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EC-sponsored Research on Safety of Genetically Modified Organisms - A Review of Results
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imageimageimage Plant  microbes
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image Use of marker/reporter genes in microbial ecology

Background and objectives

The deliberate release of genetically modified micro-organisms (GMMs) (e.g. biofertilisers and biopesticides) is regulated to ensure that the risk of hazardous consequences is kept to a minimum. Specific genes can be used as markers for monitoring of bacterial survival or as reporters for monitoring gene expression. There are a variety of specific marker and reporter genes being developed, each with particular advantages and disadvantages. Marker techniques provide significant advantages for risk assessment studies compared to traditional methodologies and, when used in combination with other approaches, provide information that is essential for risk assessment of GMMs. This information arises directly from tracking of GMMs in dedicated risk assessment studies but also through more fundamental studies designed to increase our understanding of microbial ecology.

We have therefore chosen to monitor progress on novel approaches for monitoring GMMs in nature and their biosafety aspects and to promote information and technology exchange between laboratories having expertise with different marker genes, reporter genes and monitoring methods. We aimed to propose standardised test-methods for risk assessment, to review and develop the use of reporter genes for monitoring gene activity in specific environments and to promote coordination of scientific efforts between academic and regulatory authorities in the handling of biosafety data and development of monitoring tools.

Folsomia candida (Collembola) fed with gfp-tagged Escherichia coli. image Folsomia candida (Collembola) fed with gfp-tagged Escherichia coli. (Epifluorescence microscopy image, contributed by Christoph Tebbe.)
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A barley seed inoculated with the gfp-tagged biocontrol strain, Pseudomonas chlororaphis MA342. image A barley seed inoculated with the gfp-tagged biocontrol strain, Pseudomonas chlororaphis MA342. A layer of gfp-tagged green fluorescent bacterial cells is seen underneath the barley seed coat. (Fluorescence stereomicroscopy image, contributed by Janet Jansson.)

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Colonisation of the gfp-tagged biocontrol strain Pseudomonas chlororaphis MA 342 of the barley seed surface. image Colonisation of the gfp-tagged biocontrol strain Pseudomonas chlororaphis MA 342 of the barley seed surface. (3-D rendering of confocal microscopy image, contributed by Janet Jansson.)
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Colonies of two bacteria, tagged with different luc genes. image Colonies of two bacteria, tagged with different luc genes. The colonies are grown on the same agar plate and the different bacterial strains are distinguished on the basis of the color of light emitted by the respective luciferase enzymes. (Contributed by Antonio Palomare.)
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Example of a field release of luc-tagged Sinorhizobium meliloti. image Example of a field release of luc-tagged Sinorhizobium meliloti. (Contributed by Christoph Tebbe.)



Approach and methodology

During the course of the project, the partners met at a series of workshops to discuss and write reports on marker genes, reporter genes, monitoring methods and biosafety. At each workshop, the participants met in a series of work sessions to discuss and make recommendations about key aspects of the various topics. At the end of each workshop a report was formulated which was later revised and published as an “opinion” of the workshop participants. In addition, we sponsored two international conferences on marker/reporter genes in microbial ecology. At the conferences, the state-of-the-art of markers and reporters and examples of their application in research were presented via a series of oral and poster presentations.
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A micropipette tip filled with luc-tagged bacteria that emit light (bioluminescence) upon addition of substrate. image A micropipette tip filled with luc-tagged bacteria that emit light (bioluminescence) upon addition of substrate. (Contributed by Antonio Palomares.)
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Intracellular colonisation of the gfp-tagged biocontrol strain Pseudomonas chlororaphis MA 342 of the barley seed coat cells. image Intracellular colonisation of the gfp-tagged biocontrol strain Pseudomonas chlororaphis MA 342 of the barley seed coat cells. (Confocal microscopy image contributed by Janet Jansson.)


Main findings and outcome

The first workshop on “Marker Genes” resulted in a report that described the characteristics of available marker systems, assessed approaches used in the construction of marked strains and analysed their use in a hypothetical “test case” release experiment.

The second workshop, on "Reporter Genes", resulted in a report that included factors to consider when choosing a reporter gene, in addition to practical aspects such as choice of host and finding environmentally inducible promoters in host cells. Applications of target genes and promoters were also described, along with recommendations for future improvements.

The report from the third workshop, on “monitoring methods” focused on the methodology used in applying marker and reporter genes to assess the impact of environmental conditions on specific microbes and microbial communities, the presence and abundance of organisms and the metabolic activity of these organisms.

