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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 sequenced bacteria has been hampered by the lack of available tools for making high- throughput chromosomal manipulations in a systematic manner that can be applied across diverse species. In this work, we highlight the use of synthetic biological tools to assemble custom suicide vectors with reusable and interchangeable DNA “parts” to facilitate chromosomal modification at designated loci. These constructs enable an array of downstream applications including gene replacement and creation of gene fusions with affinity purification or localization tags. We employed this approach to engineer chromosomal modifications in a bacterium that has previously proven difficult to manipulate genetically, Desulfovibrio vulgaris Hildenborough, to generate a library of over 700 strains. Furthermore, we demonstrate how these modifications can be used for examining metabolic pathways, protein-protein interactions, and protein localization. The ubiquity of suicide constructs in gene replacement throughout biology suggests that this approach can be applied to engineer a broad range of species for a diverse array of systems biological applications and is amenable to high-throughput implementation.

Authors:
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Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
Earth Sciences Division
OSTI Identifier:
1052164
Report Number(s):
LBNL-5207E
DOE Contract Number:  
DE-AC02-05CH11231
Resource Type:
Journal Article
Journal Name:
Applied&Environmental Microbiology
Additional Journal Information:
Journal Volume: 77; Journal Issue: 22; Related Information: Journal Publication Date: 2011
Country of Publication:
United States
Language:
English
Subject:
54 ENVIRONMENTAL SCIENCES; 58 GEOSCIENCES; 59 BASIC BIOLOGICAL SCIENCES; Functional Genomics; Microbial Chromosomal Engineering; Obligate Anaerobic Bacteria

Citation Formats

Chhabra, S.R., Butland, G., Elias, D., Chandonia, J.-M., Fok, V., Juba, T., Gorur, A., Allen, S., Leung, C.-M., Keller, K., Reveco, S., Zane, G., Semkiw, E., Prathapam, R., Gold, B., Singer, M., Ouellet, M., Sazakal, E., Jorgens, D., Price, M., Witkowska, E., Beller, H., Hazen, T.C., Biggin, M., Auer, M., Wall, J., and Keasling, J. Generalized schemes for high throughput manipulation of the Desulfovibrio vulgaris Hildenborough genome. United States: N. p., 2011. Web.
Chhabra, S.R., Butland, G., Elias, D., Chandonia, J.-M., Fok, V., Juba, T., Gorur, A., Allen, S., Leung, C.-M., Keller, K., Reveco, S., Zane, G., Semkiw, E., Prathapam, R., Gold, B., Singer, M., Ouellet, M., Sazakal, E., Jorgens, D., Price, M., Witkowska, E., Beller, H., Hazen, T.C., Biggin, M., Auer, M., Wall, J., & Keasling, J. Generalized schemes for high throughput manipulation of the Desulfovibrio vulgaris Hildenborough genome. United States.
Chhabra, S.R., Butland, G., Elias, D., Chandonia, J.-M., Fok, V., Juba, T., Gorur, A., Allen, S., Leung, C.-M., Keller, K., Reveco, S., Zane, G., Semkiw, E., Prathapam, R., Gold, B., Singer, M., Ouellet, M., Sazakal, E., Jorgens, D., Price, M., Witkowska, E., Beller, H., Hazen, T.C., Biggin, M., Auer, M., Wall, J., and Keasling, J. Fri . "Generalized schemes for high throughput manipulation of the Desulfovibrio vulgaris Hildenborough genome". United States. https://www.osti.gov/servlets/purl/1052164.
@article{osti_1052164,
title = {Generalized schemes for high throughput manipulation of the Desulfovibrio vulgaris Hildenborough genome},
author = {Chhabra, S.R. and Butland, G. and Elias, D. and Chandonia, J.-M. and Fok, V. and Juba, T. and Gorur, A. and Allen, S. and Leung, C.-M. and Keller, K. and Reveco, S. and Zane, G. and Semkiw, E. and Prathapam, R. and Gold, B. and Singer, M. and Ouellet, M. and Sazakal, E. and Jorgens, D. and Price, M. and Witkowska, E. and Beller, H. and Hazen, T.C. and Biggin, M. and Auer, M. and Wall, J. and Keasling, J.},
abstractNote = {The ability to conduct advanced functional genomic studies of the thousands of sequenced bacteria has been hampered by the lack of available tools for making high- throughput chromosomal manipulations in a systematic manner that can be applied across diverse species. In this work, we highlight the use of synthetic biological tools to assemble custom suicide vectors with reusable and interchangeable DNA “parts” to facilitate chromosomal modification at designated loci. These constructs enable an array of downstream applications including gene replacement and creation of gene fusions with affinity purification or localization tags. We employed this approach to engineer chromosomal modifications in a bacterium that has previously proven difficult to manipulate genetically, Desulfovibrio vulgaris Hildenborough, to generate a library of over 700 strains. Furthermore, we demonstrate how these modifications can be used for examining metabolic pathways, protein-protein interactions, and protein localization. The ubiquity of suicide constructs in gene replacement throughout biology suggests that this approach can be applied to engineer a broad range of species for a diverse array of systems biological applications and is amenable to high-throughput implementation.},
doi = {},
journal = {Applied&Environmental Microbiology},
number = 22,
volume = 77,
place = {United States},
year = {2011},
month = {7}
}