Introduction to Biotechnology

1 Introduction to Biotechnology
The term biotechnologist will be used to describe a person who deals with
fermentation and microbial systems. This term was coined because a
biotechnologist is a professional who integrates and applies a number of
disciplines such as microbiology, genetics, biochemistry, chemistry, pharmacy,
food technology, mathematics and engineering in his or her work.
When designing or working with fermentations, the biotechnologists
understanding of the above disciplines needs to be sufficient enough to allow the
manipulation of process conditions in a rational manner. For instance, when
biotechnologists are modelling fermentation systems, they integrate their
knowledge of biological concepts (microbiology, biochemistry and genetics) with
their mathematical and engineering skills. The idea of integrating biodisciplines
and engineering is summarised by Figure 1.

Chemistry

Biochemistry

Pharmacy

Microbiology

Biotechnology
Food Technology
Engineering

Genetics
Mathematics

Figure 1: The discipline of biotechnology, an integration of other disciplines.

This study is an ideal example that shows the integration between disciplines and
in particular, it looks at the production of a recombinant protein hormone via
fermentation while relying on the integration of the engineering, microbiology and
biochemistry disciplines. Details of the fermentation are explained in the following
sections.

1.1 The Biotechnological Process

A typical biotechnological process is similar to a chemical process in many ways.
For instance, both processes generally have a reaction step, followed by a
separation step. Yet the biotechnological process exhibits clear distinctions in
that it relies on live cells or organisms to carry out the reaction. These may be
thought of as bio-catalysts. The steps required in a typical biotechnological
process are shown in Figure 2.

Introduction to Biotechnology

Bio-catalyst
Selection and
Manipulation

Raw materials

Bio-reactor

Product
Separation and
Purification

Product
formulation

Process Control

Figure 2: Schematic showing the typical steps in a biotechnological process.

The bio-reactor is at the heart of the process and its performance relies on the
material input, the bio-catalyst performance and the control strategy
implemented. A typical biotechnological process runs at low temperatures and
consumes little energy. It also often utilises inexpensive raw materials for feed26.
However antibiotics and inductants can have considerable costs.
1.1.1 Bio-catalyst Selection and Manipulation
The initial step in any biotechnological process is the selection of a suitable biocatalyst. In the selection process, the biotechnologist aims to find a
microorganism that exhibits the following characteristics26:

It should be a pure culture,

Be genetically stable,
Be easily propagated,
Exhibit rapid growth characteristics,
Have good rate of product formation,
Be free of toxic byproducts and
Be amenable to genetic manipulation.

Natural selection and mutation selection, are two ways of obtaining the desired
bio-catalyst. Recent developments in the field of recombinant DNA technology
are becoming more commonplace and are widely used in the selection and
preparation of suitable bio-catalysts.
1.1.2 Natural Selection

Introduction to Biotechnology
Most microorganisms exhibit a great capacity for natural variation. Microorganism
cultures isolated from nature generally lead, once grown, to variant cultures,
some of which may be superior in their capacity to synthesize a product6. This
capacity is exploited in biotechnology where this improved variant is preserved
and utilised.
1.1.3 Mutation Selection
Once a bio-catalyst is found, it may be improved by genetic modification. The
most effective method previously used for improving the yield of product is by
way of induced mutation followed by selection of an improved variant7. Genetic
and microbiology texts21,22 offer detailed discussions of mutation and procedures
used for selection. Although mutation selection is very effective, it has been
superseded by the new techniques employed in genetic engineering because it is
not as directed and relies heavily on chance.

1.2 Recombinant DNA Technology

Recent developments in the genetic engineering techniques have presented
tools that enable the handling and manipulation of DNA, allowing more flexibility
in the bio-catalyst selection process.
DNA is a molecule found in living cells and is responsible for several complex
functions. In humans for instance, DNA determines the physical features such as
height and eye colour. Bacterial DNA, as a second example, has specialised
functions that allow the bacteria to metabolise nutrients and regulate this
metabolism. DNA is also involved in development and reproduction of an
organism.
DNA is a polymeric molecule made of four different monomeric units called nucleotides.
Each nucleotide consists of a 5-carbon sugar to which is attached a phosphate group and
one of four nitrogenous (nitrogen containing) bases - adenine, guanine, cytosine and
thymine. The structure of DNA is shown in Figure 3.
A series of nucleotides is called a gene. Different genes are responsible for
different functions. A complete collection of genes is called a genome and this
would fully represent an organisms complete DNA sequence. Genes are
organised inside the cells in coiled structures known as chromosomes. Alteration,
addition or deletion of genes may from natural or human effect, reconstruct the
chromosomes. A more important reconstruction method is by way of
recombination.

