transfer between soil bacteria as monitored by novel techniques in model
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.
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.
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
Amici A., Bazzicalupo M., Gallori E. and Rollo F., Monitoring
a genetically engineered bacterium in a freshwater environment by
rapid enzymatic amplification of synthetic DNA number-plate".
Appl Microbiol Biotechnol, 36, 1991, pp. 222-227.
January 1989 December 1990
University of Firenze (IT)
Technical University of Denmark
Institut für Bodenbiologie FAL