genetic engineering of microorganisms to improve the quality of life

1. 1
AN ESSAY SEMINAR ON
GENETIC ENGINEERING OF MICROBES TO IMPROVE THE
QUALITY OF LIFE
BY
ADEWUMI, GBEMISOLA DEBORAH
130408007
SUBMITTED TO THE DEPARTMENT OF BIOLOGICAL SCIENCES, ONDO STATE
UNIVERSITY OF SCIENCE AND TEHNOLOGY, OKITIPUPA IN PARTIAL
FULFILMENT FOR THE REQUIREMENTS OF THE AWARD OF BACHELOR OF
TECHNOLOGY (B.TECH) DEGREE IN MICROBIOLOGY.
APRIL, 2018

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CERTIFICATION
This seminar report was carried out by ADEWUMI, GBEMISOLA DEBORAH and submitted
to the Department of Biological Sciences, Faculty of Science, having met the standard as required
by the institution and approved as to contents and style by:
DR. S. FAKOYA _____________________
SEMINAR SUPERVISOR SIGN/DATE
MRS. F.M. OJO ______________________
SEMINAR COORDINATOR SIGN/DATE
DR. S. FAKOYA _____________________
HEAD OF DEPARTMENT SIGN/DATE

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DECLARATION
I, ADEWUMI, GBEMISOLA DEBORAH hereby declare that this report was written by me.
All sources of information are clearly acknowledged by means of references.
…………………………………………. ……………………………………
SIGNATURE DATE

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ACKNOWLEDGEMENTS
With a heart full of thanks, I return all glory and adoration to Almighty God for the success of this
report. I appreciate my parents for their financial and moral support, effort and care. I also
appreciate my Seminar Supervision, Dr. S. Fakoya for his effort, support and for guiding me all
through. A big thank you to those who contribute effortlessly to the success of this report, my
friends and some other people.
`

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ABSTRACT
Genetic engineering can simply be explained as the alteration of an organism's genetic, or
hereditary, material to eliminate undesirable characteristics or to produce desirable new ones.
Several works have been done on genetic engineering with major focus on its importance ranging
from increasing plant and animal food production, diagnosing disease condition, medical treatment
improvement, as well as production of vaccines and other useful drugs. Methods in this techniques
involve the selective breeding of animals and plants, hybridization (reproduction between different
strains or species), and recombinant deoxyribonucleic acid (rDNA). With the increasing global
population, the idea of Genetic Engineering on microorganism has greatly been embraced by man
to improve on his well-being.

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TABLE OF CONTENTS
TITLE PAGE .................................................................................Error! Bookmark not defined.
CERTIFICATION .......................................................................................................................... 2
DECLARATION ............................................................................................................................ 3
ACKNOWLEDGEMENTS............................................................................................................ 4
ABSTRACT.................................................................................................................................... 5
TABLE OF CONTENTS................................................................................................................ 6
LIST OF PLATE............................................................................................................................. 7
CHAPTER ONE ........................................................................................................................... 8
1.0 INTRODUCTION............................................................................................................ 8
1.1 GENETIC ENGINEERING........................................................................................... 11
1.2 HISTORY OF GENETIC ENGINEERING .................................................................. 13
CHAPTER TWO ........................................................................................................................ 15
2.0 GENETICALLY MODIFIED ORGANISMS ............................................................... 15
2.1 PROCESSES INVOLVED IN GENETIC ENGINEERING OF MICROBES ............ 17
2.2 GENE CLONING IN MICROORGANISMS ............................................................... 22
2.2.1 Methods of gene cloning…………………………………………………………….22
2.2.2 Gene Librarary. .......................................................................................................... 22
2.3 CLONING VECTORS.................................................................................................. 24
2.3.1 TYPES OF CLONING VECTORS ........................................................................... 25
CHAPTER THREE .................................................................................................................... 27
3.0 HOW DOES GENETIC ENGINEERING OF MICROORGANISMS HELPS TO
IMPROVE THE QUALITY OF LIFE ......................................................................................... 27
CHAPTER FOUR ……………………………………………………………………………...34
4.0 Advantages of Genetically modified microorganisms…………………………………34
4.1 Disadvantages of Genetically modified microorganisms……………………………...35
CHAPTER FIVE ……………………………………………………………………………....37
5.0 CONCLUSION……………………………………………………………...…………37
REFERENCES…………………………………………………………………...……38

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LIST OF PLATE
1. Processes involved in genetic engineering of microorganisms………………………19

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CHAPTER ONE
1.0 INTRODUCTION
Human life is greatly affected by three factors: deficiency of food, health problems, and environmental
issues. Food and health are basic human requirements beside a clean and safe environment. With increasing
world's population at a greater rate,human requirements for food are rapidly increasing (Batie et al.,2001).
Humans require safe-food at reasonable price. Several human related health issues across the globe cause
large number of deaths. Approximately 36 million people die each year from non-communicable and
communicable diseases, such as cardiovascular diseases, cancer, diabetes, AIDS/HIV, tuberculosis,
malaria, and several others. Despite extensive efforts being made, the current world food production is
much lower than human requirements, and health facilities are even below standard in the third-world
countries. Rapid increase in industrialization has soared up the environmental pollution and industrial
wastes are directly allowed to mix with water,which has affected aquatic marines and, indirectly, human-
beings. Therefore, these issues urge to be addressed through modern technologies.
Unlike tradition approaches to overcome agriculture, health, and environmental issues through
breeding, traditional medicines, and pollutants degradation through conventional techniques respectively,
the genetic engineering utilizes modern tools and approaches, such as molecular cloning and
transformation, which are less time consuming and yield more reliable products. For example, compared to
conventional breeding that transfers a large number of both specific and nonspecific genes to the recipient,
genetic engineering only transfers a small block of desired genes to the target through various approaches,
such as biolistic and Agrobacterium-mediated transformation. The alteration into plant genomes is brought
either by homologous recombination dependent gene targeting or by nuclease-mediated site-specific
genome modification. Recombinase mediated site-specific genome integration and oligonucleotide directed
mutagenesis can also be used. Recombinant DNA technology is playing a vital role in improving health
conditions by developing new vaccines and pharmaceuticals. The treatment strategies are also improved by
developing diagnostic kits, monitoring devices, and new therapeutic approaches. Synthesis of synthetic

