Comparison of transformation protocols in Streptococcus gordonii and evaluation of native promoter strength using a multiple-copy plasmid

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Abstract:

An active area of research in the development of Streptococcus gordonii for use as a bacterial commensal vector involves the identification and utilization of strong promoters for high-level expression of heterologous products. Escherichia coli plasmid vectors containing different streptococcal promoters often fail to become established in E. coli for unknown reasons. Therefore, it is desirable at times to transform S. gordonii, which is naturally competent, with small quantities of nascently ligated DNA without using E. coli first to amplify or screen the product. By comparing the efficiency of two methods used to induce competence in S. gordonii, it was shown that the use of a synthetic competence stimulating peptide substantially enhanced plasmid uptake by S. gordonii. We amplified the amylase-binding protein (abpA) promoter from the S. gordonii genome and, using a synthetic peptide to induce competence, directly introduced plasmid DNA containing this promoter into S. gordonii as an unamplified product of ligation. This plasmid facilitated abundant secretion of a heterologous product by S. gordonii. By assessing the levels of heterologous product secreted by two plasmid constructs, it was possible to evaluate the relative strength of two native promoters.

L’identification et l’utilisation de promoteurs forts permettant d’exprimer une grande quantité des protéines hétérologues est un domaine de recherche actif dans la perspective d’utiliser Streptococcus gordonii comme vecteur bactérien commensal. Des vecteurs plasmidiques contenant différents promoteurs du streptocoque n’arrivent souvent pas à s’établir chez Escherichia coli pour des raisons inconnues. Il est ainsi souhaitable de transformer S. gordonii qui est naturellement compétent, avec de petites quantités d’ADN ligaturé sans utiliser d’abord E. coli pour amplifier ou cribler le produit. En comparant l’efficacité de deux méthodes pour induire la compétence chez S. gordonii, l’on a montré que l’utilisation de peptides synthétiques de stimulation de la compétence augmentait substantiellement la captation du plasmide par S. gordonii. Nous avons amplifié le promoteur de abpA (amylase-binding protein) du génome de S. gordonii et, en utilisant un peptide synthétique pour induire la compétence, nous avons directement introduit l’ADN plasmidique contenant ce promoteur dans S. gordonii en tant que produit non amplifié de la ligation. Ce plasmide a facilité une sécrétion abondante d’un produit hétérologue par S. gordonii. En estimant les quantités de produit hétérologue sécrété par deux constructions plasmidiques, il a été possible d’évaluer la force relative de deux promoteurs natifs.

Document Type: Research Article

Publication date: March 1, 2007

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  • Published since 1954, this monthly journal contains new research in the field of microbiology including applied microbiology and biotechnology; microbial structure and function; fungi and other eucaryotic protists; infection and immunity; microbial ecology; physiology, metabolism and enzymology; and virology, genetics, and molecular biology. It also publishes review articles and notes on an occasional basis, contributed by recognized scientists worldwide.
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