216 Environmental Biotechnology
indigenous species which could outgrow the recombinant. The novel bacterium
may also lose its carefully engineered new capability through normal transfer of
genes given the high level of promiscuity between bacteria. A highly controlled
and contained environment such as a bioreactor may circumvent some of these
objections but it is not always practical to move the contamination to the solution
rather than the solution to the contamination. Again this involves expense and
practical considerations, not least of which are safety concerns associated with
the transport of contaminated material.
In reality, there is rarely any need to use recombinants or transgenics and
it is far more likely that the required metabolic capability will be provided by
indigenous organisms, or ones which have been trained for the task. There are,
however, some exotic and ingenious applications, and by way of illustration,
some examples are given here. The aim is to provide an overview of some of
the more frequently used technologies together with specific examples. There
are very many excellent textbooks and specialised publications which should be
consulted should a more detailed and working knowledge be required. However,
an overview of the principles of genetic engineering are given here for the benefit
of those unfamiliar with the technology.
Basic Principles of Genetic Engineering
There are endless permutations of the basic cloning procedures but they all share
some fundamental requirements. These are: the enzymes, solutions and equipment
necessary to perform the procedures; the desired piece of DNA to be transferred;
a cloning vector; and the recipient cell which may be a whole organism. For the
process to be of any measurable value, it is also essential to have some means
of determining whether or not the transfer has been successful. This is achieved
by the use of marker genes. The requirements referred to above are described in
the following sections.
Enzymes, solutions and equipment
There are many steps involved in the isolation of DNA which now have become
standard laboratory techniques. Once DNA has been isolated from an organism, it
is purified from contaminating material such as protein and is precipitated out of
aqueous solution by the addition of alcohol, for example ethanol, to approximately
70%. The DNA appears as a white, semi-transparent material, coiling out of
solution on addition of the alcohol. This may be collected by centrifugation and
dried down ready for the next stage which is usually enzyme digestion. The aim
of the next stage is to insert the DNA into the vector, for which the ends of
the DNA and the vector have to be prepared. This may be done by restriction
endonucleases which recognise specific sequences within the DNA and cut at
that site, either producing a flush or staggered end, Figure 9.1, or by incubation