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human insulin and erythropoietin by genetically modified bacteria and production of new types of
experimental mutant mice for research purposes are one of the leading examples of genetic engineering in
health. Likewise, genetic engineering strategies have been employed to tackle the environmental issues
such as converting wastes into biofuels and bioethanol, cleaning the oil spills, carbon, and other toxic
wastes,and detecting arsenic and other contaminants in drinking water. The genetically modified microbes
are also effectively used in biomining and bioremediation. The advent of recombinant DNA technology
revolutionized the development in biology and led to a series of dramatic changes. It offered new
opportunities for innovations to produce a wide range of therapeutic products with immediate effect in the
medical genetics and biomedicine by modifying microorganisms, animals, and plants to yield medically
useful substances. Most biotechnology pharmaceuticals are recombinant in nature which plays a key role
against human lethal diseases. The pharmaceutical products synthesized through recombinant DNA
technology, completely changed the human life in such a way that the U.S. Food and Drug Administration
(FDA) approved more recombinant drugs in 1997 than in the previous several years combined, which
includes anemia, AIDS, cancers (Kaposi's sarcoma,leukemia, and colorectal, kidney, and ovarian cancers),
hereditary disorders (cystic fibrosis, familial hypercholesterolemia, Gaucher's disease, hemophilia A,
severe combined immunodeficiency disease, and Turnor's syndrome), diabetic foot ulcers, diphtheria,
genital warts, hepatitis B, hepatitis C, human growth hormone deficiency, and multiple sclerosis.
Considering the plants develop multigene transfer,site-specific integration and specifically regulated gene
expression are crucial advanced approaches. Transcriptional regulation of endogenous genes, their
effectiveness in the new locations, and the precise control of transgene expression are major challenges in
plant biotechnology which need further developments for them to be used successfully.
Human life is greatly threatened by various factors, like food limitations leading to malnutrition, different
kinds of lethal diseases, environmental problems caused by the dramatic industrialization and urbanization
and many others. Genetic engineering has replaced the conventional strategies and has the greater potential
to overcome such challenges. In line with this, we have detailed the limitations of genetic engineering and

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possible future directions for researchers to surmount such limitations through modification in the current
genetic engineering strategies. For thousands of years, man has purposely manipulated the evolution
of other organisms; farmers have used selective breeding to improve their livestock and crops. As
a result, we have cows that produce more milk, hens that lay more eggs, sheep with better wool,
and disease resistant plants with higher productivity. To widen his imagination, man has furthered
his quest for knowledge through biological manipulation and technology to develop Genetically
Modified Organisms (GMO). This has been through the use of Genetically Modified
microorganisms (GMM) particularly bacteria and fungi due to their small size and ease of
manipulation. Though products of GMM have largely been accepted and consumed by the
population, their utilization remains questionable as regards the role and implication in the
ecosystem. Human genetic engineering relies heavily on science and technology. It was developed
to help end the spread of diseases (Kaisier et al., 2003). With the advent of genetic engineering,
scientists can now change the way genomes are constructed to terminate certain diseases that occur
as a result of genetic mutation. Today genetic engineering is used in fighting problems such as
cystic fibrosis, diabetes, and several other diseases. Another deadly disease now being treated with
genetic engineering is the "bubble boy" disease (Severe Combined Immunodeficiency). Clearly,
one of the greatest benefits of this field is the prospect of helping cure illness and diseases in
unborn children. Having a genetic screening with a fetus can allow for treatment of the unborn.
This is a clear indication that genetic engineering has the potential to improve the quality of life
and allow for longer life span.

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1.1 GENETIC ENGINEERING
Genetic engineering, also called genetic modification or genetic manipulation, is the direct
manipulation of an organism's genes using biotechnology (Zughart et al., 2011). It is a set of
technologies used to change the genetic makeup of cells, including the transfer of genes within
and across species boundaries to produce improved or novel organisms. New DNA is obtained by
either isolating or copying the genetic material of interest using recombinant DNA methods or by
artificially synthesising the DNA. A construct is usually created and used to insert this DNA into
the host organism. The first recombinant DNA molecule was made by Paul Berg in 1972 by
combining DNA from the monkey virus SV40 with the lambda virus. As well as inserting genes,
the process can be used to remove, or "knock out ", genes. The new DNA can be inserted randomly,
or targeted to a specific part of the genome. The first GMO was a bacterium generated by Herbert
Boyer and Stanley Cohen in 1973. Rudolf Jaenisch created the first GM animal when he inserted
foreign DNA into a mouse in 1974. The first company to focus on genetic engineering, Genentech,
was founded in 1976 and started the production of human proteins. Genetically engineered human
insulin was produced in 1978 and insulin-producing bacteria were commercialised in 1982.
Genetically modified food has been sold since 1994, with the release of the Flavr Savr tomato. The
Flavr Savr was engineered to have a longer shelf life, but most current GM crops are modified to
increase resistance to insects and herbicides. GloFish, the first GMO designed as a pet, was sold
in the United States in December 2003. In 2016 salmon modified with a growth hormone were
sold. Genetic engineering has been applied in numerous fields including research, medicine,
industrial biotechnology and agriculture. In research GMOs are used to study gene function and
expression through loss of function, gain of function, tracking and expression experiments. By
knocking out genes responsible for certain conditions it is possible to create animal model

