Background and objectives

The effects of releasing bacteria with engineered traits into the open environment are still largely unknown. One reason for this is the difficulties encountered in monitoring these bacteria due to the fact that many bacterial species cannot be cultured in vitro. Since engineered genes can move to a novel host via transformation, transduction or conjugation, difficulties in monitoring ‘foreign’ DNA pose serious problems. Another important issue for the biosafety of the use of genetically modified organisms (GMOs) concerns the life span of the ‘foreign’ DNA, once it is present in the environment. These difficulties in monitoring GMOs introduced into the environment, raise serious concerns over the validity of GMO risk assessments and the biosafety of their use.

The first aim of this project was to develop highly sensitive monitoring technology for ‘foreign’ DNA, by applying a type of “ID plate” to engineered DNA sequences that were introduced into bacteria. The second aim was to examine the use of ‘suicide’ genes as a means of limiting the life span of the GMO. The third aim was to develop realistic laboratory environments, from which samples could be taken to test for the presence of the GMO. The ID plate allows unambiguous identification of the engineered gene independently from the bacteria carrying it, providing a means of specifically monitoring the movement of introduced genes.


Approach and methodology

Synthetic oligonucleotides were used to construct a specific DNA sequence, the ID sequence. This sequence was inserted into a Tn5 plasmid to deliver it to the bacterial chromosome. The gef family of suicide genes from Escherichia coli was used to obtain a regulatory suicide gene and was tested in several different bacteria. Sterile and non-sterile soil microcosms were established. Genetically engineered plasmids were introduced into a strain of Pseudomonas bacteria, and were released into soil microcosms in a closed laboratory environment and monitored for a few months. DNA was extracted from environmental samples including both soil and water and the presence of GMOs was monitored by polymerase chain reaction (PCR) amplification of the target sequence.
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Main findings and outcome

A strain of Pseudomonas putida carrying the ID sequence was constructed. In laboratory experiments, bacteria were resuspended in soil and water and the presence of the bacteria with the target sequence was successfully monitored by PCR amplification using primers specific for the ID tag. The sensitivity of the PCR amplification was demonstrated to be higher than the traditional plating method.

The gef genes were found to be highly effective suicide genes and were functional in a broad spectrum of bacteria, notably in P. putida. Strains containing these suicide gene grew under optimal growth conditions, however, under non-growth conditions, viable bacteria were almost completely eliminated. Alternative bacterial systems in which these suicide genes could function are also being developed. It was found that different bacterial strains expressing the gef genes had different survival rates, so several experiments will need to be carried out to determine the optimal species. P. putida carrying foreign DNA sequences were found to survive much more readily than any of the E. coli strains tested.

Conclusions

This technique of ‘tagging’ modified genes to monitor GMOs released into the environment, should prove particularly useful due to the fact that it is more sensitive than other methods that are employed. This should provide a means of improving risk assessments of the biosafety of GMOs introduced into the environment. Furthermore, the ID sequence has also been proposed to be useful for patenting purposes, as it could be used to mark a particular gene construct, allowing the owner of the patent to identify ‘their’ gene in any sample tested. Functional laboratory microcosms were also developed and have proved to be a practical way of studying this model system. Finally, this study also showed the feasibility of the development and implementation of a safe bacterial system for the introduction of foreign DNA, using bacteria which have a limited life span.