Introduction to Biotechnology

Figure 3: A space filling model showing the structure of the DNA double helix [21].

Recombination of DNA allows the variation and diversity of living organisms. The
joining of the males sperm cell with the females egg cell is a natural
recombination process. The DNA of the male joins and mixes with the females
DNA resulting in an offspring cell with a combination of both parents. This is
evident in the offsprings physical appearance, which exhibits features that are
similar to both parents.
Natural recombination is now mimicked by biotechnologists allowing the
reconstruction of DNA molecules in test tubes. In this process, the construction of
a new DNA molecule takes place from two different sources. The techniques and
procedures associated with this are known as recombinant DNA technology and
are the specialty of the geneticist. Today, this technology is commonly known as
gene cloning.
1.2.1 Gene Cloning
Genes are cloned by isolating a piece of DNA from an organism and inserting it
into a cloning vector to make a recombinant DNA molecule. This molecule is then
introduced into the target organism carrying with it the cloned gene(s) which
allow(s) the organism to carry out new functions or improve existing ones. An
organism carrying foreign genes is known as recombinant organism. The steps
required for cloning a gene are illustrated in Figure 4. Here the gene of interest is
integrated into a vector DNA molecule using cut-and-paste techniques facilitated
by special enzymes. The combination of the vector and the gene is called a
recombinant molecule. This molecule is introduced into the desired host
organism by transformation, where the effect of the gene is amplified and the
host acquires the new characteristics brought about by the new gene.

Introduction to Biotechnology

Figure 4: Recombinant plasmid construction and cloning [21].

1.2.2 DNA Isolation

Before cloning a particular gene, the source DNA must be isolated and the gene
identified. Isolation usually is a process consisting of three steps9: 1) cell
disruption, 2) removal of cell debris, proteins and enzymes and 3) purification
and concentration of the DNA. Once the DNA is isolated, the gene to be cloned
is cut out of the DNA by the use of restriction endonuclease enzymes. These are
special enzymes that cleave DNA at certain base sequences called restriction
sites. They merely act like special scissors that cut out the desired gene. Since
their discovery in the 1960s, hundreds have been identified. The next step is to
paste the excised gene into a cloning vector before transferring to host cell.

Introduction to Biotechnology

1.2.3 Cloning Vectors and Gene Cloning

Typical cloning vectors include plasmids, transposons, cosmids, phages, shuttle
vectors and artificial chromosomes. Because they are most commonly used and
because this present work uses one, features of plasmid vectors are discussed
here. A plasmid is an extrachromosomal genetic element that replicates
autonomously inside the host cell. It is circular and double-stranded in structure.
To be useful, a plasmid ought to have the following qualities9:
1) single restriction site for a number of given enzymes,
2) High copy number of plasmids in a cell (presence of more than one plasmid in
the cell at any one time; approaching 100 copies), and
3) Selectable marker (this tells us that the cell has the plasmid, E.g. antibiotic
resistance marker).
The desired gene is joined to the
appropriate
vector
forming
a
recombinant molecule, which is then
introduced in a process called
transformation
into
the
host
organism. Transformation is the
process of introducing new DNA into
a host cell. The recombinant DNA
molecule is mixed with the
appropriate host cell. The host cell is
made permeable by addition of
calcium chloride and incubated for a
short time allowing the new DNA to
be taken up into the cell. Once the
DNA is inside, the cell begins to
exhibit the features associated with
the new DNA. The introduction of a
DNA plasmid into a bacterial host is
illustrated in Figure 5.

Figure 5: The transformation

process. The recombinant plasmid
molecule is introduced into the host
cell [21].