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organisms of human diseases. As well as producing hormones, vaccines and other drugs genetic
engineering has the potential to cure genetic diseases through gene therapy. The same techniques
that are used to produce drugs can also have industrial applications such as producing enzymes for
laundry detergent, cheeses and other products. Genetic engineering is a process that alters the
genetic make-up of an organism by either removing or introducing DNA (Christer et al., 2011).
Unlike traditionally animal and plant breeding, which involves doing multiple crosses and then
selecting for the organism with the desired phenotype, genetic engineering takes the gene directly
from one organism and inserts it in the other. This is much faster, can be used to insert any genes
from any organism (even ones from different domains) and prevents other undesirable genes from
also being added. Genetic engineering could potentially fix severe genetic disorders in humans by
replacing the defective gene with a functioning one. It is an important tool in research that allows
the function of specific genes to be studied. Drugs, vaccines and other products have been
harvested from organisms engineered to produce them. Crops have been developed that aid food
security by increasing yield, nutritional value and tolerance to environmental stresses. The DNA
can be introduced directly into the host organism or into a cell that is then fused or hybridized with
the host. This relies on recombinant nucleic acid techniques to form new combinations of heritable
genetic material followed by the incorporation of that material either indirectly through a vector
system or directly through micro-injection, macro-injection or micro-encapsulation. Genetic
engineering does not normally include traditional breeding, in vitro fertilization, induction of
polyploidy, mutagenesis and cell fusion techniques that do not use recombinant nucleic acids or a
genetically modified organism in the process. However, some broad definitions of genetic
engineering include selective breeding. Cloning and stem cell research, although not considered
genetic engineering, are closely related and genetic engineering can be used within them. Synthetic

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biology is an emerging discipline that takes genetic engineering a step further by introducing
artificially synthesized material into an organism. Plants, animals or micro-organisms that have
been changed through genetic engineering are termed genetically modified organisms or GMOs.
If genetic material from another species is added to the host, the resulting organism is called
transgenic. If genetic material from the same species or a species that can naturally breed with the
host is used the resulting organism is called cisgenic. If genetic engineering is used to remove
genetic material from the target organism the resulting organism is termed a knockout organism.
1.2 HISTORY OF GENETIC ENGINEERING
As early as 1865, the idea of genetics was raised by Mendel while monitoring the inheritance
pattern of organisms from one generation to another. It took about 35 years for other researchers
to grasp its significance until the 1900s where there was a steady progress in understanding the
genetic make-up of all living things ranging from microorganism to humans. In 1920, a major step
in human control over genetic traits was taken when Muller and Stadler discovered that radiation
can induce mutations in animals and plants. European Journal of Biotechnology and Bioscience
Later in 1930 and 1949s, several new methods of chromosome and gene manipulation were
discovered, such as the use of colchicine to achieve a doubling in chromosome number and other
techniques to induce gene mutations using chemicals such as nitrogen mustard and ethyl methane
sulphonate. This was closely followed by the discovery of double helix structure of DNA
(deoxyribonucleic acid), the chemical substance of heredity, by James Watson and Francis Crick
in 1953. Since then, there was an explosive progress in the field of genetics. In the mid-1970s, the
public of the Western world was astonished to learn that scientists had recently invented ways to
move pieces of genetic material, the very blueprint of life, from one species to another. The earliest

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of such discoveries was the transfer of a gene for antibiotic resistance from certain bacteria species
to Escherichia coli by researcher at Stanford University in 1975. This introduced the era of genetic
engineering so-called “genetic revolution” which extended from bacteria to plants, mammals and
ultimately human cells. Supporters and opponents of genetic engineering were just as divided
about the basic ethics or morality of the technology as they were about its practical implications.
This first wave of concern died down during the 1980s as genetically modified microorganisms
were released into the environment and no disasters occurred. Guidelines were later established by
the American National Institute of Health to control possible hazardous effects of GMOs. On the
contrary, these guidelines were progressively weakened in subsequent years, despite substantial
records of abuses, accidental releases and other “minor” scandals. For example, a researcher at
Montana State University introduced the Dutch elm disease into a new area while testing the
toxicity of genetically modified bacteria on fungi. As the twenty-first century begins, genetic
engineering has taken over the traditional biotechnology industry so completely that many people
now use the terms genetic engineering and biotechnology interchangeably.

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` CHAPTER TWO
2.0 GENETICALLY MODIFIED ORGANISMS
An organism that is generated through genetic engineering is considered to be genetically modified
(GM) and the resulting entity is a genetically modified organism (GMO). Genetically modified
organisms (GMOs) are organisms that have been altered using genetic engineering methods.
Although genetic engineering is a common and essential practice in biotechnology, its specific use
in crops is controversial. The key steps involved in genetic engineering are identifying a trait of
interest, isolating that trait, inserting that trait into a desired organism, and then propagating that
organism. Methods for genetic manipulation have rapidly improved over the last century from
simple selective breeding, to inserting genes from one organism into another, to more recent
methods of directly editing the genome. A genetically modified organism (GMO) is any organism
whose genetic material has been altered using genetic engineering techniques (i.e., a
genetically engineered organism) (Hopkins et al., 2003). GMOs are used to produce many
medications and genetically modified foods and are widely used in scientific research and the
production of other goods. The term GMO is very close to the technical legal term, 'living modified
organism', defined in the Cartagena Protocol on Biosafety, which regulates international trade in
living GMOs (specifically, "any living organism that possesses a novel combination of genetic
material obtained through the use of modern biotechnology"). A more specifically defined type of
GMO is a "transgenic organism." This is an organism whose genetic makeup has been altered by
the addition of genetic material from an unrelated organism. This should not be confused with the
more general way in which "GMO" is used to classify genetically altered organisms, as typically
GMOs are organisms whose genetic makeup has been altered without the addition of genetic
material from an unrelated organism.

