Download The purpose of modifying the genetic properties of any organism is

Title:
An Assessment of Genetically Engineered Micro-organisms for
Release into the Environment.
Submitted To:
Michael Broaders
Submitted By:
Clare Bowens
Laura Greally
Genevieve O’Malley
Date:
January 1999
Environmental Science and Technology, Year 4
1
Table of Contents:
1.0
Introduction
Page 1
2.0
Examples and uses of engineered micro-organisms
Page 14
3.0
Risk assessment of genetically engineered micro-organisms
Page 35
4.0
Release and monitoring of GEMs in the environment
Page 45
5.0
Legislation regarding GEMs
Page 54
6.0
Conclusion
Page 57
References
Page 58
2
1.0 INTRODUCTION.
Biotechnology, in the simplest and broadest sense, is a series of enabling technologies
that involve the manipulation of living organisms or their subcellular components to
make or modify products, improve plants or animals, or to develop micro-organisms
for specific uses.
One of the principal tools of biotechnology is genetic engineering, also known as
recombinant DNA technology. Genetic engineering is the transfer of a gene from one
organism, the donor, into another, the recipient. Genetic engineering is a relatively
new and rapidly developing technology which has opened up almost limitless
possibilities for influencing the genetic makeup of living organisms. While this may
lead to many useful and exciting developments in industry, conservation, and
medicine, it may also have the potential for producing considerable ecological or
human problems.
This project addresses the rapidly growing area of genetic engineering of microorganisms, especially bacteria, through a discussion of all facets of the technology.
3
1.1 FUNDAMENTALS OF GENETICS.
In both eukaryotic and prokaryotic cells, the molecule that serves as the ultimate
agent of chemical control is deoxyribonucleic acid ( DNA ). DNA is a long threadlike
molecule composed of subunits, it’s overall structure being referred to as a double
helix. DNA molecules can be very long, sometimes containing more than a hundred
million subunits called nucleotides. It has been found that each nucleotide is
composed of three parts; a flattened ring structure called a base, a sugar ring called
deoxyribose, and a phosphate. Alternating sugars and phosphates form the backbone
of the DNA. The bases are located between the backbones of the DNA strands, and
they lie perpendicular to the long axis of the strands. As the backbones of the two
strands wind around each other, they form a double helix, leading to the popular
expression for DNA. The bass tend to stack one on top of the other, like steps in a
spiral staircase. There are four different bases, (abbreviated A, T, G, and C), the
letters stand for adenine, thymine, guanine, and cytosine which are their chemical
names. Since each nucleotide contains only one base, the nucleotides can also be
identified by the same four letters. These four nucleotides are precisely ordered in
DNA, and it is through this arrangement of nucleotides that cells store information.
Extensive examination of DNA has led to the identification of three rules govern
DNA structure. First a single DNA strand does not have branches. Consequently, the
information is stored in a simple line. Second, the ends of a DNA strand are
chemically different. Thus a strand of DNA has directionality. Third when two DNA
strands come together and form a double helix, bases must fit together in a precise
way. Whenever an A occurs in one strand a T must occur opposite it in the other
strand. Likewise G always aligns opposite C. Only when the bases are properly
paired will the two DNA strands fit together. This third rule is called complimentary
4
base pairing and allows the two strands to act as templates for the formation of new
strands.
Genes are discrete stretches of nucleotides that contain information specifying the
sequence of amino acids in proteins. It takes three nucleotides to specify a particular
amino acid, that is, specific nucleotide triplets or codons correspond to specific amino
acids. Specific combinations of nucleotides also signal the beginning and end of a
gene. A gene is a portion of DNA molecule composed of a specific series of
nitrogenous bases that either chemically codes for the production of a specific protein
or RNA molecule or serves as an operator in controlling the transcription of RNA
within an operon unit. The DNA of E. coli, one of the most thoroughly investigated
nucleoids, contains about 5 × 106 base pairs. That amounts to approximately 5000
genes, many of which have been identified in their proper sequence.
An organism’s DNA constitutes a catalogue of genes known as the genotype of the
organism. The expression of those genes will result in a certain collection of
characteristics known as the phenotype. Although the phenotype of an organism
consists of its observable characteristics, the genotype is not visible because it is the
DNA chemical code (formula) of an organism. There is not always a total expression
of the genotype. Particular genes may not be expressed for a variety of reasons. In
some cases the physical environment will determine if certain genes are expressed.
For example if lactose is not supplied to a bacterial population that can metabolise
that sugar, that part of the phenotype will not be expressed because the presence of
lactose is required to induce the formation of the enzymes needed for the sugar’s
breakdown.
DNA is very stable, thus it is an excellent molecule to serve as the transmitter of
chemical codes through generations. The stability of DNA and it’s resistance to
5
change ensures the continuation of a species even though alterations regularly occur
in it’s gene structure. Any permanent change in a nitrogenous base sequence of DNA
is called a mutation.
1.2 FUNDAMENTALS OF BACTERIA.
Bacteria are the simplest organisms found in most natural environments. Bacteria
replicate quickly by simply dividing in two by binary fission. When food is plentiful
‘survival of the fittest’ generally means survival of those that can divide the fastest.
The ability to divide quickly enables populations of bacteria to adapt rapidly to
changes in their environment.
In nature, bacteria live in a wide variety of ecological niches and they show a
corresponding richness in their underlying biochemical composition. Two distinctly
related groups are recognised, the eubacteria, which are the commonly encountered
forms that inhabit soil, water, and larger living organisms; and the archaebacteria,
which are found in such environments as bogs, ocean depths, and hot acid springs.
Despite their relative simplicity, bacteria have survived for longer than any other
organisms and are still the most abundant type of cell on earth.
Family Relationships Between Present-Day Bacteria.
