treated with cytosol from control cells showed the greatest cut activity in the OriC region, the β‐lactamase gene and the poly(A) region following the luciferase gene. Additional regions of cut activity include the SV40 promoter and the β‐lactamase poly(A) termination sequence. Both cytosolic nucleases and the S1 nuclease showed substantial activity at the bacterial origin of replication ( OriC ). Conclusions High‐throughput plasmid Sequencing revealed regions of the luciferase plasmid DNA sequence that are sensitive to cytosolic nuclease degradation. This provides new targets for improving plasmid and/or polymer design to optimize the likelihood of protein expression. Copyright © 2014 John Wiley & Sons, Ltd.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/106947/1/jgm2761.pd
Bielinska, Anna U. - One of the best experts on this subject based on the ideXlab platform.
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Quantification of cytosolic plasmid DNA degradation using high‐throughput Sequencing: implications for gene delivery
'Wiley', 2014Co-Authors: Rattan Rahul, Bielinska, Anna U., Banaszak Holl, Mark M.Abstract:Background Although cytosolic DNA degradation plays an important role in decreasing transgene expression, the plasmid degradation pattern remains largely unexplored. Methods Illumina Dye Sequencing was employed to provide degradation site information for S1 and cytosolic nucleases. S1 nuclease provided a positive control for a comparison between the agarose gel method and Sequencing approaches. Results The poly(A) region between the β‐lactamase gene and the cytomegalovirus (CMV) promoter was identified as the most likely cut site for polyplex‐treated cytosol. The second most likely site, at the 5' end of the β‐lactamase gene, was identified by gel electrophoresis and Sequencing. Additional sites were detected in the OriC region, the SV40/poly(A) region, the luciferase gene and the CMV promoter. Sequence analysis of plasmid treated with cytosol from control cells showed the greatest cut activity in the OriC region, the β‐lactamase gene and the poly(A) region following the luciferase gene. Additional regions of cut activity include the SV40 promoter and the β‐lactamase poly(A) termination sequence. Both cytosolic nucleases and the S1 nuclease showed substantial activity at the bacterial origin of replication ( OriC ). Conclusions High‐throughput plasmid Sequencing revealed regions of the luciferase plasmid DNA sequence that are sensitive to cytosolic nuclease degradation. This provides new targets for improving plasmid and/or polymer design to optimize the likelihood of protein expression. Copyright © 2014 John Wiley & Sons, Ltd.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/106947/1/jgm2761.pd
Anni Djurhuus - One of the best experts on this subject based on the ideXlab platform.
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Microbial oceanography of southern hemisphere seamounts and hydrothermal vents
2015Co-Authors: Anni DjurhuusAbstract:Microbial biogeography is being increasingly more studied, both in terms of genetic divisions and 'ecotype' variation. This thesis investigates the regional (100-1000s km) and local (10s m) distribution and diversity of microorganisms around hydrothermal vents and seamounts at the East Scotia Ridge and the Southwest Indian Ridge. Microbial communities were characterized using Illumina Dye Sequencing to de- termine taxon richness and diversity and flow cytometry to obtain cell counts. In addition I investigated the physicochemical environment (nutrients, organic carbon, salinity and temperature) in which the microorganisms persist. Typical deep-sea microorganisms were abundant at vents and below the euphotic zone on the seamounts. The surface layer of the seamounts contained typical open-ocean photoautotrophic organisms. Microbial communities were correlated to or- ganic carbon on both hydrothermal vents and seamounts. With microorganisms possi- bly having a large influence on carbon sequestration into the deep-sea from hydrothermal vents. On a local scale the hydrothermal vents had a relatively higher abundance of chemosynthetic Epsilonproteobacteria and the Gammaproteobacteria family SUP05, which were closely correlated to the redox potential in the vent effluent. This was prominent in both plumes from the East Scotia Ridge and the Southwest Indian Ridge, which also had distinct microbial community structures. Across the Southwest Indian Ocean the microbial communities were firstly segregated by depth. However, on a regional scale their physical environment primarily divided the microbial communities into three biological regimes the sub-tropical, con- vergence zone and the sub-Antarctic. The microbial community structure and biogeography is influenced by steep environmental gradients, displaying a distance-decay relationship between sampling locations. With environmental conditions persisting at different scales, from local (10s m) around the hydrothermal vents to regional (100-1000 km) between the seamounts, driving the microbial community patterns. The same horizontal and vertical patterns for microorganisms and metazoans points to fundamental differences throughout all parts of the ecosystem/food web.