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Genetically modified microorganisms include;
Bacteria
Bacteria were the first organisms to be modified in the laboratory, due to the relative ease of
modifying their genetics. They continue to be important model organisms for experiments in
genetic engineering. In the field of synthetic biology, they have been used to test various synthetic
approaches, from synthesizing genomes to creating novel nucleotides. These organisms are now
used for several purposes, and are particularly important in producing large amounts of pure
human proteins for use in medicine (Harhoff et al.,2001). Genetically modified bacteria are used to
produce the protein insulin to treat diabetes. Similar bacteria have been used to produce
biofuels, clotting factors to treat haemophilia, and human growth hormone to treat various forms
of dwarfism.
Virus
In 2017 researchers genetically modified a virus to express spinach defensin proteins. The virus
was injected into orange trees to combat citrus greening disease that had reduced orange
production 70% since 2005.
Fungi and Other microbes
In addition, various genetically engineered micro-organisms are routinely used as sources
of enzymes for the manufacture of a variety of processed foods. These include alpha-amylase from
bacteria, which converts starch to simple sugars, chymosin from bacteria or fungi, which clots
milk protein for cheese making, and pectinesterase from fungi, which improves fruit juice clarity.

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2.1 PROCESSES INVOLVED IN GENETIC ENGINEERING OF
MICROORGANISMS
The processes involved include the following;
1. Isolation
2. Cutting
3. Ligation and Insertion
4. Transformation
5. Expression
• Isolation: involves the isolation of a specific gene from a donor and the isolation of plasmid
from bacterial cell. Isolation of a specific gene from a donor involves breaking open the
cells of the donor to release the DNA and isolate the gene of interest e.g. insulin producing
gene, cells are broken open using chemicals and enzymes e.g. washing up liquid donor
DNA is extracted then genetic probe is added. A DNA probe consists of a small fragment
of DNA labelled with an enzyme, a radioactive tag or a fluorescent dye tag (Batie et al.,
2001). The probe will bind to a complementary DNA sequence by base pairing. Identifying
the presence and location of the gene of interest claim is for the resultant organism and is
built on earlier research. Genetic screens can be carried out to determine potential genes
and further tests then used to identify the best candidates. The development of microarrays,
transcriptomes and genome sequencing has made it much easier to find suitable genes. The
next step is to isolate the candidate gene. The cell containing the gene is opened and the
DNA is purified. A vector is needed to transfer gene into the host cell, plasmids or viruses
are vectors. Plasmids are small, circular, self-replicating, extrachromosomal pieces of
DNA that occur naturally the plasmid is isolated from the bacterial cell. The plasmid will

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act as a vector for carrying a new gene i.e. the gene from the donor will be inserted into the
plasmid DNA. Universality of genetic code Plasmid will produce the polypeptide coded
for by the donor DNA
• Cutting: Restriction enzymes act as molecular scissors and cut DNA at specific sites called
restriction sites DNA molecule is cut in half lengthwise and joined with a strand from
another organism or perhaps even another species to form a recombinant DNA molecule.
The DNA is cut into shorter fragments through the use of restriction enzymes and the ends
of the fragments are usually produced such that they have affinity to complementary ends
of other DNA fragments and will seek those out of the target DNA (Metcalfe et al., 1996).
Some restriction enzymes generate blunt ends, cutting across both strands of DNA while
others generate a staggered cut, producing “sticky ends.” These ends anneal by hydrogen
bonding to similar ends on by hydrogen bonding to similar ends on another DNA segment
cut with the same restriction enzyme. The cell containing the gene is opened and the DNA
is purified. The gene is separated by using restriction enzymes to cut the DNA into
fragments or polymerase chain reaction (PCR) to amplify up the gene segment. These
segments can then be extracted through gel electrophoresis. If the chosen gene or the donor
organism's genome has been well studied it may already be accessible from a genetic
library. If the DNA sequence is known, but no copies of the gene are available, it can also
be artificially synthesised.
• Ligation: This is the process of rejoining cut fragments of DNA and forming artificial
recombinant molecules.Once isolated the gene is ligated into a plasmid that is then inserted
into a bacterium. The plasmid is replicated when the bacteria divide, ensuring unlimited
copies of the gene are available. Before the gene is inserted into the target organism it must

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be combined with other genetic elements. These include a promoter and terminator region,
which initiate and end transcription. A selectable marker gene is added, which in most
cases confers antibiotic resistance, so researchers can easily determine which cells have
been successfully transformed (Harhoff et al., 2001). The gene can also be modified at this
stage for better expression or effectiveness. These manipulations are carried out using
recombinant DNA techniques, such as restriction digests, ligations and molecular cloning.
• Insertion: Inserting DNA into the host genome. There are a number of techniques
available for inserting the gene into the host genome (B et al., 2001). Some bacteria can
naturally take up foreign DNA. This ability can be induced in other bacteria via stress (e.g.
thermal or electric shock), which increases the cell membrane's permeability to DNA; up-
taken DNA can either integrate with the genome or exist as extrachromosomal DNA. DNA
is generally inserted into animal cells using microinjection, where it can be injected through
the cell's nuclear envelope directly into the nucleus, or through the use of viral vectors. In
plants the DNA is often inserted using Agrobacterium-mediated recombination, taking
advantage of the Agrobacteriums T-DNA sequence that allows natural insertion of genetic
material into plant cells. Other methods include biolistics, where particles of gold or
tungsten are coated with DNA and then shot into young plant cells, and electroporation,
which involves using an electric shock to make the cell membrane permeable to plasmid
DNA. Due to the damage caused to the cells and DNA the transformation efficiency of
biolistics and electroporation is lower than agrobacterial transformation and
microinjection.
• Transformation: Recombinant DNA is then introduced into bacterial cell. As only a single
cell is transformed with genetic material, the organism must be regenerated from that single

