Chemistry Reference
In-Depth Information
to be a microorganism with great industrial potential. Therefore, this chapter focuses on how
to take advantage of
Streptomyces
for producing industrially valuable enzymes.
Lastly, like other technologies, there are pros and cons about the application of recombi-
nant DNA technology and protein engineering. It is important to control these technologies
so that they can be used in beneficial ways. Regulations and guidelines for evaluating the
use of GMOs and general issues regarding safety as well as environment are introduced and
discussed.
2.2
RECOMBINANT DNA TECHNOLOGY
Recombinant DNA technologies involve isolating a target gene, connecting it with a carrier,
transforming it into another organism and use that organism to propagate the gene product.
This process is also called 'cloning'. There are a few elemental factors that need to be
considered in order to succeed in cloning a gene:
Donor organism
: A donor is the organism that provides the gene of interest. It can be
derived from mammalian sources, plants or, in most cases, from microorganisms. The
choice of a proper donor depends on the specific requirements for the property of the gene
product. The donor can be found through either a screening process designed for locating
the gene of interest or through a DNA matching process performed on genome DNA with
known partial gene sequence information.
Host organism
: A host is the organism in which the target gene can be replicated,
transcribed and further translated into products of interest. In most cases, the host organism
is chosen because of its easy reproduction. It can also be mammalian cells, plant cells
or an insect system. Choosing the correct host organism is very important for successful
expression of a target gene. Although, unfortunately, there are no definite rules in this
respect, it has been generally established that homologous gene expression, that is genes
of one origin expressed in the same or closely related organism, is the better choice.
Nevertheless, a vast number of heterologous expressions succeeded in the bacterium
E. coli
support the conclusion that it is a rather universal host. Choosing a host cannot be
separated from choosing a vector. The choice of a host-vector system as a set should be the
consideration for the purpose of each target expression. Table 2.1 lists some commonly
used host-vector systems such as
E
.
coli
(pUC18/19),
Bacillus subtilis
(pUB110) and
Streptomyces lividans
(pIJ702).
Vector
: A vector is the carrier, as mentioned above, which is capable of carrying the target
gene and replicating it in the corresponding host cells and has the property to integrate
into the chromosome of the host. The most commonly used vector is closed double-helix
circular plasmid DNA. It usually contains an
ori
region for replication and typically some
antibiotic-resistant genes as selection markers and multiple restriction sites for splicing
and joining it with target genes. It can also be single-stranded phage DNA or cosmid
DNA. Figure 2.1 shows a typical plasmid map of pIJ702, a commonly used vector for
Streptomyces
hosts.
12
A shuttle vector is a vector with the characteristic of functioning in
multiple hosts of different genus.
Target gene
: A target gene can be isolated from the donor organism by 'shotgun cloning' (a
method which will be described later) or by using primers designed from known sequences
of the target gene. These sequences can be deduced from N-terminal or internal amino