ANCESTRAL PROKARYOTES, which gave rise to:
1) ARCHAEBACTERIA ( PROCARYOTES ).
Anaerobic bacteria living in hot acid conditions,( e.g., sulphur bacteria ).
Bacteria living in extreme salt conditions. ( extreme halophiles ).
Anaerobic bacteria that reduce CO2 to methane. (methanogens ).
2)EUBACTERIA ( PROCARYOTES ).
Gram positive bacteria.
6
Green photosynthetic bacteria.
Cyanobacteria ( blue green algae ).
Purple photosynthetic bacteria.
Nonphotosynthetic gram negative bacteria.
Spirochites.
1.3 BASIC STRUCTURE OF A BACTERIAL CELL.
All bacterial cells are procaryotic. Procaryotic cells are smaller than eucaryotic cells
and are typically the size of a chloroplast or a mitochondrion. All procaryotic cells
are surrounded by a cell wall which gives support and protection to the cell and is
made of a variety of polysaccharides. Bacterial cell walls contain large amounts of
substances known as peptioglycans, which, as their name suggests, are made up of
molecules in which peptides and sugars are combined. These form long, branched,
cross linked chains and make the wall very strong.
Many bacteria have a thick layer of jelly like material surrounding them called a
capsule which protects the bacterium from attack from viruses and antibodies. The
capsule is made of polysaccharides which absorb water to form a slimy material.
Beneath the cell wall is a cell surface membrane. This has a very similar structure to
that of eucaryotic cells, being made up of a phospholipid bilayer in which protein
molecules float.
The cytoplasm often contains large numbers of ribosomes. These are made up of
ribosomal RNA and protein and are the site of protein synthesis.
The DNA of bacteria is a single, large, circular molecule. This is unlike the DNA of
eucaryotes, which is linear rather than circular and is usually made up of several
molecules each of which forms a chromosome. Procaryotic DNA does not form
7
chromosomes , also as there is no nuclear envelope in prokaryotic cells, the DNA lies
free in the cytoplasm.
1.4 BACTERIAL GENE TRANSFER.
‘Horizontal gene transfer’ is the term given to the process of movement of genetic
material from one bacteria to another. The term ‘horizontal’ gene transfer is used to
distinguish it from the ‘vertical’ transfer occurring between a parent and it’s
offspring. Genes travel between independent bacteria more often than was once
assumed, by one of three processes, namely, conjugation, transformation and
transduction.
CONJUGATION:
Conjugation was the first mechanism of gene transfer studied extensively as a way
bacteria might disseminate genetic material in nonlaboratory arenas. The process was
identified in 1946 by Joshua Lederberg and Edward Tatum during their studies of E.
coli. Conjugation in prokaryotes is the transfer of genes from one cell to another by
direct contact. The genes that control the process are located on an extra
chromosomal piece of DNA called a plasmid. A plasmid is a small circular piece of
naked DNA that is self replicating and contains a limited number of genes, about 40.
Plasmids often carry genes that enhance the chances of survival in hostile
circumstances. For example, in addition to including the genes needed for their own
replication and transfer, they often harbour genes for proteins that enable bacteria to
evade destruction by antibiotics, to degrade toxic compounds such as PCBs or to
transform mercury or other heavy metals into less noxious forms.
Plasmids that control such characteristics as fertility are called F factors and those that
contain genes for transferable drug resistance are known as R factors. Many Gram-
8
negative bacteria have been shown to contain F factors that enable them to form pairs
and mate by conjugation. Gram-negative bacteria that contain F factors are
designated F+, or males, since they serve as donors. Those that lack the F factor are
designated F-, or females, since they serve as recipients. When the F factor is donated
to an F- cell, the female becomes a male or F+.
The first stage in plasmid-controlled conjugation involves the attachment of two cells.
A donor bacterium attaches an appendage called a pilus, which is a filamentous
structure extending from the cell wall of the F+ Cell, to a recipient bacterium that
displays a receptor for the pilus; then the pilus retracts, drawing together the donor
and recipient. Generally many donors extend pili at about the same time, and several
donor cells can converge on a recipient at once. Consequently, extension of pili
causes bacterial cells to aggregate into clusters. After contact has been made between
the two cells a conjugation tube is formed between them and as that tube is formed
the plasmid is replicated inside the donor cell. This process takes place in the same
way that the host nucleoid is replicated. One of the F-factor plasmids remains
attached to the inner surface of the F+ cell, the other plasmid is free to move through
the conjugation tube into the recipient cell. After the transfer has been completed and
the cells separate both the recipient and the donor contain plasmids. The percentage
of F factor containing bacteria in a population increases if the micro-organisms are
crowded into close contact. Plasmid controlled conjugation occurs more easily and
successfully within the Gram-negative enteric species normally found in the intestinal
tract. Those bacteria show a great amount of genetic variety. Bacterial populations
that lack such close contact usually have a lower rate of conjugation, fewer F factors
and less genetic variety.
9
Although conjugation in this exact form is not known to exist among the Grampositive bacteria, a conjugation like process has been identified in the Gram-positive
Streptococcous faecalis. In that process, the recipient cell excretes a protein
compound that is a cell clumping agent. The presence of this so-called sex
pheromone ( i.e. a chemical released into the environment that triggers behavioural
processes in some other individual ) causes plasmid containing donor cells to
synthesise another substance that becomes located on the donor cell surfaces. The
aggregation substance can recognise a binding compound on the recipient cells, and
the two can bind together. Such behaviour facilitates aggregation or clumping so that
conjugation can occur.
TRANSFORMATION.