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cell (Kaisier et al., 2003). In plants this is accomplished through the use of tissue culture. In
animals it is necessary to ensure that the inserted DNA is present in the embryonic stem
cells. Bacteria consist of a single cell and reproduce clonally so regeneration is not
necessary. Selectable markers are used to easily differentiate transformed from
untransformed cells. These tests can also confirm the chromosomal location and copy
number of the inserted gene. The presence of the gene does not guarantee it will be
expressed at appropriate levels in the target tissue so methods that look for and measure
the gene products (RNA and protein) are also used. These include northern hybridisation,
quantitative RT-PCR, Western blot, immunofluorescence, ELISA and phenotypic analysis.
The new genetic material can be inserted randomly within the host genome or targeted to
a specific location. The technique of gene targeting uses homologous recombination to
make desired changes to a specific endogenous gene. This tends to occur at a relatively low
frequency in plants and animals and generally requires the use of selectable markers. The
frequency of gene targeting can be greatly enhanced through genome editing. Genome
editing uses artificially engineered nucleases that create specific double-stranded breaks at
desired locations in the genome, and use the cell’s endogenous mechanisms to repair the
induced break by the natural processes of homologous recombination and non-homologous
end-joining.
• Expression: Expression is getting the organism with the recombinant DNA to produce the
desired protein. When the protein is produced in large amounts it is isolated and purified.

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PLATE 1: PROCESSES INVOLVED IN GENETIC ENGINEERING OF
MICROORGANISMS
SOURCE: Napompeth, 1996

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2.2 GENE CLONING IN MICROORGANISMS
Gene cloning is the process in which a gene of interest is located and copied (cloned) out of DNA
extracted from an organism (Batie et al., 2001). When DNA is extracted from an organism, all of
its genes are extracted at one time. This DNA, which contains thousands of different genes. The
genetic engineer must find the one specific gene that encodes the specific protein of interest. Since
there is no way to locate a gene by visibly looking at all of the DNA, scientists make gene libraries
to catalogue the organism's DNA. The gene the scientist is looking for is selected from this library.
2.2.1 Methods of gene cloning
The step following DNA extraction of an organism is the construction of a library to organize the
DNA. A gene library can be defined as a collection of living bacteria colonies that have been
transformed with different pieces of DNA from the organism that is the source of the gene of
interest. If a library is to have a colony of bacteria for every gene, it will consist of tens of
thousands of colonies or clones.
2.2.2 Gene Library
Constructing a gene library requires not only the extracted DNA, but also restriction enzymes
and a plasmid.
Step 1: DNA extracted from an organism, with the gene of interest, is cut into gene-size pieces
with restriction enzymes. These enzymes read the nucleotide sequence of the DNA and recognize
specific sequences. The enzymes then cut the DNA sequence by breaking the bonds between
nucleotides in a DNA strand.

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Step 2: Bacterial plasmids are cut with the same restriction enzyme. Plasmids are small circles of
DNA in bacterial cells that are naturally present in addition to the bacteria's other DNA.
Step 3: The gene-sized DNA and cut plasmids are combined into one test tube. Some of the
enzyme cut DNA pieces will combine with the cut plasmids and form recombinant DNA (or DNA
in a 'new combination').
Step 4: The recombinant plasmids are then transferred into bacteria using either electroporation
or heat shock. Electroporation uses mild pulses of electricity to disrupt the cell walls of the
bacterium and create small holes. The plasmids are small enough to pass through the holes into
the cell. Heat shock works in a similar fashion. However, rather than using electricity to create
holes in the bacterium, it is done by alternating the temperature between hot and cold.
Step 5: The bacteria is grown on a culture dish and allowed to grow into colonies. All the colonies
on all the plates (cultures) are called a gene library.
Step 6: The gene library is then screened in order to discover which bacterial colony is making
copies of the one gene they are interested in. Library screening identifies colonies, which have
that particular gene. Screening can be based on detecting the DNA sequence of the cloned gene,
detecting a protein that the gene encodes, or the use of linked DNA markers. Therefore before
library screening can be done, the scientist must know either the DNA sequence of the gene, or a
very similar gene, the protein that the gene produces, or a DNA marker that has been mapped
very close to the gene. When the bacteria multiply and replicate the recombinant DNA, the
number of gene copies also increases, making gene or protein detection easier.

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When the bacteria colony containing the desired gene is located, the bacteria can be propagated
to make millions of copies of the recombinant plasmid that contains the gene. The plasmids can
be extracted for the next steps of genetic engineering, gene modification, and transformation.
Gene cloning is also important because copies of a gene are needed for these procedures.
2.3 CLONING VECTORS
A cloning vector is a small piece of DNA, taken from a virus, a plasmid, or the cell of a higher
organism, that can be stably maintained in an organism, and into which a foreign DNA fragment
can be inserted for cloning purposes(Christer et al., 2011). The vector therefore contains features
that allow for the convenient insertion or removal of a DNA fragment to or from vector, for
example by treating the vector and the foreign DNA with a restriction enzyme that cuts the DNA.
DNA fragments thus generated contain either blunt ends or overhangs known as sticky ends, and
vector DNA and foreign DNA with compatible ends can then be joined together by molecular
ligation. After a DNA fragment has been cloned into a cloning vector, it may be
further subcloned into another vector designed for more specific use.
There are many types of cloning vectors, but the most commonly used ones are genetically
engineered plasmids. Cloning is generally first performed using Escherichia coli, and cloning
vectors in E. coli include plasmids, bacteriophages (such as phage λ), cosmids, and bacterial
artificial chromosomes (BACs). Some DNA, however, cannot be stably maintained in E. coli, for
example very large DNA fragments, and other organisms such as yeast may be used. Cloning
vectors in yeast include yeast artificial chromosomes (YACs). A vector has three distinctive
features;

