Genetic Manipulation - Environmental Biotechnology: Theory and Application - page 221

the Western blot which is slightly different in that the material electrophoresed
is protein, which is probed with antibodies against the anticipated protein, rather
than nucleic acid, as is the case in Southern and Northern blots.
Recombinant Bacteria
GE of microorganisms for use in environmental biotechnology has tended to focus
on the expansion of metabolic pathways either to modify the existent metabolic
capability or to introduce new pathways. This has various applications, from
the improved degradation of contaminants, to the production of enzymes for
industry, thus making the process less damaging to the environment. One such
experimental example taken from 'clean technology' with potential for the man-
ufacturing industry, is a strain of Escherichia coli into which was engineered
some 15 genes originating from Pseudomonas . These were introduced to con-
struct a pathway able to produce indigo for the dyeing of denim, commented on
by Bialy (1997). The traditional method requires the use of toxic chemicals with
the associated safety measures and inherent pollution problems. Similar tech-
nologies were investigated in the early 1980s, by Amgen in the USA and Zeneca
in the UK, but were not pursued due to questionable profitability. Whether or
not this route will ultimately be taken up by industry still remains to be seen
(BMB, 1995).
Recombinant Yeast
Yeast, being unicellular eukaryotes, has become popular for cloning and
expressing eukaryotic genes. It is fairly simple to propagate, some species
being amenable to culture in much the same way as bacteria. Yeast cells are
surrounded by a thick cell wall which must be removed to permit entry of
DNA into the cell. There are several types of plasmid vector available for GE,
some of which have been constructed to allow replication in both bacteria and
yeast (Beggs, 1981). All have a region which permits integration into the host
yeast genome by recombination. This occurs by alignment of the sequences
complementary between the host genome and the incoming plasmid DNA. Two
crossover events then take place which effectively swap over a piece of host
DNA with the plasmid DNA. A similar process occurs in the construction of
recombinant Baculoviruses.
Recombinant Viruses
The insect virus, Baculovirus, has been shown to be the method of choice for the
over expression of genes in many applications of molecular biology. The viral
genome is large relative to bacterial plasmids and so DNA manipulations are
normally carried out on a plasmid maintained in Escherichia coli . Introduction