Although conjugation was the first mechanism of bacterial gene transfer to be studied
extensively in the environment, it was not the earliest to be identified. The study of
gene transfer among bacteria began in 1928, when British bacteriologist Frederick
Griffith observed that nonvirulent pneumococcal bacteria became virulent when
injected into mice along with dead virulent pneumococcus. Griffith concluded that
the initially nonvirulent bacteria picked up a ‘ transforming ’ agent from the dead
virulent bacteria and thus became potent enough to kill the mice. That transforming
agent is now known to be DNA that was released into the surrounding medium when
the dead bacteria fell apart.
Transformation in bacterial cells may be defined as the process whereby a recipient
cell takes in a segment of naked DNA from the environment which may have been
released from a donor cell while it was alive or after it died. Not all bacterial cells
have the ability to take in naked DNA. These organisms with the genetic ability to do
so are called competent cells. Competent cells may operate naturally, or they may be
10
artificially stimulated to take in DNA if the environment in which the culture is being
grown is altered.
Trans formation occurs in three stages. After release by a donor cell, a large segment
of DNA is first bound to a special receptor site on the surface of the competent cell.
The segment is then cut into smaller, more manageable pieces by a DNAase enzyme
released by the recipient. Finally, the attached segment of DNA is actively moved
into the cell where it is prepared for recombination.
Thus the essential factors in the process of transformation are:
* The freed DNA must remain stable.
* The potential recipient cell display specialised surface proteins that bind to the
DNA and internalise it. ( i.e. be compent )
Until recently scientists assumed that transformation would not occur in most places,
because free DNA would not be stable in soil or water. However, studies by Michael
Lorenz and Wilfried Wackernagel have demonstrated that free DNA can become
stable by associating with soil components and that this DNA can be taken up by
competent cells. Newer investigations indicate that plasmid DNA has at times been
transferred by transformation in river water and in the epilithion on river stones.
Ultimately it can be said that transformation plays an important role in forming new
gene combinations and creating genetic variety in micro-organisms.
TRANSDUCTION.
The third method of bacterial gene transfer is called transduction. In transduction,
bacteriophages ( viruses that infect bacteria ) pick up genetic material from one
bacterial cell and deposit it in another. Bacteriophages have a lytic cycle during
which the virus adsorbs to the surface of the host cell and injects its DNA through the
outer covering. Once inside the nucleic acid takes command of the host’s metabolism
11
to synthesise more virus particles. After the synthesis is complete the host cell is
ruptured to free new bacteriophages which go on to infect other cells. The lytic cycle
takes place very quickly ( about 40 minutes ) and there is no delay from the time of
initial penetration to lysis of the host. During the lytic cycle the DNA of the host is
broken down into small segments that are about the same size as the virus nucleic
acid. In the case of certain types of bacteriophages, a small segment of host DNA is
sometimes incorporated during assembly of the viron into the virus protein coat in
place of the phage genome.
Laboratory experiments indicate that some bacteriophages can apparently infect
several species and even genera of bacteria, suggesting that they might broadcast
bacterial genes well beyond the locale where they first took up the genes.
12
1.5 HOW GENETIC ENGINEERING IS CARRIED OUT.
The process of genetic engineering involves three basic stages, each of which have
several smaller steps.
( 1 ) The desired gene in the donor organism is identified and isolated. It is then
cloned.
( 2 ) Copies of the genes are inserted into vectors usually a virus or a bacterial
plasmid. The vector is also cloned so that many new vectors containing the required
gene are produced.
( 3 ) The gene is inserted into the recipient organism by the vector.
IDENTIFYING AND ISOLATING THE GENE:
This may be done by extracting all the DNA from a cell and then using enzymes
called restriction endonucleases to break it down into smaller fragments. These
fragments are then inserted into a vector which produces many copies of each
fragment. These sets of DNA fragments are called genomic libraries.
To identify the fragment of DNA containing the desired gene a probe is used. A gene
probe is a length of single stranded DNA containing the complimentary base
sequence to the gene you are interested in. The DNA of the probe is ‘labelled’ in
some way, often by using a radioactive isotope of phosphorus, as a component of its
phosphate groups. After the DNA in the cell is cut into pieces using restriction
endonucleases, the pieces can be separated using gel electrophoresis. The gel,
containing the DNA fragments, is then soaked in sodium hydroxide solution, which
breaks the double stranded DNA apart so that it now consists of single strands. A
nitro-cellulose sheet is then placed on the gel, and the single stranded DNA fragments
stick to it, in the same pattern as on the gel.
13
The nitro-cellulose sheet, containing the single stranded DNA molecules, is incubated
with the probe. The single stranded probe will base pair with the gene you are
looking for , because it has a complimentary base sequence.
X-ray film is then placed over the nitro-cellulose sheet. It will darken where the
radioactivity from the probe affects it, so you can tell exactly where the required gene
is.
INSERTING THE GENE INTO A VECTOR.
In biology the term vector is used for a agent which can carry something from one
organism to another. In genetic engineering, a vector transfers DNA from one
organism to another. Plasmids are usually chosen as vectors in genetic engineering.
To insert a piece of DNA into a plasmid, the plasmid is cut open using a restriction
endonuclease, which make staggered cuts in the DNA, leaving a short length of
unpaired bases at each end. These are called sticky ends. If the length of DNA to be
inserted was produced using the same restriction endonuclease, then it too will have
sticky ends and they will have the same base sequences as those on the plasmid. If
the broken plasmid and the required DNA are mixed, the sticky ends will stick
together, as complementary bases pair. Another enzyme, called DNA ligase, is used
to join the sugar-phosphate backbones of the plasmid and the inserted DNA together.
This new DNA molecule is called recombinant DNA.
