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Highly fluorescent GFP(m)(2+)‐based genome integration‐proficient promoter probe vector to study Mycobacterium tuberculosis promoters in infected macrophages

Study of activity of cloned promoters in slow‐growing Mycobacterium tuberculosis during long‐term growth conditions in vitro or inside macrophages, requires a genome‐integration proficient promoter probe vector, which can be stably maintained even without antibiotics, carrying a substrate‐independen...

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Autores principales: Roy, Sougata, Narayana, Yeddula, Narayanaswamy Balaji, Kithiganahalli, Ajitkumar, Parthasarathi
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Blackwell Publishing Ltd 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3815276/
https://www.ncbi.nlm.nih.gov/pubmed/21958386
http://dx.doi.org/10.1111/j.1751-7915.2011.00305.x
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author Roy, Sougata
Narayana, Yeddula
Narayanaswamy Balaji, Kithiganahalli
Ajitkumar, Parthasarathi
author_facet Roy, Sougata
Narayana, Yeddula
Narayanaswamy Balaji, Kithiganahalli
Ajitkumar, Parthasarathi
author_sort Roy, Sougata
collection PubMed
description Study of activity of cloned promoters in slow‐growing Mycobacterium tuberculosis during long‐term growth conditions in vitro or inside macrophages, requires a genome‐integration proficient promoter probe vector, which can be stably maintained even without antibiotics, carrying a substrate‐independent, easily scorable and highly sensitive reporter gene. In order to meet this requirement, we constructed pAKMN2, which contains mycobacterial codon‐optimized gfp(m)(2+) gene, coding for GFP(m)(2+) of highest fluorescence reported till date, mycobacteriophage L5 attP‐int sequence for genome integration, and a multiple cloning site. pAKMN2 showed stable integration and expression of GFP(m)(2+) from M. tuberculosis and M. smegmatis genome. Expression of GFP(m)(2+), driven by the cloned minimal promoters of M. tuberculosis cell division gene, ftsZ (MtftsZ), could be detected in the M. tuberculosis/pAKMN2‐promoter integrants, growing at exponential phase in defined medium in vitro and inside macrophages. Stable expression from genome‐integrated format even without antibiotic, and high sensitivity of detection by flow cytometry and fluorescence imaging, in spite of single copy integration, make pAKMN2 useful for the study of cloned promoters of any mycobacterial species under long‐term in vitro growth or stress conditions, or inside macrophages.
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spelling pubmed-38152762014-02-12 Highly fluorescent GFP(m)(2+)‐based genome integration‐proficient promoter probe vector to study Mycobacterium tuberculosis promoters in infected macrophages Roy, Sougata Narayana, Yeddula Narayanaswamy Balaji, Kithiganahalli Ajitkumar, Parthasarathi Microb Biotechnol Research Articles Study of activity of cloned promoters in slow‐growing Mycobacterium tuberculosis during long‐term growth conditions in vitro or inside macrophages, requires a genome‐integration proficient promoter probe vector, which can be stably maintained even without antibiotics, carrying a substrate‐independent, easily scorable and highly sensitive reporter gene. In order to meet this requirement, we constructed pAKMN2, which contains mycobacterial codon‐optimized gfp(m)(2+) gene, coding for GFP(m)(2+) of highest fluorescence reported till date, mycobacteriophage L5 attP‐int sequence for genome integration, and a multiple cloning site. pAKMN2 showed stable integration and expression of GFP(m)(2+) from M. tuberculosis and M. smegmatis genome. Expression of GFP(m)(2+), driven by the cloned minimal promoters of M. tuberculosis cell division gene, ftsZ (MtftsZ), could be detected in the M. tuberculosis/pAKMN2‐promoter integrants, growing at exponential phase in defined medium in vitro and inside macrophages. Stable expression from genome‐integrated format even without antibiotic, and high sensitivity of detection by flow cytometry and fluorescence imaging, in spite of single copy integration, make pAKMN2 useful for the study of cloned promoters of any mycobacterial species under long‐term in vitro growth or stress conditions, or inside macrophages. Blackwell Publishing Ltd 2012-01 2011-12-14 /pmc/articles/PMC3815276/ /pubmed/21958386 http://dx.doi.org/10.1111/j.1751-7915.2011.00305.x Text en Copyright © 2011 The Authors. Microbial Biotechnology © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd
spellingShingle Research Articles
Roy, Sougata
Narayana, Yeddula
Narayanaswamy Balaji, Kithiganahalli
Ajitkumar, Parthasarathi
Highly fluorescent GFP(m)(2+)‐based genome integration‐proficient promoter probe vector to study Mycobacterium tuberculosis promoters in infected macrophages
title Highly fluorescent GFP(m)(2+)‐based genome integration‐proficient promoter probe vector to study Mycobacterium tuberculosis promoters in infected macrophages
title_full Highly fluorescent GFP(m)(2+)‐based genome integration‐proficient promoter probe vector to study Mycobacterium tuberculosis promoters in infected macrophages
title_fullStr Highly fluorescent GFP(m)(2+)‐based genome integration‐proficient promoter probe vector to study Mycobacterium tuberculosis promoters in infected macrophages
title_full_unstemmed Highly fluorescent GFP(m)(2+)‐based genome integration‐proficient promoter probe vector to study Mycobacterium tuberculosis promoters in infected macrophages
title_short Highly fluorescent GFP(m)(2+)‐based genome integration‐proficient promoter probe vector to study Mycobacterium tuberculosis promoters in infected macrophages
title_sort highly fluorescent gfp(m)(2+)‐based genome integration‐proficient promoter probe vector to study mycobacterium tuberculosis promoters in infected macrophages
topic Research Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3815276/
https://www.ncbi.nlm.nih.gov/pubmed/21958386
http://dx.doi.org/10.1111/j.1751-7915.2011.00305.x
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