1.2.4 Gene Expression

The rate of plasmid-encoded gene expression is important because of the direct
influence on gene-product levels. Inserting a gene into a cloning vector does not
ensure that expression will be successful. In order for a cloned gene to be
expressed, the DNA must get transcribed and translated. A promoter site on the
plasmid initiates transcription, while a terminator site induces cessation of
transcription. Promoter and terminator sites merely act as signals functioning to
turn the gene on or off. Cloned genes usually do not carry these DNA sites

Introduction to Biotechnology
without which they can not be transcribed. To deal with this, the gene to be
cloned is inserted in the correct location between these sites. To produce large
amount of gene product, it is necessary to use a promoter that supports
transcription at a high level.
1.2.5 Recombinant DNA Technology in Biotechnology
The overall efficiency of the biotechnology process is improved with the aid of the
new recombinant techniques. The benefits of recombinant DNA technology to the
biotechnology process lie in the bio-catalyst selection stage and include26:
1. Custom design of products with desirable characteristics,
2. Increase of a yield of a particular product,
3. Improve the efficiency of the production with respect to energy utilisation,
4. Transfer of a particular activity to a more desirable host, and
5. Minimise the purification steps required.
Today it is possible to introduce mammalian genes into bacteria for the largescale production of products encoded by those genes26. The impact of this will be
felt by the pharmaceutical industry in particular with the production of insulin,
growth hormone and vaccines.
Clearly bio-catalyst selection is an area receiving a great deal of attention with
optimism in the development of newer high-producing stable bio-catalysts.

1.3 Recombinant Protein Production

The emergence of recombinant DNA technology enabled the production of many
important proteins that were previously extremely difficult to produce in large
commercial scale quantities8. An example of a recombinant protein is porcine
growth hormone (pGH), the production of which is studied in this thesis. Porcine
growth hormone is a naturally occurring protein produced in pigs by their pituitary
glands and is responsible for normal growth and muscle development. Advances
in recombinant DNA technology now permit the large-scale production of
synthetic analogues of pGH. pGH plays an important role in regulating skeletal
growth, protein and fat metabolism of pigs. It is used in the pig farming industry to
improve the efficiency of converting feed into muscle tissue.
1.3.1 Cloning the Porcine Growth Hormone
The DNA coding for pGH was cloned into the vector plasmid pET3a (Figure 6).
The recombinant plasmid pET3a-pGH was transformed into E.coli strain BL21
DE3 for expression. pGH is produced intracellularly in the form of insoluble
inclusion bodies. The formation of inclusion bodies inside E.coli cells is promoted
by hydrophobic, ionic, and in some cases covalent interactions (i.e. disulfide
bonds) between protein molecules8.

Introduction to Biotechnology

Figure
7
shows
electron
micrographs of E.coli cells having
inclusion bodies. The cells with
inclusion bodies show bulging
around where the inclusion bodies
are found, while cells without
inclusion bodies show a different
morphology, with pointy ends in
some
cases.
The
genetic
engineering
designed
the
production of pGH by the bacteria
to be induced by the addition of
IPTG (inducer) and repressed by
glucose.

Figure 6: Cloning of the pGH gene into the

pET3a plasmid system. The restriction
enzymes BamH I and Nde I were used to
cut the pGH and the plasmid DNA.

Figure 7: Intracellular inclusion bodies in sectioned transmission electron

micrographs of recombinant E.coli. Arrows point to inclusion bodies [3,8].

Introduction to Biotechnology
1.3.2 Production of pGH
Production of pGH can takes place by fermentation of the E.coli culture growing
on glucose as a substrate. The fermentations can be carried out in Chemap
fermenter (Figure 8) This fermenter system is capable of automatic sterilisation
and allows the on-line monitoring of temperature, pH and DO. Previous work has
demonstrated success in the production of pGH. Kollaras15 performed nine
fermentations and found optimum temperature and pH to be 34-36C and 6.9
respectively.
Downstream processing of the fermentation broth takes place in the bioseparation plant. This consists of a series of unit operations aimed at the
separation and purification of the product. Figure 9 shows a view of the bioseparation plant. For optimisation purposes, the fermenter and bio-separation
plant should be considered as one process as they interact heavily with each
other to produce the final product.

Figure 8: The 14-litre Chemap Fermenter.

Introduction to Biotechnology

Figure 9: The bio-separations plant for product separation and purification.

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Introduction to Biotechnology
7. Benson HJ., 1984, Microbiological applications a laboratory manual in general
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Introduction to Biotechnology

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