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Genetic tools for constructing genetically engineered microorganisms with high predictability in performance and behaviour

The project considers scientific approaches to constructing genetically engineered microorganisms (GEM) of high predictability in performance and behaviour in ecological microcosms, soils, rhizospheres and river sediments. Initial research objectives are the development of:
specialized vectors to obtain gene expression under environmental conditions and stable maintenance of engineered traits;
barriers to gene transfer;
analysis and performance of killing functions and adaptation of conditional suicide systems to suicide circuits and improvement of TOL plasmid regulatory elements;
construction of polychlorinated biphenyl (PCB) degraders;
validation tests under laboratory conditions and in microcosms.

For the first objective the following 2 actions have been undertaken; construction of transposon probes to detect promoters responsive to growth phase and a study of 2 nonantibiotic selection markers; resistance to silver salts and to the herbicide glyphosate have been conducted. For the second objective the colicin E production immunity lysis system was used as a source of killing functions for biological containment. The experimental work devoted to the third objective has been aimed at designing a suitable biological containment system to be used in concert with the degradation of xenobiotics. Efforts have been directed to the construction of PCB degrading strains of pseudomonas fluorescens which are good colonizers of the sugar beet rhizosphere and p putida KT 2442.

Work to date has produced the following milestones:
a number of useful tools have been designed to obtain an efficient expression system under environmental starvation conditions;
new nonantibiotic markers for tracking herbicide and heavy metal resistance in GEMs are being developed;
the TOL meta cleavage pathway regulatory elements, the Pm promoter, and the xyls regulator have been coupled to the gef gene whose gene products kill cells when overexpressed;
Pseudomonas putida bacteria beari ng a recombinant TOL plasmid and the suicide function under Pm/Xyls control have been shown to function both in the laboratory and in soils;
the GEM F113PCB was sucessfully constructed using a suicide delivery system (pDDPCB);
the PCB phenotype was stably maintained both in vitro and after growth in a nonsterile soil during 4 weeks;
colonization of sugar beet roots by the GEM was monitored and was found to be similar to colonization of the parent in both BP amended and nonamended soil;
biologically contained strain with biodegradative potential.

Reported by

Consejo Superior de Investigaciones Cientificas
Profesor Albareda 1
18008 Granada
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