INSERTING THE VECTOR INTO THE REQUIRED ORGANISM:
The plasmids can now be inserted into bacteria. This can be done by mixing them
together so that transformation can take place. Cells can be made competent by
treating the bacteria with a solution which makes the cells more likely to take up the
plasmids. To determine the bacteria which have taken up a plasmid from those who
14
have not, the bacteria can be grown with an antibiotic whose resistance gene is known
to be carried on the plasmid.
The transformed bacteria are now grown on a large scale. Each time a bacterial cell
divides, the plasmid inside it also divides and replicates the gene giving rise to the
expression of the desired characteristic, or synthesis of a desired product for example
a hormone.
15
2.0 Examples and Uses of engineered micro-organisms
In the past a full understanding of any biological process could be achieved only
when there has been a detailed analysis of gene structure and function. This analysis
was undertaken by making mutants, studying their properties, mapping them and
generating hypotheses for future testing.
Hypothesising what has happened at the DNA level is no longer necessary: The genes
now can be cloned and sequenced and the location and nature of the mutation
identified precisely, be it base change, deletion or addition.
Because of the speed and precision of the techniques of gene manipulation, biologists
now are making major advances in the analysis of fundamental but much more
complex biological systems. Examples range from the control of mitosis and devision
of individual cells to the differentiation and development of whole animals. These
studies are being facilitated by the impact of gene manipulation on biochemical
methods.
In 1970 Escherichia coli molecules; a normally innocuous commensal occupant of the
human gut, were manipulated in vitro. By inserting a piece of DNA of interest into a
vector molecule, i.e. a molecule with a bacterial origin of replication, when the whole
recombinant construction is introduced into a bacterial host cell, a large number of
identical copies is produced. Together with the rapid growth of bacterial colonies all
derived from a single original cell bearing the recombinant vector, in a short time
(e.g. a few hours) a large amount of the DNA of interest is produced. This can be
purified from contaminating bacterial DNA easily and the resulting product is said to
have been "cloned".
In the cell, proteins play a key role because they are intermediaries between gene and
phenotype. Traditionally proteins have been purified from cell extracts and their
16
properties studied in-vitro. However, the behaviour of a purified protein in the test
tube (“in-vitro biochemistry”) may be quite different from that of the same protein in
the complex milieu of the cell. Now it is possible to do “in-vivo biochemistry” by
under - or overproducing natural and mutant protein inside the cell and studying their
effects on key cellular processes. The traditional approach also presupposes that
enough of the protein is made in the cell for it to be made in the first place. However,
many key cellular proteins are made transiently and at very low levels, e.g. proteins
involved in cell division, lymphokines, etc. In principle, using recombinant DNA
technology it is possible to produce any protein in quantity. Impact of this goes far
beyond understanding cellular processes: many of them have commercial value as
pharmaceuticals.
17
2.1 Which Cloning host to use?
There is a wide range of cloning hosts and theoretically, any one could be used to
overproduce a protein of interest. So, what governs the ultimate choice?
If overproduction is all that is required then it will be convenience. More often than
not, though, the deciding factor will be the degree of authenticity required. Ideally a
recombinant-derived protein would have the same amino acid sequence, the same
post-translational modifications, the same three dimensional structure and the same
range of biological activities as its natural counterpart. In practice this is difficult to
achieve and what deviation from the ideal is acceptable depends on the use to which
the protein will be put. For an enzyme to be used as a detergent -additive the key
parameters will be specific activity and stability. For a therapeutic protein which will
be administered parentally the criteria are much more stringent.
None of the cloning systems currently available are ideal. Each has its advantages
and disadvantages.
The following table shows the advantages and disadvaantages of the cloning hostss
noww in use.
18
Comparison of different organisms as cloning hosts
Organism
Advantage
Disadvantage
Escherichia
Ease of manipulation
Promoters and gene
regulation well understood
Many high-expression
vectors available
Easy to culture on a large
scale
Already used in the
manufacture of insulin,
interferon and human
somatotrophin
Many proteins naturally
exported into growth
medium
Non-pathogenic
Easy to culture
Some Bacillus enzymes
excreted at high level
(> 5 gl-1)
Widely used industrial
organism which is easy to
culture
Glycosylates proteins
Can get export into growth
medium of heterologous
proteins
High-level expression
systems developed
Large surface area to
volume ratio should favour
protein export
Have been used in
microbiology for over 40
years
Large surface area to
volume ratio should favour
protein export
Widely used in industrial
microbiology
Good expression systems
been developed
Do not usually get export of
proteins into growth
medium
Overexpressed foreign
proteins often form
aggregates (’inclusions’) of
denatured protein
Many foreign proteins
rapidly degraded
Many post-translational
modifications do not occur
Still not much known about
gene regulation
Good, high-level expression
vectors lacking
High-level export of
heterologous proteins not
achieved
Bacillus subtilis
Saccharomyces cerevisiae
Filamentous fungi
Actinomycetes
19
Much still to be learned
about control of gene
expression
Post-translational
modifications of proteins not
necessarily the same as
those in the animal cell
Heterologous proteins can
form inclusions
Promoters/gene regulation
poorly understood but may
be similar to yeast
Good expression systems
lacking rheology of
fermentations important
Promoters/gene regulation
still poorly understood
Rheology of fermentations
important
The purpose of modifying the genetic properties of any organism is to make it capable
of producing new substances or performing new functions. Genetic modification is
also central to the development of new gene therapy treatments to combat serious
disease and disability. Increasingly, genetically modified products (i.e. Products
consisting of or containing GMOs) are been released into the environment as seeds
and crops, entering the food-chain as “novel” foods, and been used in human
medicines.
