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

Kanno, AI, Goulart, C, Rofatto, HK, Oliveira, SC, Leite, LC and McFadden, J (2016) New Recombinant Mycobacterium bovis BCG Expression Vectors: Improving Genetic Control over Mycobacterial Promoters. Applied and Environmental Microbiology, 82 (8). pp. 2240-2246.

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Abstract

The expression of many antigens, stimulatory molecules, or even metabolic pathways in mycobacteria such asMycobacterium bovisBCG orM. smegmatiswas 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 PL5promoter, originally from mycobacteriophage L5. These promoters were cloned upstream of the enhanced green fluorescent protein reporter gene, and recombinantM. smegmatisbacteria 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 bothM. smegmatisandM. 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 theSchistosoma mansoniantigen 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.

Item Type: Article
Subjects : Biosciences
Divisions : Faculty of Health and Medical Sciences > School of Biosciences and Medicine
Authors :
AuthorsEmailORCID
Kanno, AIUNSPECIFIEDUNSPECIFIED
Goulart, CUNSPECIFIEDUNSPECIFIED
Rofatto, HKUNSPECIFIEDUNSPECIFIED
Oliveira, SCUNSPECIFIEDUNSPECIFIED
Leite, LCUNSPECIFIEDUNSPECIFIED
McFadden, JUNSPECIFIEDUNSPECIFIED
Date : 15 April 2016
Funders : BBSRC
Identification Number : 10.1128/AEM.03677-15
Copyright Disclaimer : Copyright © 2016 Kanno et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
Related URLs :
Depositing User : Symplectic Elements
Date Deposited : 12 Jul 2016 10:00
Last Modified : 05 Aug 2016 12:46
URI: http://epubs.surrey.ac.uk/id/eprint/811170

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