Retrotransfert et mobilisation de plasmides à large spectre d'hôtes.

Doctorat en Sciencesinfo:eu-repo/semantics/nonPublishe

Rétrotransfert et mobilisation de plasmides à large spectre d'hôtes

Doctorat en Sciencesinfo:eu-repo/semantics/nonPublishe

Rétrotransfert et mobilisation de plasmides à large spectre d'hôtes

Doctorat en Sciencesinfo:eu-repo/semantics/nonPublishe

Separate-component-stabilization system for protein and DNA production without the use of antibiotics.

Plasmid instability is a significant concern in the industrial utilization of microorganisms for protein or DNA production. Here we report on the development of a new and highly effective stabilization system based on the use of the ccd antidote/poison genes. For the first time, we separated the antidote gene from the poison gene: localizing the former in the plasmid and integrating the latter in the bacterial chromosome. We show that this separate-component-stabilization (SCS) strategy: (i) allows for perfect stabilization without the use of antibiotics; (ii) increases three to five times the recombinant protein production levels; and (iii) does not require any specific modification of the protein production process or culture medium. We illustrate that point by using the classical T7 promotor (i.e. used in most expression systems). Finally, we demonstrate that the SCS system increases by five the yield in DNA production, a result especially important for the design and production of gene therapy constructs void of any antibiotic resistance gene.Evaluation StudiesJournal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

The art of selective killing: plasmid toxin/antitoxin systems and their technological applications.

Journal ArticleReviewinfo:eu-repo/semantics/publishe

Automated discovery and phylogenetic analysis of new toxin-antitoxin systems.

Although often viewed as elements "at the service of" bacteria, plasmids exhibit replication and maintenance mechanisms that make them purely "selfish DNA" candidates. Toxin-antitoxin (TA) systems are a spectacular example of such mechanisms: a gene coding for a cytotoxic stable protein is preceded by a gene coding for an unstable antitoxin. The toxin being more stable than the antitoxin, absence of the operon causes a reduction of the amount of the latter relative to the amount of the former. Thus, a cell exhibiting a TA system on a plasmid is 'condemned' either not to loose it or to die.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Interaction between the RP4 coupling protein TraG and the pBHR1 mobilization protein Mob

It is currently believed that interaction between the relaxosome of a mobilizable plasmid and the transfer machinery of the helper conjugative plasmid is mediated by a TraG family coupling protein. The coupling proteins appear as an essential determinant of mobilization specificity and efficiency. Using a two-hybrid system, we demonstrated for the first time the direct in vivo interaction between the coupling protein of a conjugative plasmid (the TraG protein of RP4) and the relaxase of a mobilizable plasmid (the Mob protein of pBHR1, a derivative of the broad host range plasmid pBBR1). This interaction was confirmed in vitro by an overlay assay and was shown to occur even in the absence of the transfer origin of pBHR1. We showed that, among 11 conjugative plasmids tested, pBHR1 is efficiently mobilized only by plasmids encoding an IncP-type transfer system. We also showed that the RP4 TraG coupling protein is essential for mobilization of a pBBR1 derivative and is the element that allows its mobilization by R388 plasmid (IncW) at a detectable frequency.Journal ArticleResearch Support, Non-U.S. Gov'tFLWINinfo:eu-repo/semantics/publishe

Method for the selection of recombinant clones comprising a sequence encoding an antidote protein to a toxic molecule

info:eu-repo/semantics/publishe

Plasmid poison/antidote systems: functions and technological applications

info:eu-repo/semantics/publishe

Direct selection cloning vectors adapted to the genetic analysis of Gram-negative bacteria and their plasmids

A range of specific and unusual biological pathways are found in Gram-negative bacteria. It is possible to express the genes involved in these processes in Escherichia coli, however, some genes prove lethal when cloned into high copy number vectors in common usage. Conversely, various genetic functions remain silent in E. coli and require to be transferred into their original host for expression and subsequent analysis. To facilitate the cloning and the characterisation of bacterial genes, we have constructed CcdB 'positive-selection' vectors that possess one or more of the following properties: (i) low or medium copy number; (ii) narrow or broad replication host range; (iii) conjugational mobilisation. In this communication, we illustrate the use of these new cloning tools and analyse the CcdB toxicity in different bacterial species.SCOPUS: ar.jinfo:eu-repo/semantics/publishe