The application and hypothetical benefits of genetic engineering of micro-organisms
cross into many different areas in everyday life. The following is a summary of the
examples and uses of genetic engineering:
2.2 Healthcare using Transgenic Micro-organisms
Genetic engineering is very important in medicine. Commercial products that have
been made via genetic engineering and been given the approval for diagnosing and
treating disease include: Humulin (rDNA - derived from human insulin), human
growth hormone, alpha interferon, erythropoietin and tissue plasma activator. Work
is now been done at several research facilities towards developing vaccines against
influenza, AIDS, polio, herpes viruses, cholera, Rocky Mountain spotted fever, and
against several human diarrheal diseases.
Transgenic micro-organisms are micro-organisms that have had their genetic make-up
altered by transferring itself into a gene from another species. As a result it
manufactures a protein that it would not normally produce.
Once a gene has been isolated it is relatively easy to move it into a bacterium. Once
in place the bacterium can manufacture the protein coded by the gene. Placing the
human insulin gene into the bacteria has been successfully used commercially to
20
produce human insulin, a vital drug for people with diabetes. Another example is the
yeast cell that have DNA incorporated so that they manufacture a Hepatitis B vaccine.
Patents for genetically altered micro-organisms are now routinely granted by the US,
European and Japanese Patent offices.
21
Current status of recombinant proteins used in healthcare.
Protein
Size Structure
Expression
System
Clinical
indications
Comments
Human Insulin
Two peptide
chains. A 21
amino acids
long, and B 30
amino acids
long
E.coli
Juvenile onset
diabetes
Human
Somatotrophin
191 amino acids
E. coli
Pituitary
dwarfism
IFN-α2
166 amino acids
E. coli
Hairy cell
leukaemia
Prophylaxis of
common cold
IFN - Y
143 amino acids
glycosylated
E. coli
Treatment of
cancers
Treatment of
viral diseases
Thrombosis
A and B chains
made separately
as fusion
proteins and
joined invitro.
Already on
market.
If used in
treatment of
osteoporosis
then market size
will be much
larger.
Has additional
methione
residue at Nterminus, but
technology for
removing this
now
Available
already on
market
Over 80%
success in
treatment of
hairy cell
leukaemia
Success with
other cancers
lower and more
variable market
size may be
limited
Unpleasant (flu
like) side effects
Already on
market
In clinical trials
Tissue
plasminogen
activator
E.coli
Yeast
Animal cells
22
Animal cell
culture most
effective
Way of
producing active
enzyme on the
market
Relaxin
53 amino acids
Insulin-like (two
protein chains)
E. coli
Facilitates
childbirth
Prepares
endometrium for
parturition and
reduces foetal
distress
Pig relaxin
shown to be
clinically
effective
α1- Antitrypsin
394 amino acids
Glycoylated
E. Coli
Yeast
Treatment of
emphysema
Prevents
cumulative
damage to lung
tissue caused by
leukocyte
elastase
In clinical trials
on the market
Interleukin - 2
133 amino acids
E. Coli
Animal cells
Treatment of
cancer
Tumour necrosis
factor
157 amino acids
E.coli
Animal cells
Treatment of
cancer
Human serum
albumin
582 amino acids
17 disulphide
bridges
Yeast
Plasma
replacement
therapy
Normally
obtained from
plasma
But now
concern over
potential
contamination
with AIDS virus
Hepatitis B
Surface antigen
226 amino acids
(monomer)
Yeast
Mammalian
cells
Vaccination
Monomer selfassembles into a
structure
resembling virus
particles
Now on market
23
2.3 Agriculture
Never before has there been such a demand been placed on the worlds agriculture.
The global population has been expanding each year. Recombinant DNA technology
is been applied to increase yields, to increase resistance to disease or pollution and to
create new crops that can utilise previously wasted resources.
A popular method for introducing DNA into uses a strain of bacteria found in soil
(Agrobacterium tumefaciens). In their natural state, the bacteria infect plant cells,
inserting part of their DNA and causing cancer - like growths. New genes can be
inserted into a section of the bacterium’s DNA, to transfer the genes to plant cells. As
tobacco plants are particularly susceptible to infection by this bacteria, much of the
fundamental research has been carried out on them, with the intention of using the
knowledge gained to help develop food crops.
Nitrogen Fixation
A major area of research is dedicated to finding ways of moving nitrogen - fixing
genes (NIF genes) into agricultural crops. Nitrates are vital nutrients for most plants,
and some bacteria are particularly good at creating them by biochemically reducing
nitrogen. The genes which produce the necessary NIF enzymes have been isolated,
sequenced and cloned in E.coli. Now scientists are looking for ways of placing them
into cells of crops such as wheat or rice. This would enable these crops effectively to
fertilise themselves, saving much money and increasing yields. Placing the gene in
the crops grown in developing countries would have an enormous impact on their
ability to grow food, as currently they cannot afford the nitrogenous fertilisers used in
more affluent countries.
The Following are two examples of genetically engineered insecticides used in
agriculture:
24
• Genetic engineering enables a new strain of Bacillus thuringiensis to be produced
with increased potency and wider host spectra than the original strain. This is
released as an insecticide on crops, ornamentals, forest trees and stored grains.
The engineered cells produce a crystal which can have highly specific toxic
properties which kill many insects.
• Viruses can also be used as insecticides to control insect pests. Baculoviruses are
naturally occurring and they only affect a few species of insect. They infect
arthropods, but have no effect on vertebrates or plants or they do not pollute the
environment or cause adverse reactions in soil or water. Because they are slow to
exert their effect they are usually superseded by chemical insecticides. This can be
altered by genetic engineering to improve their speed thus rendering them a viable
option for insecticides.
2.4 Food and Drink
Description of the use of genetically engineered techniques in the food industry.
Bacteria can be designed to grow on virtually any energy-rich molecules. Some have
been adapted to use methane gas as a nutrient and others grow successfully on paper
pulp. This is already providing a new source of protein to the food industry. The
growing bacteria can be harvested and their protein purified. These proteins may be
of particular value as the numbers of people eating a vegetarian diet increase.
