Cargando…
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...
Autores principales: | , , , , , , , , , , , |
---|---|
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 |
_version_ | 1782480591516598272 |
---|---|
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. |
format | Online Article Text |
id | pubmed-4049104 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | American Society of Microbiology |
record_format | MEDLINE/PubMed |
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 |
work_keys_str_mv | AT jainparas specializedtransductiondesignedforprecisehighthroughputunmarkeddeletionsinmycobacteriumtuberculosis AT hsutsungda specializedtransductiondesignedforprecisehighthroughputunmarkeddeletionsinmycobacteriumtuberculosis AT araimasayoshi specializedtransductiondesignedforprecisehighthroughputunmarkeddeletionsinmycobacteriumtuberculosis AT biermannkarolin specializedtransductiondesignedforprecisehighthroughputunmarkeddeletionsinmycobacteriumtuberculosis AT thalerdavids specializedtransductiondesignedforprecisehighthroughputunmarkeddeletionsinmycobacteriumtuberculosis AT nguyenandrew specializedtransductiondesignedforprecisehighthroughputunmarkeddeletionsinmycobacteriumtuberculosis AT gonzalezpabloa specializedtransductiondesignedforprecisehighthroughputunmarkeddeletionsinmycobacteriumtuberculosis AT tufariellojoannm specializedtransductiondesignedforprecisehighthroughputunmarkeddeletionsinmycobacteriumtuberculosis AT kriakovjordan specializedtransductiondesignedforprecisehighthroughputunmarkeddeletionsinmycobacteriumtuberculosis AT chenbing specializedtransductiondesignedforprecisehighthroughputunmarkeddeletionsinmycobacteriumtuberculosis AT larsenmichelleh specializedtransductiondesignedforprecisehighthroughputunmarkeddeletionsinmycobacteriumtuberculosis AT jacobswilliamr specializedtransductiondesignedforprecisehighthroughputunmarkeddeletionsinmycobacteriumtuberculosis |