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 An origin of replication: which is the sequence that the host cell recognizes and which
allows it to make copies of the vector and the cloned DNA.
 Multiple cloning sites: Sites where the cloned DNA can insert.
 Selectable marker: Specific DNA sequence that is used by Biologists to tell if the clone
has entered the cell (transformation), it will enable the Scientist to select host cells that
have been transformed by the cloning vectors. It is usually an antibiotic resistance gene.
2.3.1 TYPES OF CLONING VECTORS
 Plasmid vectors
 Bacterial artificial chromosome (BAC)
 Yeast artificial chromosome (YAC)
• Plasmid vectors: Plasmids are autonomously replicating circular extra-chromosomal
DNA (Zughart et al., 2011). They are the standard cloning vectors and the ones most
commonly used. Most general plasmids may be used to clone DNA insert of up to 15 kb
in size. One of the earliest commonly used cloning vectors is the pBR322 plasmid. Other
cloning vectors include the pUC series of plasmids, and a large number of different cloning
plasmid vectors are available. Many plasmids have high copy number, for
example pUC19 which has a copy number of 500-700 copies per cell, and high copy
number is useful as it produces greater yield of recombinant plasmid for subsequent
manipulation. However low-copy-number plasmids may be preferably used in certain
circumstances, for example, when the protein from the cloned gene is toxic to the cells.

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• Bacterial Artificial Chromosome (BAC): is a DNA construct, based on a functional
fertility plasmid (or F-plasmid), used for transforming and cloning in bacteria, usually E.
coli (Batie et al., 2001). F-plasmids play a crucial role because they contain partition genes
that promote the even distribution of plasmids after bacterial cell division. The bacterial
artificial chromosome's usual insert size is 150–350 kbp. A similar cloning vector called
a PAC has also been produced from the DNA of P1 bacteriophage. BACs are often used
to sequence the genome of organisms in genome projects, for example the Human Genome
Project. A short piece of the organism's DNA is amplified as an insert in BACs, and then
sequenced. Finally, the sequenced parts are rearranged in silico, resulting in the genomic
sequence of the organism. BACs were replaced with faster and less laborious sequencing
methods like whole genome shotgun sequencing and now more recently next-gen
sequencing.
• Yeast Artificial Chromosome: are genetically engineered chromosomes derived from the
DNA of the yeast, Saccharomyces cerevisiae, which is then ligated into a bacterial plasmid.
By inserting large fragments of DNA, from 100–1000 kb, the inserted sequences can be
cloned and physically mapped using a process called chromosome walking. This is the
process that was initially used for the Human Genome Project, however due to stability
issues, YACs were abandoned for the use of Bacterial artificial chromosomes (BAC). The
primary components of a YAC are the ARS, centromere, and telomeres from S. cerevisiae.
Additionally, selectable marker genes, such as antibiotic resistance and a visible marker,
are utilized to select transformed yeast cells. Without these sequences, the chromosome
will not be stable during extracellular replication, and would not be distinguishable from
colonies without the vector.

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CHAPTER THREE
3.0 HOW DOES GENETIC ENGINEERING OF MICROORGANISMS `
HELPS TO IMPROVE THE QUALITY OF LIFE
GMMs have been widely used to produce numerous molecules required by the pharmaceutical,
agroalimentary and chemical industries. Producing these molecules involves culturing the
microorganism responsible for producing the required molecule in a fermenter containing a
suitable nutritive medium under defined conditions. This operation is generally performed in a
confined atmosphere and, in theory, does not cause microorganisms to be released into the
environment. There are several ways in which GMM has helped to improve the quality of life and
have generally been classified into five categories;
• Chemical industries; producing bioactive molecules
• Agro-alimentary; producing fermented foods
• The environment; various uses in agriculture, for pollution control, etc.
• Medicine; producing microbes and substances for therapeutic purposes (e.g. live
vaccines, pharmaceuticals)
• Research; gaining fundamental knowledge
a. Chemical industry for producing bioactive molecules
In the chemical industry, GMMs have been used to produce many molecules such as enzymes,
organic acids and biofuels produced by these microorganisms. Majority of these products serve as
reagents for other industries (Siedler et al., 1998). Certain enzymes produced are used in
agroalimentary industries to digest food products and catalyze the synthesis of other products such
as alcohol or organic acid in brewery industries. Other products of the chemical industries act as

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final products for consumption by man or its environment. These include alcohol, amino acids,
vitamins or fuel used by automobiles.
b. Agro-alimentary for producing fermented foods
A vast number of fermented foodstuffs we consume have genetically been modified. In the western
world, most foodstuffs such as Bread, wine, cheese, butter, crème fraiche, yoghurts, kefir,
fermented meats (dry-cured sausage, salami) and fermented vegetables (sauerkraut, olives) are
produced by the action of an extremely varied microbial flora. Some of these fermented foods can
either be produced from a complex and little known microbial flora that may be categorised as
wild flora found in raw materials and the environment. This includes some unpasteurized cheeses,
beers and sourdough bread. Others are made from industrial starter cultures of simpler composition
and identified flora usually been manipulated genetically (industrial floral) and may include many
cheeses made from pasteurized milk (Lomedico, 1982). Lastly, other fermented foodstuffs contain
both complex wild flora and industrial flora. The organoleptic component of genetically modified
foodstuff serves an additional advantage of genetically modified microbial flora over the wild
flora. Recombinant DNA technology has major uses which made the manufacturing of novel
enzymes possible which are suitable in conditions for specified food-processing. Several important
enzymes including lipases and amylases are available for the specific productions because of their
particular roles and applications in food industries. Microbial strains production is another huge
achievement that became possible with the help of recombinant DNA technology. A number of
microbial strains have been developed which produce enzyme through specific engineering for
production of proteases. Certain strains of fungi have been modified so that their ability of
producing toxic materials could be reduced. Lysozymes are the effective agents to get rid of
bacteria in food industries. They prevent the colonization of microbial organisms. It is suitable