Benefits:
• Growing farm animals to supply us with protein is a very inefficient use of energy
as It can take between 10 and 20 Kg of protein in feeds to produce 1 Kg of meat
protein. However, bacteria are much more efficient.
25
• Waste materials such as pulped newspapers could form the basic nutrient supply
for new bacteria.
Risks:
• Care will need to be taken that the bacteria do not contain proteins to which people
are allergic.
• If a microbe is designed to digest cellulose efficiently, care will be needed in the
way that it is contained. Were viable cellulose-digesting bacteria to escape into the
environment, they could devastate anything made of paper or wood.
Microbes in food production:
Microbes are used in the production of food ingredients with biotechnological
methods and production of additives; such as, sugar substitutes, fat substitutes, colour,
and flavours by micro-organisms or through cell culture techniques.
Recombinant DNA technology has also come to the help of food manufacturers who
need to know whether a product is safe to eat or whether it is contaminated with
pathogenic bacteria. A series of gene probes have been built that carry sequences
which can specifically identify the presence of a wide variety of different food
pathogens.
Some bacteria, such as Listeria monocytogenes, are only dangerous if they are alive.
Whilst conventional tests take one to three days to give results, gene probes can do
the job in a few hours. Gene probes for sections of DNA in L. Monocytogenes have
been built, and these can be used to see whether the bacteria are present. But as DNA
last for thousands of years once the bacteria are dead, this will not distinguish
between dead and live organisms. However, a new variety of probes has been built,
this time coding for the mRNA sequence. As mRNA is only present in living cells
this will only detect bacteria that are alive.
26
Industrial cheese production uses a lot of an enzyme called chymosin (commonly
called rennin) to coagulate the protein casein, found in milk. Traditionally this
enzyme is obtained from suckling calves when they are slaughtered for veal.
However, the numbers of calves being slaughtered is decreasing, but the quantity of
chymosin required for cheese making is increasing. Bacteria are modified by
transforming them with the inclusion of a gene that causes chymosin production. Such
bacterial chymosin is used in the production of vegetarian cheese.
2.5 Environment
Microbes can be designed to grow on many waste materials to produce useful
materials such as food, they can also be usefully employed to control pollution.
Bacteria have been designed to break up oil slicks
Bacteria have been designed to destroy noxious gases released form factories. Fumes
are pumped through pipes running under gravel or wood chippings, which supply a
large surface area on which the bacteria can grow. As the gases leave the pipes they
pass through the filter-bed and the bacteria ingest and destroy the noxious
components.
2.6 Waste Management
As populations concentrate in urban areas, treating garbage and other waste has
become more difficult. Waste management or bioremediation is becoming more of a
concern. Recent developments in biotechnology are providing new ways to clean up
industrial wastes and yielding efficient new production methods that are less polluting
27
than traditional processes. Biotechnology can even help convert industrial wastes
into useful products.
Treating waste water.
The following is a synopsis of a feasibility study carried out on the use of genetically
engineered micro-organisms in wastewater treatment.
Feasibility of wastewater Treatment using Genetically Engineered Micro-organisms
Introduction
Removal of xenobiotic compounds, such as synthetic polymers, aromatic compounds,
haloaromatic compounds and so on, has become a major issue of biological
wastewater treatment in recent years. As biodegradation of xenobiotic compounds
depends on specific micro-organisms, it is necessary to make corresponding
degradation micro-organisms dominant in the wastewater treatment process by
acclimation or enrichment cultures. However, as degrading micro-organisms
generally have lower growth rates than other wastewater micro-organisms and cannot
exhibit their degradation activity fully in the mixed substrates/micro-organisms
process, it is difficult to keep them dominant and to improve degradation rates of
xenobiotic compounds in an actual wastewater treatment system. Genetic engineering
or molecular breeding, which has recently developed in the biotechnology field, is an
attractive and effective way for solving the above-mentioned problems. For example,
it may be possible to create genetically engineered micro-organisms (GEMs) which
can grow either fast or flocculently with high degradation activity in order to enhance
the degradation rate of xenobiotics. This approach will be considered as a future
technology to develop an advanced wastewater treatment process.
Discussion
• Increase of degradation Activity by Genetic Engineering:
28
Simultaneous degradation of salicylate and phenol by genetically engineered P.
Putida.
By introducing the recombinant plasmid containing the nahG gene the GEM P. Putida
PpG1064(pHF400) was created, capable of mineralising salicylate in addition to
benzoate and phenol, which are degraded via the ortho cleavage pathway coded on
the chromosome of the host strain PpG1064. On the other hand the wild strain, P.
PG1064 (NAH), carries the nahG coded on the naturally-occurring plasmid NAH,
which also codes the meta cleavage pathway. In degradation tests both strains
degraded salicylate and phenol simultaneously. The degradation rates of the GEM
were higher than those of the wild strain. Especially the salicylate degradation rate of
the GEM was 2.3 times higher than the rate of the wild strain. The differences in the
substrate degradation rates between the GEM and the wild strain seem to depend on
the differences in the metabolic pathways and their transcriptional and translational
controls.
• Stability of the Recombinant Plasmid in the recipient:
In wastewater treatment using GEMs, stability of the recombinant plasmids is one of
the most important problems. In a continuous culture of GEMs without selective
pressure such as antibiotics or xenobiotics, plasmids generally are not stabily
maintained and the plasmid-free segregants tend to be dominant. As it is difficult to
keep selective pressure consistently in the wastewater treatment process because of
both quality and quantity fluctuation of influent, it is necessary to select the hostplasmid systems having high genetic stability.
