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Title: Generalized Schemes for High Throughput Manipulation of the Desulfovibrio vulgaris Hildenborough Genome.

Abstract

The ability to conduct advanced functional genomic studies of the thousands of 38 sequenced bacteria has been hampered by the lack of available tools for making high39 throughput chromosomal manipulations in a systematic manner that can be applied across 40 diverse species. In this work, we highlight the use of synthetic biological tools to 41 assemble custom suicide vectors with reusable and interchangeable DNA parts to 42 facilitate chromosomal modification at designated loci. These constructs enable an array 43 of downstream applications including gene replacement and creation of gene fusions with 44 affinity purification or localization tags. We employed this approach to engineer 45 chromosomal modifications in a bacterium that has previously proven difficult to 46 manipulate genetically, Desulfovibrio vulgaris Hildenborough, to generate a library of 47 662 strains. Furthermore, we demonstrate how these modifications can be used for 48 examining metabolic pathways, protein-protein interactions, and protein localization. The 49 ubiquity of suicide constructs in gene replacement throughout biology suggests that this 50 approach can be applied to engineer a broad range of species for a diverse array of 51 systems biological applications and is amenable to high-throughput implementation.

Authors:
 [1];  [1];  [2];  [1];  [1];  [3];  [1];  [4];  [1];  [3];  [1];  [5];  [3];  [1];  [1];  [1];  [1];  [4];  [1];  [1] more »;  [4];  [1];  [1];  [1];  [1];  [3];  [1] « less
  1. Lawrence Berkeley National Laboratory (LBNL)
  2. ORNL
  3. University of Missouri
  4. University of California, San Francisco
  5. University of Missouri, Columbia
Publication Date:
Research Org.:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
1028745
DOE Contract Number:  
DE-AC05-00OR22725
Resource Type:
Journal Article
Journal Name:
Applied and Environmental Microbiology
Additional Journal Information:
Journal Volume: 77; Journal Issue: 21; Journal ID: ISSN 0099-2240
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES; AFFINITY; BACTERIA; BIOLOGICAL PATHWAYS; BIOLOGY; DESULFOVIBRIO; DNA; ENGINEERS; FUNCTIONALS; GENES; IMPLEMENTATION; MODIFICATIONS; PROTEINS; PURIFICATION; STRAINS; VECTORS; ENGIMA; PCAP; Desulfovibrio; D. vulgaris; protein complex

Citation Formats

Chhabra, Swapnil, Butland, Gareth, Elias, Dwayne A, Chandonia, John-Marc, Fok, Olivia, Juba, tom, Gorur, A., Allen, S., Leung, C. M., Keller, Kim, Reveco, S., Zane, Mr. Grant M., Semkiw, Elizabeth M., Prathapam, R., Gold, B., Singer, Mary, Ouellet, M., Sazakal, E. D., Jorgens, Dominique, Price, Morgan N., Witkowska, Ewa, Beller, Harry R., Hazen, Terry, Biggin, Mark D., Auer, Dr. Manfred, Wall, Judy D., and Keasling, Jay. Generalized Schemes for High Throughput Manipulation of the Desulfovibrio vulgaris Hildenborough Genome.. United States: N. p., 2011. Web. doi:10.1128/AEM.05495-11.
Chhabra, Swapnil, Butland, Gareth, Elias, Dwayne A, Chandonia, John-Marc, Fok, Olivia, Juba, tom, Gorur, A., Allen, S., Leung, C. M., Keller, Kim, Reveco, S., Zane, Mr. Grant M., Semkiw, Elizabeth M., Prathapam, R., Gold, B., Singer, Mary, Ouellet, M., Sazakal, E. D., Jorgens, Dominique, Price, Morgan N., Witkowska, Ewa, Beller, Harry R., Hazen, Terry, Biggin, Mark D., Auer, Dr. Manfred, Wall, Judy D., & Keasling, Jay. Generalized Schemes for High Throughput Manipulation of the Desulfovibrio vulgaris Hildenborough Genome.. United States. https://doi.org/10.1128/AEM.05495-11
Chhabra, Swapnil, Butland, Gareth, Elias, Dwayne A, Chandonia, John-Marc, Fok, Olivia, Juba, tom, Gorur, A., Allen, S., Leung, C. M., Keller, Kim, Reveco, S., Zane, Mr. Grant M., Semkiw, Elizabeth M., Prathapam, R., Gold, B., Singer, Mary, Ouellet, M., Sazakal, E. D., Jorgens, Dominique, Price, Morgan N., Witkowska, Ewa, Beller, Harry R., Hazen, Terry, Biggin, Mark D., Auer, Dr. Manfred, Wall, Judy D., and Keasling, Jay. 2011. "Generalized Schemes for High Throughput Manipulation of the Desulfovibrio vulgaris Hildenborough Genome.". United States. https://doi.org/10.1128/AEM.05495-11.
@article{osti_1028745,
title = {Generalized Schemes for High Throughput Manipulation of the Desulfovibrio vulgaris Hildenborough Genome.},
author = {Chhabra, Swapnil and Butland, Gareth and Elias, Dwayne A and Chandonia, John-Marc and Fok, Olivia and Juba, tom and Gorur, A. and Allen, S. and Leung, C. M. and Keller, Kim and Reveco, S. and Zane, Mr. Grant M. and Semkiw, Elizabeth M. and Prathapam, R. and Gold, B. and Singer, Mary and Ouellet, M. and Sazakal, E. D. and Jorgens, Dominique and Price, Morgan N. and Witkowska, Ewa and Beller, Harry R. and Hazen, Terry and Biggin, Mark D. and Auer, Dr. Manfred and Wall, Judy D. and Keasling, Jay},
abstractNote = {The ability to conduct advanced functional genomic studies of the thousands of 38 sequenced bacteria has been hampered by the lack of available tools for making high39 throughput chromosomal manipulations in a systematic manner that can be applied across 40 diverse species. In this work, we highlight the use of synthetic biological tools to 41 assemble custom suicide vectors with reusable and interchangeable DNA parts to 42 facilitate chromosomal modification at designated loci. These constructs enable an array 43 of downstream applications including gene replacement and creation of gene fusions with 44 affinity purification or localization tags. We employed this approach to engineer 45 chromosomal modifications in a bacterium that has previously proven difficult to 46 manipulate genetically, Desulfovibrio vulgaris Hildenborough, to generate a library of 47 662 strains. Furthermore, we demonstrate how these modifications can be used for 48 examining metabolic pathways, protein-protein interactions, and protein localization. The 49 ubiquity of suicide constructs in gene replacement throughout biology suggests that this 50 approach can be applied to engineer a broad range of species for a diverse array of 51 systems biological applications and is amenable to high-throughput implementation.},
doi = {10.1128/AEM.05495-11},
url = {https://www.osti.gov/biblio/1028745}, journal = {Applied and Environmental Microbiology},
issn = {0099-2240},
number = 21,
volume = 77,
place = {United States},
year = {Sat Jan 01 00:00:00 EST 2011},
month = {Sat Jan 01 00:00:00 EST 2011}
}