29. 29
agent for food items including fruits, vegetables, cheese, and meat to be stored as it increases their
shelf life. The inhibition of food spoiling microorganisms can be carried out through immobilized
lysozyme in polyvinyl alcohol films and cellulose. Lysozyme impregnation of fish skin gelatin
gels increase the shelf life of food products and inhibit different food spoiling bacterial growth.
Exopolysaccharides of Staphylococcus and E. coli can be hydrolyzed with the use of DspB which
is engineered from T7. This ability of DspB causes a declination in the bacterial population.
Biofilms related to food industries can be removed by the combining activity of serine proteases
and amylases. Staphylococcus aureus, Salmonella infantis, Clostridium perfringens, B. cereus,
Campylobacter jejuni, L. monocytogenes, Yersinia enterocolitica, and some other food spoiling
microorganisms can be inhibited by glucose oxidase. It is also considered one of the most
important enzymes in food industry to kill wide range of foodborne pathogens. With the invention
of HBV vaccine production in plants, the oral vaccination concept with edible plants has gained
popularity. Lysozymes are the effective agents to get rid of bacteria in food industries. They
prevent the colonization of microbial organisms. It is suitable agent for food items including fruits,
vegetables, cheese, and meat to be stored as it increases their shelf life. The inhibition of food
spoiling microorganisms can be carried out through immobilized lysozyme in polyvinyl alcohol
films and cellulose. Lysozyme impregnation of fish skin gelatin gels increase the shelf life of food
products and inhibit different food spoiling bacterial growth. Biofilms related to food industries
can be removed by the combining activity of serine proteases and amylases. S. aureus, Salmonella
infantis, Clostridium perfringens, B. cereus, Campylobacter jejuni, L. monocytogenes, Yersinia
enterocolitica, and some other food spoiling microorganisms can be inhibited by glucose oxidase.
It is also considered one of the most important enzymes in food industry to kill wide range of
foodborne pathogens. Derivation of recombinant proteins being used as pharmaceuticals came into

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practice from first plant recently and many others are through to be used for more production of
similar medically important proteins. These processes and predictions are helpful to improve crop
production and resistance to either environmental or microbial stresses. Genetic modification is
needed in facilitating gene by gene introduction of well-known characters. It allows access to
extended range of genes from an organism. Potato, beans, eggplant, sugar beet, squash, and many
other plants are being developed with desirable characters, for example, tolerance of the herbicide
glyphosate, resistance to insects, drought resistance, disease and salt tolerance. Nitrogen
utilization, ripening, and nutritional versatility like characters have also been enhanced.
c. Environmental protection in pollution control and Agriculture
Microorganisms extensively exploit their environment in search for food and protection to enhance
their survival. In return, they generate substances useful to man and eliminate other substance not
needed by man (Batie et al., 2001). Microorganisms come into play in many pollution control
processes, the most common of which is sewage treatment, a process that involves highly complex
wild flora. Methods for controlling pollution of more specific compound (hydrocarbons, slurry,
various pesticides, etc.) have also been developed and involve selected flora, which is less complex
(in terms of diversity). However, the action of this flora is far from optimal and therefore requires
genetic improvement. Numerous GMMs with properties that are compatible with the process
(resistance to the substrate to be biodegraded, good establishment in the environment, etc.) have
been developed. In the agricultural sector, microbial strains are used to enhance the growth of
plants and crop protection by enriching the soils with valuable nutrients. In the same way as above,
it has been necessary to develop genetically recombinant strains to optimise these processes.
Strains of Sinorhizobium meliloti that have been genetically improved to enable nitrogen fixation

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by the plant have been used since 1997 to seed legume crops. Similarly, pesticides using other
genetically improved species (Agrobacterium radiobacter) are used in soils.
d. Medicine - producing microbes and substances for therapeutic purposes
In the pharmaceutical industry, many molecules (such as antibiotics or vitamin B12) are produced
by microorganisms which synthesize them naturally (Metcalfe etal.,1996). There are also numerous
molecules whose gene has been cloned in microorganisms (e.g. human insulin, growth hormone,
Hepatitis B vaccine). All these molecules have been marketed for many years and are part of
developed countries' daily therapeutic arsenal (recombinant insulin has been produced since 1983).
Owing to their ability to survive or pass through human and animal mucosa, microorganisms can
be used to treat or prevent certain diseases. For example, a strain of Lactobacillus jensenii has been
modified to secrete the CD4 protein used by the HIV virus in the vaginal mucosa to penetrate
lymphocytes. This secreted protein also traps viruses. Recombinant DNA technology has wide
spectrum of applications in treating diseases and improving health conditions. Gene therapy is an
advanced technique with therapeutic potential in health services. The first successful report in field
of gene therapy to treat a genetic disease provided a more secure direction toward curing the
deadliest genetic diseases. Many different cancers including lung, gynecological, skin, urological,
neurological, and gastrointestinal tumors, as well as hematological malignancies and pediatric
tumors, have been targeted through gene therapy. Inserting tumor suppressor genes to
immunotherapy, oncolytic virotherapy and gene directed enzyme prodrug therapy are different
strategies that have been used to treat different types of cancers. The p53, a commonly transferred
tumor suppressor gene, is a key player in cancer treating efforts. In some of the strategies, p53
gene transfer is combined with chemotherapy or radiotherapy. The most important strategies that
have been employed until now are vaccination with tumor cells engineered to express