• Model analysis of plasmid instability:
29
In general, it is observed that the stability of plasmid depends on two major factors,
the probability of plasmid loss due to segregation during cell devision and the
difference in the specific growth rate between the GEMs and plasmid-free cells.
In order to create a GEM having high stability, it is important to choose the host strain
whose specific growth rate is hardly affected (decreased) by plasmid maintenance.
• Increase of Ecological Stability of GEMs in the Wastewater treatment system:
Ecological stability means how long GEMs have been staying in the mixed flora (for
example in activated sludge) and maintaining xenobiotics degradation activity. So
ecological stability is an important a problem as genetic genetic stability in the actual
wastewater treatment process. Select a GEM which has high genetic stability and
apply it to a wastewater treatment system, a GEM with low ecological stability may
disappear from the process. It is considered that immobilisation is one of the easiest
methods to maintain GEMs in activated sludge. But the cost of the immobilising
materials are so expensive that the immobilisation method would be hard to apply on
a large scale. It was proposed that the floc-forming micro-organisms should be use as
a recipient for solving both ecological stability and the cost forming problem. Since
the floc-forming GEM is expected not to be washed out from the activated sludge
reactor even if it cannot grow fast in wastewater, we can strengthen the ecological
stability and degrade xenobiotic compounds continuously using the floc-forming
GEM.
Conclusion
The degradation rate, the genetic stability, and the ecological stability of GEMs were
investigated and discussed synthetically. The availability of the application of
xenobiotic-degrading GEMs was confirmed from the experimental results
demonstrating the extension of catabolic range and the increase of the degradation
30
activity by genetic engineering. It seems to be useful to use the isolates from the
activated sludge, especially the floc-forming bacteria as a recipient in actual process.
In conclusion, it is suggested that an advanced wastewater treatment process using
GEMs will be expected in the near future.
Cleaning up chemicals
Biotechnology is providing environmentally acceptable methods of modifying or
destroying chemical wastes so they are no longer toxic to the environment. This
usually involves finding bacteria or other microbes that can digest the target
pollutants. If necessary, these organisms can be genetically engineered to provide
strains with better containment-degrading potential than their natural counterparts.
An example is the research being carried out at old military dumps where TNT (2,4,6trinitrotoluene) explosive is being made safe by using white rot fungi to degrade the
dangerous explosives to harmless products. Genetically engineered bacteria are also
used to detect the presence of TNT in soil. This is useful in the detection of land
mines left over from various wars. The bacteria can be genetically engineered to
glow in the presence of certain compounds, in this case explosives. Biotechnologies
using bacteria hinge on the micro-organisms ability to metabolise and break down
organic compounds or transform heavy metals. Luminescence - glowing in visible
light and fluorescence - glowing in ultraviolet light are rare in bacteria, however
using genetic engineering the chromosomes in the bacteria can be modified to make
the bacteria glow in the presence of certain chemicals. This technique can be applied
to bioremediation e.g. the bacteria, when applied to soil, would glow if the soil was
contaminated with solvents like toluene or xylene. TNT is closely related to these
solvents chemically, so the technique was adapted to fluoresce in its presence. The
31
experimental plan was to spray a solution of genetically engineered Pseudomonas
over a field. Land mines and unexploded shells have a tendancy, over time, to leak
the explosives into the adjoining earth. When the Pseudomonas contacts the
explosives and starts metabolising it, it triggers the gene that elicits the UV glow.
Land mines leak the explosive chemicals in the parts per million range, which suits
these bacteria. Vegetation also tends to take up the chemicals, so the bacteria
glowing on the vegetation could even localise the explosives more. Mines are most
often placed in roadways and open fields where troops are likely to tread, and those
places would be ideal for the bacteria. Places where these bacteria would not work
would include rice paddies and other wet areas, which would disperse the bacteria and
rough jungle and snow. These techniques have not yet been used in the field,
however the have been proven in the laboratory and are still in the early stages of
development.
A genetically engineered strain of E.coli "E.coli K-12" is well characterised as to
metal biosorptions.
Treating petroleum Sludge and Oil spills.
Oil sludge, normally discharged into the sea from oil refineries, contains toxic
compounds that are a major threat to the marine ecology. All forms of aquatic life are
adversely affected, and contaminated fish, when eaten by humans, present a serious
health hazard.
Biotechnology, however, has shown that particular species of bacteria and fungi,
normally found in soil, can protect the marine environment by breaking down various
types of hydrocarbons, the main component of petroleum. To be effective in cleaning
up oils spills, however, micro-organisms must be able to withstand the marine
32
environment- for example they need to survive in high salt concentrations and to
grow at low temperatures. Genetic engineering can now introduce these
characteristics into "oil-eating micro-organisms".
Kajima, an US company is an active participant in a research project undertaken by
the Marine Biotechnology Institute (MBI), Tokyo based, with its main aim "to
harness micro-organisms as a force for restoring ocean areas contaminated by crude
oil and other organic solvents". These researchers are also dealing with the problem,
that among the hundreds of different hydrocarbons which make up crude oil, there are
many that cannot be decomposed by ordinary micro-organisms. These researchers
are working to isolate microbes which can effectively decompose such hard-todegrade elements.
Through the application of biotechnical methods, enzyme bioreactors are being
investigated to pretreat certain components of disposable serviceware or food waste
and allow their removal through the sewage system rather through the solid waste
disposal mechanisms or will allow their conversion to biofuel for operating
generators.
