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Genetic tools for constructing genetically-engineered microorganisms (GEMs) with high predictability in performance and behavior in ecological microcosms, soils, rhizosperes and river sediments

Objective

The objectives are the development of genetic tools for constructing GEMs with high predictability in performance and behaviour in ecological microcosms, soils, rhizospheres and river sediments.
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.
The development of GEMs for biotechnological applications, involving their deliberate release, particularly in the agricultural and environmental protection sectors, is growing in importance. Concerns about possible risks, mainly the lack of predictability of GEMs behaviour in natural habitats, transfer of recombinant information to other organisms, and impact on natural populations, has become apparent.
The goal of our current work is to make available a collection of genetic tools and resources for engineering GEMs with a high degree of ecological predictability. Specific approaches are being developed to assure the stable inheritance of cloned genes, their efficient expression where and when desired, death of the GEM when its application is completed, and depression of the level of lateral transfer of recombinant genes of the GEM to indigenous bacteria. As a model microorganism we use Pseudomonas bacteria engineered to degrade toxic aromatic chemicals (alkyltoluenes and alkyl- and chlorobenzoates). Within the context of this project "new" bacteria able to eliminate polychlorinated biphenyls (PCBs) will be constructed. The construction of such bacteria will involve the transference of a genetic module containing PCB-degradation pathway of a PCB-degrading bacteria that survives poorly in soil to indigenous gram-negative ones from the plant root rhizosphere, which are more likely to survive in soils and to emove PCB's more efficient in situ.

Coordinator

CONSEJO SUPERIOR DE INVESTIGACIONES CIENTIFICAS
Address
1,Profesor Albareda 1
18008 Granada
Spain

Participants (3)

Danmarks Tekniske Universitet
Denmark
Address

2800 Lyngby
GBF - NATIONAL CENTRE FOR BIOTECHNOLOGY
Germany
Address
Mascheroder Weg 1
38124 Braunschweig
UNIVERSITY COLLEGE CORK, NATIONAL UNIVERSITY OF IRELAND, CORK
Ireland
Address
Western Road Biomerit
30 Cork