The fourth workshop focused on biosafety. Two main issues were defined with respect to the biosafety of marker and reporter genes: 1) the safety of the marker/reporter genes themselves and 2) how marker/reporter genes can be used for biosafety assessment of GMMs. A consensus was reached that the marker/reporter DNA itself presents no additional risks to a GMM. It is only the gene product(s) of introduced genes that may potentially pose a hazard. Therefore, the safety and utility of the gene products were addressed in the report. In particular, the risks with use of antibiotic resistance genes as markers were thoroughly evaluated. We concluded that antibiotic resistance genes are valuable tools for the construction and monitoring of GMOs in laboratory, greenhouse and small-scale field trials. Based on our knowledge of field released GMMs we believe that the hazards of non-clinical antibiotic resistance determinants used to date are negligible. However, it is prudent at this stage to continue to use good judgement in the choice and utilisation of these markers. Antibiotic resistance genes are not recommended for use in large-scale and commercial applications, and alternative markers are available and should be used when possible in place of antibiotic resistance markers.

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Conclusions

Most of the markers available to date are safe for use in the laboratory or field-released organisms. However, the use of genes encoding resistance to clinically-important antibiotics in large-scale environmental releases should be avoided. We now have methods to monitor GMMs and their impact to provide factual information. These tools should be more widely implemented to assist in regulatory decision-making.


Major publications

Jansson J.K., van Elsas J.D. and Bailey M.J. (eds.), Tracking genetically-engineered micro-organisms, Biotechnology Intelligence Unit 2, Landes Bioscience, EUREKAH.COM, Austin, Texas, USA, 2000, 164 pp.

De Lorenzo V. et. al., Marker genes as tags for monitoring micro-organisms in nature – An opinion, EC Biotechnology Programme, DGXII, 1998.

Bailey M. et. al., Reporter genes for monitoring microbial cell activity and/or the environment - An opinion, EC Biotechnology Programme, DGXII, 2000.

de Bruijn F.J. et. al., Monitoring methods for specific micro-organisms and microbial communities in nature - An opinion, EC Biotechnology Programme, DGXII, 2001.

Atlas R. et. al., Biosafety aspects of marker and reporter genes used for monitoring micro-organisms in nature - An opinion, EC Biotechnology Programme, DGXII, 2001.

Microbial Ecology, Volume 41, Special issue on marker/reporter genes in microbial ecology (MAREP): Molecular tools to study in situ microbial activity, 2001.

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imageConcerted action
 

Contract number
BIO4-CT96-0434

Period
November 1996 – December 1999

Coordinator
J. Jansson
Södertörns högskola
Huddinge (SE)


Project website address
http://www.sh.se/marep/
marep.html

 
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Partners


J.D. van Elsas
Plant Research International (formerly IPO-DLO)
Wageningen (NL)

K. Gustafsson
Swedish National Chemicals Inspectorate
Solna (SE)

S. Molin
Technical University of Denmark
Lyngby (DK)

E. Top, W. Verstraete
University of Ghent (BE)

J. Prosser, A. Glover,
K. Killham

University of Aberdeen (UK)

M. Mergeay
Laboratory of Genetics and Biotechnology (SCK/VITO)
Mol (BE)

J. Vanderleyden
Katholieke Universiteit Leuven
Heverlee (BE)

C. Tebbe
Bundesforschungsanstalt für Landwirtschaft (FAL)
Braunschweig (DE)

W. Selbitschka,
A. Pühler

Universität Bielefeld (DE)

A.J. Palomares
University of Seville (ES)

V. de Lorenzo
CSIC
Centro Nacional de Biotecnología
Madrid (ES)

O. Nybroe
Royal Veterinary and Agricultural University
Frederiksberg (DK)

M. Romantschuk,
K. Lindström

University of Helsinki (FI)

M. Karp
University of Turku (FI)

J.A.W. Morgan
Horticulture Research International
Wellesbourne (UK)

G. Stewart, P. Hill
University of Nottingham
Loughborough (UK)

K. Jørgensen
Finnish Environment Agency
Helsinki (FI)

E.M.H. Wellington
University of Warwick
Coventry (UK)

V. Torsvik
University of Bergen (NO)

M.J. Bailey
Natural Environment Research Council (NERC)
Oxford (UK)

M.P. Nuti
Universitá di Pisa (IT)

J. Oliver
University of North Carolina at Charlotte (US)

S. Lindow
University of California
Berkeley (US)

F. de Bruijn
Michigan State University
East Lansing (US)

R. Atlas
University of Louisville (US)

 
 
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