Publication

Construction of pDYN6902, a new Streptomyces integrative expression vector designed for cloning sequences interfering with Escherichia coli viability

Julien Hiblot
2015
Journal Articles

Abstract

We describe here the construction of a new version of the Escherichia coli-Streptomyces shuttle cloning vector pIJ6902, which carries the original thiostrepton-inducible PtipA as well as the integrative and cloning systems, but with the E. coil low copy number replication control of the F (fertility) factor. The rationale of this construct was to provide a shuttle cloning vector to enable the cloning of genes whose expression is toxic in E. coli while keeping the efficient regulated expression system for Streptomyces as its ascendant vector pIJ06902. This new vector named pIJ6902 (10,975 bp) showed a copy-number reduced 12 times compared to that of pDYN6902 and indeed a decreased expression of the cloned gene. It was also shown to allow the successful cloning in E. coli of a deleterious gene, i.e. I-Scel meganuclease encoding gene. (C) 2015 Elsevier Inc. All rights reserved.

Official source

https://infoscience.epfl.ch/entities/publication/d18b4db9-2bac-472b-b1ae-0661d872288c

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Julien Hiblot
2015
Journal Articles

Abstract

Official source

https://infoscience.epfl.ch/entities/publication/d18b4db9-2bac-472b-b1ae-0661d872288c

About this result

Related concepts (32)

Molecular cloning

Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms. The use of the word cloning refers to the fact that the method involves the replication of one molecule to produce a population of cells with identical DNA molecules. Molecular cloning generally uses DNA sequences from two different organisms: the species that is the source of the DNA to be cloned, and the species that will serve as the living host for replication of the recombinant DNA.

Cloning vector

A cloning vector is a small piece of DNA that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes. The cloning vector may be DNA taken from a virus, the cell of a higher organism, or it may be the plasmid of a bacterium. The vector contains features that allow for the convenient insertion of a DNA fragment into the vector or its removal from the vector, for example through the presence of restriction sites.

Multiple cloning site

A multiple cloning site (MCS), also called a polylinker, is a short segment of DNA which contains many (up to ~20) restriction sites - a standard feature of engineered plasmids. Restriction sites within an MCS are typically unique, occurring only once within a given plasmid. The purpose of an MCS in a plasmid is to allow a piece of DNA to be inserted into that region. An MCS is found in a variety of vectors, including cloning vectors to increase the number of copies of target DNA, and in expression vectors to create a protein product.

Related publications (32)

Construction of pDYN6902, a new Streptomyces integrative expression vector designed for cloning sequences interfering with Escherichia coli viability

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