A New Large-DNA-Fragment Delivery System Based on Integrase Activity from an Integrative and Conjugative Element.

Détails

ID Serval
serval:BIB_064586E230F3
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Institution
Titre
A New Large-DNA-Fragment Delivery System Based on Integrase Activity from an Integrative and Conjugative Element.
Périodique
Applied and Environmental Microbiology
Auteur⸱e⸱s
Miyazaki R., van der Meer J.R.
ISSN
1098-5336 (Electronic)
ISSN-L
0099-2240
Statut éditorial
Publié
Date de publication
2013
Peer-reviewed
Oui
Volume
79
Numéro
14
Pages
4440-4447
Langue
anglais
Notes
Publication types: Journal Article Publication Status: ppublish
Résumé
During the past few decades, numerous plasmid vectors have been developed for cloning, gene expression analysis, and genetic engineering. Cloning procedures typically rely on PCR amplification, DNA fragment restriction digestion, recovery, and ligation, but increasingly, procedures are being developed to assemble large synthetic DNAs. In this study, we developed a new gene delivery system using the integrase activity of an integrative and conjugative element (ICE). The advantage of the integrase-based delivery is that it can stably introduce a large DNA fragment (at least 75 kb) into one or more specific sites (the gene for glycine-accepting tRNA) on a target chromosome. Integrase recombination activity in Escherichia coli is kept low by using a synthetic hybrid promoter, which, however, is unleashed in the final target host, forcing the integration of the construct. Upon integration, the system is again silenced. Two variants with different genetic features were produced, one in the form of a cloning vector in E. coli and the other as a mini-transposable element by which large DNA constructs assembled in E. coli can be tagged with the integrase gene. We confirmed that the system could successfully introduce cosmid and bacterial artificial chromosome (BAC) DNAs from E. coli into the chromosome of Pseudomonas putida in a site-specific manner. The integrase delivery system works in concert with existing vector systems and could thus be a powerful tool for synthetic constructions of new metabolic pathways in a variety of host bacteria.
Pubmed
Web of science
Open Access
Oui
Création de la notice
11/08/2013 8:33
Dernière modification de la notice
20/08/2019 12:28
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