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Specialized Transduction Designed for Precise High-Throughput Unmarked Deletions in Mycobacterium tuberculosis

Specialized transduction has proven to be useful for generating deletion mutants in most mycobacteria, including virulent Mycobacterium tuberculosis. We have improved this system by developing (i) a single-step strategy for the construction of allelic exchange substrates (AES), (ii) a temperature-se...

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Autores principales: Jain, Paras, Hsu, Tsungda, Arai, Masayoshi, Biermann, Karolin, Thaler, David S., Nguyen, Andrew, González, Pablo A., Tufariello, Joann M., Kriakov, Jordan, Chen, Bing, Larsen, Michelle H., Jacobs, William R.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society of Microbiology 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4049104/
https://www.ncbi.nlm.nih.gov/pubmed/24895308
http://dx.doi.org/10.1128/mBio.01245-14
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author Jain, Paras
Hsu, Tsungda
Arai, Masayoshi
Biermann, Karolin
Thaler, David S.
Nguyen, Andrew
González, Pablo A.
Tufariello, Joann M.
Kriakov, Jordan
Chen, Bing
Larsen, Michelle H.
Jacobs, William R.
author_facet Jain, Paras
Hsu, Tsungda
Arai, Masayoshi
Biermann, Karolin
Thaler, David S.
Nguyen, Andrew
González, Pablo A.
Tufariello, Joann M.
Kriakov, Jordan
Chen, Bing
Larsen, Michelle H.
Jacobs, William R.
author_sort Jain, Paras
collection PubMed
description Specialized transduction has proven to be useful for generating deletion mutants in most mycobacteria, including virulent Mycobacterium tuberculosis. We have improved this system by developing (i) a single-step strategy for the construction of allelic exchange substrates (AES), (ii) a temperature-sensitive shuttle phasmid with a greater cloning capacity than phAE87, and (iii) bacteriophage-mediated transient expression of site-specific recombinase to precisely excise antibiotic markers. The methods ameliorate rate-limiting steps in strain construction in these difficult-to-manipulate bacteria. The new methods for strain construction were demonstrated to generalize to all classes of genes and chromosomal loci by generating more than 100 targeted single- or multiple-deletion substitutions. These improved methods pave the way for the generation of a complete ordered library of M. tuberculosis null strains, where each strain is deleted for a single defined open reading frame in M. tuberculosis.
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spelling pubmed-40491042014-06-12 Specialized Transduction Designed for Precise High-Throughput Unmarked Deletions in Mycobacterium tuberculosis Jain, Paras Hsu, Tsungda Arai, Masayoshi Biermann, Karolin Thaler, David S. Nguyen, Andrew González, Pablo A. Tufariello, Joann M. Kriakov, Jordan Chen, Bing Larsen, Michelle H. Jacobs, William R. mBio Research Article Specialized transduction has proven to be useful for generating deletion mutants in most mycobacteria, including virulent Mycobacterium tuberculosis. We have improved this system by developing (i) a single-step strategy for the construction of allelic exchange substrates (AES), (ii) a temperature-sensitive shuttle phasmid with a greater cloning capacity than phAE87, and (iii) bacteriophage-mediated transient expression of site-specific recombinase to precisely excise antibiotic markers. The methods ameliorate rate-limiting steps in strain construction in these difficult-to-manipulate bacteria. The new methods for strain construction were demonstrated to generalize to all classes of genes and chromosomal loci by generating more than 100 targeted single- or multiple-deletion substitutions. These improved methods pave the way for the generation of a complete ordered library of M. tuberculosis null strains, where each strain is deleted for a single defined open reading frame in M. tuberculosis. American Society of Microbiology 2014-06-03 /pmc/articles/PMC4049104/ /pubmed/24895308 http://dx.doi.org/10.1128/mBio.01245-14 Text en Copyright © 2014 Jain et al. http://creativecommons.org/licenses/by-nc-sa/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license (http://creativecommons.org/licenses/by-nc-sa/3.0/) , which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Jain, Paras
Hsu, Tsungda
Arai, Masayoshi
Biermann, Karolin
Thaler, David S.
Nguyen, Andrew
González, Pablo A.
Tufariello, Joann M.
Kriakov, Jordan
Chen, Bing
Larsen, Michelle H.
Jacobs, William R.
Specialized Transduction Designed for Precise High-Throughput Unmarked Deletions in Mycobacterium tuberculosis
title Specialized Transduction Designed for Precise High-Throughput Unmarked Deletions in Mycobacterium tuberculosis
title_full Specialized Transduction Designed for Precise High-Throughput Unmarked Deletions in Mycobacterium tuberculosis
title_fullStr Specialized Transduction Designed for Precise High-Throughput Unmarked Deletions in Mycobacterium tuberculosis
title_full_unstemmed Specialized Transduction Designed for Precise High-Throughput Unmarked Deletions in Mycobacterium tuberculosis
title_short Specialized Transduction Designed for Precise High-Throughput Unmarked Deletions in Mycobacterium tuberculosis
title_sort specialized transduction designed for precise high-throughput unmarked deletions in mycobacterium tuberculosis
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4049104/
https://www.ncbi.nlm.nih.gov/pubmed/24895308
http://dx.doi.org/10.1128/mBio.01245-14
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