32. 32
immunostimulatory molecules, vaccination with recombinant viral vectors encoding tumor
antigens and vaccination with host cells engineered to express tumor antigens. Recombinant DNA
approaches have recently contributed its role through heterologous expression, where the enzyme's
genetic information is expressed in vitro or in vivo, through the transfer of gene. Comparatively
conventional vaccines have lower efficacy and specificity than recombinant vaccine. A fear free
and painless technique to transfer adenovirus vectors encoding pathogen antigens is through nasal
transfer which is also a rapid and protection sustaining method against mucosal pathogens. This
acts as a drug vaccine where an anti-influenza state can be induced through a transgene expression
in the airway. In vitro production of human follicle-stimulating hormone (FSH) is now possible
through recombinant DNA technology. FSH is considerably a complex heterodimeric protein and
specified cell line from eukaryotes has been selected for its expression. Assisted reproduction
treatment through stimulating follicular development is an achievement of recombinant DNA
technology. A large number of patients are being treated through r-FSH. Most interestingly r-FSH
and Luteinizing Hormone (LH) recombination was made successful to enhance the ovulation and
pregnancy. As an important component of alternative medicine, traditional chines medicines play
a crucial role in diagnostics and therapeutics. These medicines associated with theories which are
congruent with gene therapy principle up to some extent. These drugs might be the sources of a
carriage of therapeutic genes and as coadministrated drugs. Transgenic root system has valuable
potential for additional genes introduction along with the Ri plasmid. It is mostly carried with
modified genes in A. rhizogenes vector systems to enhance characteristics for specific use. The
cultures became a valuable tool to study the biochemical properties and the gene expression profile
of metabolic pathways. The intermediates and key enzymes involved in the biosynthesis of
secondary metabolites can be elucidated by the turned cultures.

33. 33
e. Research gaining fundamental knowledge
Another no less important use of GMMs is in research laboratories, as they enable us to better
understand how microorganisms function. Numerous genes belonging to a wide variety of
microbial species have therefore been cloned and have given rise to thousands of GMM strains
used as research material by researchers. In Europe today, genetically modified microorganism are
mainly used to produce molecules in fermenters. In this case, the microorganisms are in fact
maintained in a confined atmosphere which theoretically prevents their release into the natural
environment. They are used to produce the molecules used in the pharmaceutical, agro-alimentary
and chemical industries.

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CHAPTER 4
4.0 ADVANTAGES OF GENETICALLY MODIFIED MICROORGANISMS (GMOs)
The principles of beneficence clear states that any activity undertaken by man for his use or the
environmental must be beneficial to him, his community as well as the environment. Genetic
engineering has been very instrumental in; improving human well-being and supplied us with
products that alleviate illness, clean up the environment, increase crop yields, among other
practical benefits to humanity and the ecosystem. The socioeconomic benefits are not neglected.
Most countries with this advance technology have fully been empowered with riches creating
economic and political stability. This is well elaborated above on the applications of GMM.

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4.1 DISADVANTAGES OF GENETICALLY MODIFIED MICROORGANISMS (GMOs)
 It may affect human health causing diseases: Certain diseases have as well been created
be researchers trying to manipulate microorganism. This was the case of Dutch elm disease
into a new area while testing genetically modified bacteria in fungi. Most microbes used in
food, chemical industrials are generally nonpathogenic (Napompeth, 1996). However,
genetic manipulation of these microorganisms may lead to the development of virulent
form which may be pathogenic causing diseases to humans, plants and animals. Also,
genetic manipulation of pathogenic strains to less virulent forms in the development of
vaccines against certain diseases may develop to more virulent forms. The issue is even
more crucial when it comes to the development of biological weapons: in this case, the
primary objective is the creation of new pathogens against which an army or an enemy
country is not able to defend itself. An area of research involved the modification of the
cowpox virus so that it might cross species barriers and infect other species, such as
humans. One of the viruses developed demonstrated an increased pathogenicity. Such
GMOs threaten to escape the control of scientists and to have unpredictable consequences
on animal and human species.
 It disrupt the ecological balance exterminating certain species, cause drug and
herbicides resistance: Microorganism within the ecosystem exists as numerous diverse
species living within particular ecological niches. Genetic manipulation of microorganisms
may lead to the emergence of more adapted forms which may better adapted to a new
environment, may colonise it, thus greatly disrupting the ecological balance, whether
microbial, plant or animal. Such a problem is genuinely conceivable and was apparent even
before the arrival of GMMs. Some cases are already known in which microorganisms have

36. 36
found themselves in a new ecological niche as a result of (generally accidental) human
intervention. They have subsequently colonise this niche, disrupting it to a great extent. A
well-known example of this involves the toxigenic unicellular alga Chrysochromulina
polylepis which, because of human activity (the release of nitrogenous substances into the
sea), invaded part of the North Sea and the English Channel, leading to significant health
problems as it produces toxins which are pathogenic for humans.
 It lowers the genetic and ecological diversity: The ecosystem is a very diverse with
numerous microbial species. This diversity plays a very important role in managing the
ecosystem. The Adoption of GMMs may reduce the genetic diversity as well as ecological
diversity of microbial flora. GMM may be more adapted to the environment and compete
out the local strain within their genetic variant.

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CHAPTER FIVE
5.0 CONCLUSION
Genetic engineering is an important development in science that has made the human life much
easier. In recent years, it has advanced strategies for biomedical applications such as genetic
diseases, diabetes, and several plants disorders especially viral and fungal resistance. The role of
Genetic engineering in making environment clean (phytoremediation and microbial remediation)
and enhanced resistance of plants to different adverse acting factors (drought, pests, and salt) has
been recognized widely. The improvements it brought not only in humans but also in plants and
microorganisms are very significant. The challenges in improving the products at gene level
sometimes face serious difficulties which are needed to be dealt for the betterment of the
recombinant DNA technology future. In pharmaceuticals, especially, there are serious issues to
produce good quality products as the change brought into a gene is not accepted by the body.
Moreover, in case of increasing product it is not always positive because different factors may
interfere to prevent it from being successful. Considering health issues, the recombinant
technology is helping in treating several diseases which cannot be treated in normal conditions,
although the immune responses hinder achieving good results. The improvements it brought not
only in humans but also in plants and microorganisms are very significant and also helps to
improve the quality of life.

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