New Recombinant Mycobacterium bovis BCG Expression Vectors: Improving Genetic Control over Mycobacterial Promoters.

Kanno, AI; Goulart, C; Rofatto, HK; Oliveira, SC; Leite, LCC; McFadden, J

New Recombinant Mycobacterium bovis BCG Expression Vectors: Improving Genetic Control over Mycobacterial Promoters.

Kanno, AI Goulart, C

Rofatto, HK Oliveira, SC Leite, LCC McFadden, J

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Publisher:: AMER SOC MICROBIOLOGY
Publication Type:: Journal Article
Citation:: Appl Environ Microbiol, 2016, 82, (8), pp. 2240-2246
Issue Date:: 2016-04

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Field	Value	Language
dc.contributor.author	Kanno, AI
dc.contributor.author	Goulart, C https://orcid.org/0000-0003-2981-1934
dc.contributor.author	Rofatto, HK
dc.contributor.author	Oliveira, SC
dc.contributor.author	Leite, LCC
dc.contributor.author	McFadden, J
dc.date.accessioned	2022-03-22T23:59:43Z
dc.date.available	2016-01-25
dc.date.available	2022-03-22T23:59:43Z
dc.date.issued	2016-04
dc.identifier.citation	Appl Environ Microbiol, 2016, 82, (8), pp. 2240-2246
dc.identifier.issn	0099-2240
dc.identifier.issn	1098-5336
dc.identifier.uri	http://hdl.handle.net/10453/155472
dc.description.abstract	The expression of many antigens, stimulatory molecules, or even metabolic pathways in mycobacteria such as Mycobacterium bovis BCG or M. smegmatis was made possible through the development of shuttle vectors, and several recombinant vaccines have been constructed. However, gene expression in any of these systems relied mostly on the selection of natural promoters expected to provide the required level of expression by trial and error. To establish a systematic selection of promoters with a range of strengths, we generated a library of mutagenized promoters through error-prone PCR of the strong PL5 promoter, originally from mycobacteriophage L5. These promoters were cloned upstream of the enhanced green fluorescent protein reporter gene, and recombinant M. smegmatis bacteria exhibiting a wide range of fluorescence levels were identified. A set of promoters was selected and identified as having high (pJK-F8), intermediate (pJK-B7, pJK-E6, pJK-D6), or low (pJK-C1) promoter strengths in both M. smegmatis and M. bovisBCG. The sequencing of the promoter region demonstrated that it was extensively modified (6 to 11%) in all of the plasmids selected. To test the functionality of the system, two different expression vectors were demonstrated to allow corresponding expression levels of the Schistosoma mansoni antigen Sm29 in BCG. The approach used here can be used to adjust expression levels for synthetic and/or systems biology studies or for vaccine development to maximize the immune response.
dc.format	Electronic-Print
dc.language	eng
dc.publisher	AMER SOC MICROBIOLOGY
dc.relation.ispartof	Appl Environ Microbiol
dc.relation.isbasedon	10.1128/AEM.03677-15
dc.rights	info:eu-repo/semantics/openAccess
dc.subject.classification	Microbiology
dc.subject.mesh	Animals
dc.subject.mesh	Antigens, Helminth
dc.subject.mesh	Artificial Gene Fusion
dc.subject.mesh	Cloning, Molecular
dc.subject.mesh	Gene Expression
dc.subject.mesh	Genes, Reporter
dc.subject.mesh	Genetic Vectors
dc.subject.mesh	Green Fluorescent Proteins
dc.subject.mesh	Helminth Proteins
dc.subject.mesh	Membrane Glycoproteins
dc.subject.mesh	Mutagenesis
dc.subject.mesh	Mycobacteriophages
dc.subject.mesh	Mycobacterium bovis
dc.subject.mesh	Mycobacterium smegmatis
dc.subject.mesh	Plasmids
dc.subject.mesh	Polymerase Chain Reaction
dc.subject.mesh	Promoter Regions, Genetic
dc.subject.mesh	Schistosoma mansoni
dc.subject.mesh	Sequence Analysis, DNA
dc.subject.mesh	Animals
dc.subject.mesh	Schistosoma mansoni
dc.subject.mesh	Mycobacterium smegmatis
dc.subject.mesh	Mycobacterium bovis
dc.subject.mesh	Mycobacteriophages
dc.subject.mesh	Membrane Glycoproteins
dc.subject.mesh	Helminth Proteins
dc.subject.mesh	Green Fluorescent Proteins
dc.subject.mesh	Antigens, Helminth
dc.subject.mesh	Cloning, Molecular
dc.subject.mesh	Polymerase Chain Reaction
dc.subject.mesh	Sequence Analysis, DNA
dc.subject.mesh	Gene Expression
dc.subject.mesh	Mutagenesis
dc.subject.mesh	Genes, Reporter
dc.subject.mesh	Genetic Vectors
dc.subject.mesh	Plasmids
dc.subject.mesh	Artificial Gene Fusion
dc.subject.mesh	Promoter Regions, Genetic
dc.title	New Recombinant Mycobacterium bovis BCG Expression Vectors: Improving Genetic Control over Mycobacterial Promoters.
dc.type	Journal Article
utslib.citation.volume	82
utslib.location.activity	United States
pubs.organisational-group	/University of Technology Sydney
pubs.organisational-group	/University of Technology Sydney/Faculty of Science
pubs.organisational-group	/University of Technology Sydney/Faculty of Science/School of Life Sciences
utslib.copyright.status	open_access	*
dc.date.updated	2022-03-22T23:59:39Z
pubs.issue	8
pubs.publication-status	Published online
pubs.volume	82
utslib.citation.issue	8

Abstract:

The expression of many antigens, stimulatory molecules, or even metabolic pathways in mycobacteria such as Mycobacterium bovis BCG or M. smegmatis was made possible through the development of shuttle vectors, and several recombinant vaccines have been constructed. However, gene expression in any of these systems relied mostly on the selection of natural promoters expected to provide the required level of expression by trial and error. To establish a systematic selection of promoters with a range of strengths, we generated a library of mutagenized promoters through error-prone PCR of the strong PL5 promoter, originally from mycobacteriophage L5. These promoters were cloned upstream of the enhanced green fluorescent protein reporter gene, and recombinant M. smegmatis bacteria exhibiting a wide range of fluorescence levels were identified. A set of promoters was selected and identified as having high (pJK-F8), intermediate (pJK-B7, pJK-E6, pJK-D6), or low (pJK-C1) promoter strengths in both M. smegmatis and M. bovisBCG. The sequencing of the promoter region demonstrated that it was extensively modified (6 to 11%) in all of the plasmids selected. To test the functionality of the system, two different expression vectors were demonstrated to allow corresponding expression levels of the Schistosoma mansoni antigen Sm29 in BCG. The approach used here can be used to adjust expression levels for synthetic and/or systems biology studies or for vaccine development to maximize the immune response.

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http://hdl.handle.net/10453/155472