研究テーマ
Development of a Method for the Detection of Genetically Engineered Microorganisms
担当：M2 UPADHYE Rahul
Abstract:
We know that a Genetically Engineered Microorganisms (GEM) outperforms its wild-type strain either by the over-expression of the existing gene, or imparting new feature (like: substrate specificity, enzyme or other metabolite synthesis) by the insertion of a foreign gene in the original strain. GEMs are artificially designed microorganisms characterized by the presence of an exogenous DNA. Recently, the use of GEMs for bioremediation has become an attractive method for the accelerated degradation of pollutants. The interaction of GEMs with the environment and the biodiversity, the selective pressure acting on them after their release, and the long-term persistence effect of GEMs on the environment and human health are heavily debated topics worldwide. The present study is aimed to study and develop a detection method for detecting GEMs based on the polymerase chain reaction (PCR). Although a variety of individual methods are available to detect specific strains, however a method, which can facilitate detection of broad species, is unavailable.
Plasmids, carrying the insertion gene to be transferred into a recipient host organism, are widely used in the cloning process. The frequently used plasmid vectors, which serve as a model in the cloning process, are focused for detecting the foreign genetic material present in GEMs. A list of broad-host-range type frequently used plasmid vectors, was released by the Biodiversity Center of Japan. This frequently used plasmid vector list was subjected to grouping based on the hereditary relation of the individual plasmid. A frequently used group of plasmid vector, pUC19 derived group, was then chosen to design and experiment the detection strategy. PCR experiments were then performed with two different approaches: firstly the recombinant samples were analyzed using a vector-specific and an insertion gene-specific PCR primers and its results were compared with those of the parent strain carrying the same plasmid but with no gene insert, similarly in the second study only vector-specific primers were used and tested for a variation in the amplicon lengths as compared to that of an original plasmid vector serving as a control. Eight set of vector-specific primers and two gene-specific primers were tested on nine recombinant-plasmid bearing strains in this study. Seven out of the nine recombinant samples could be identified using one or more of the available primer sets. It is concluded from the results that using the first approach with a pair of vector-specific and gene-specific primers, a fewer detections are possible due to the limitability of the PCR products however results from the later approach, method using only the vector-specific primer set, could be applied to a wider range of recombinant strains thereby facilitating the detection of whether a strain is a genetically engineered or not. This method will be applied for the detection of unknown strains collected from various contaminated sites to distinguish them from wild-type strains (genetically unmodified strains).
References:,
Gotz A., Pukall R., Smit E., Tietze E., Prager R., Tschape H., Van Elsas J.D., Smalla K. 1996. Detection and Characterization of Broad-Host-Range Plasmids in Environmental Bacteria by PCR. Appl. Environ. Microbiol. 62: 2621-2628.
Jansson J.K. 1995. Tracking genetically engineered microorganisms in nature. Curr. Opin. Biotechnol. 6: 275-283.
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