2.7 Energy
With the techniques of biotechnology, it may be possible to improve the manner in
which micro-organisms use wastes from agriculture and forestry industries, for
growth. These materials collectively referred to as “biomass” represent a renewable
energy resource. Biotechnology is already benefiting developing countries by
providing a cheap, clean and renewable alternative to fossil fuels, but the costs of the
biomass fuels such as ethane are still high relative to fossil fuel equivalent. Biomass
fuels are greenhouse gas neutral (i.e., carbon dioxide is consumed by photosynthesis
33
during the growth of the plant, and equal amounts are released when the biomass fuel
is burned. Biomass from plant materials - such as corn stalks and wood chips - can be
broken down into smaller components resulting in the release of energy. rDNA
technology can be used to increase the supply of enzymes that micro-organisms need
for degrading biomass.
The following are two examples of genetically engineered micro-organisms that have
the capability to produce ethanol:
• The increasing use of oxygenates as fuel additives provides an opportunity for
large scale expansion of fuel ethanol production. Escherichia coli was genetically
engineered to produce ethanol from pentose and hexose sugars by inserting genes
encoding alcohol dehydrogenase and pyruvate decarboxylase from the bacterium
Zymonas mobilis. Inexpensive materials such as crude yeast autolysate and corn
steep liquor can be used effectively as nutrients for this organism.
• Xylose is one of the major fermentable sugars present in cellulosic biomass,
second only to glucose. However, Saccaromyces spp., the best sugar-fermenting
micro-organisms, are not able to metabolize xylose. Recombinant DNA plasmids
were developed that can transform Saccharomyces spp. Into xylose-fermenting
yeasts. Thus Saccaromyces spp. Effectively ferments xylose to ethanol and also
effectively utilises xylose for aerobic growth and can also coferment glucose and
xylose present in the same medium.
2.8 Biological Warfare
This is the development of biological weapons (BW) through biotechnology. The
first use of biological agents is as far back as the Romans who fouled the enemies
water supplies in order to decrease enemy numbers and lower morale.
34
“Biological warfare” is the use of disease to harm or kill an adversary’s military
forces, population, food, and livestock. This includes any living (or non-living virus)
micro-organism or bioactive substance that is produced by a micro-organism that can
be delivered by conventional warhead or even civilian means.
The biggest advantage of BW is their killing efficiency compares to using
conventional weapons. They are also cost effective. Disadvantages include the
unpredictability of its use, i.e. at its release and its unknown lifespan.
The genetically engineered micro-organisms used in BW are usually a mutant of
viruses, bacteria, rickettsia and biological toxins already used in BW, in a more
virulent strain less susceptible to current treatment.
BW’s use has decreased as history has progressed. There are efforts to have a global
ban on all kinds of biological and chemical warfare, but no one can predict how these
will turn out or how well they will work.
35
3.0 Risk Assessment (RA) of Genetically Engineered
Microorganisms
The release of genetically engineered micro-organisms into the environment, although
beneficial, also carries with it concerns about possible risks to humans, animals and
the environment. Speculation on the ultimate effects of these genetic manipulation
methods on human health and the environment tends to differ depending on the roles
of the individuals involved :
• alarm raised by concerned lay people and disenchanted scientists;
• caution urged by many observers (both scientists and non-scientists);
• and calm assurance issued by biotechnology practitioners and futurists.
3.1 Properties of Recombinant Micro-organisms
The properties of recombinant micro-organisms must be considered whether they are
intentionally or unintentionally released into the environment. These include :
1. Once bacteria are released into the environment it is not possible to remove them;
2. In general, commonly used micro-organisms are characterised by fast generation
rates;
3. Most of these micro-organisms are able to adapt to adverse environmental
conditions
4. The exchange of genetic material between different species, called horizontal gene
transfer is a very common traite of prokaryotes. For instance, bacteria are the only
organisms capable of natural transformation.
3.2 Potential risks of the release of genetically engineered micro-organisms
36
into the environment
The introduction of these genetic manipulation methods has led to apprehension,
among both scientists and the public, that these methods could, for instance, give rise
to micro-organisms with entirely unknown pathogenic properties against which there
would be no protection, or micro-organisms with highly negative environmental
effects, such as organisms rapidly degrading lignocellulosic materials. The escape of
antibiotics, is another issue for concern, as this may cause the selection of resistant
bacterial strains containing plasmids, and transfer of these plasmids into other microorganisms may bring about spreading of resistance to the antibiotic.
It has been suggested that natural micro-organisms and genetically engineered microorganisms would not normally compete in the same ecological niche. Once a
genetically engineered micro-organism intended for a task such as bioremediation has
completed its intended task (i.e. its nutrient supply is exhausted), it should be unable
to survive in already-filled niches where the natural micro-organisms are adapted to
exist. For instance, after a genetically engineered micro-organism specifically
designed to metabolize oil into harmless by-products has consumed the oil, it should
die off. However, like all natural species, a population of genetically engineered
micro-organisms is subject to natural mutations, recombination, and selective
pressures. The introduced micro-organism could, therefore, continue to exist in the
environment if it develops the capability to use new sources of food, and it would not
be driven to extinction unless it had a significant disadvantage to its competitors. To
reduce or eliminate the risks for a genetically engineered micro-organism to survive
beyond completion of the task for which it is intended, genetic weaknesses may be
engineered into the micro-organism to cause its demise after its work is done.
37
There are also many times when the establishment of persistent populations of
genetically engineered micro-organisms will be the goal of a biotechnology
introduction. Such micro-organisms must be capable of surviving for the long term in
niches previously unfilled or in which they may effectively compete with natural
species. As a result of their survivability, such biotechnology products may have a
higher probability of causing unwanted environmental effects.
That genetically engineered micro-organisms may survive past their intended period
of usefulness is not the only circumstance in which unwanted effects may arise.
Bacteria can exchange genetic material with other bacteria quite easily i.e. gene
transfer. This can occur in the process of conjugation. When transfer from
genetically engineered micro-organisms to other micro-organisms occurs, genes may
persist in the natural environment even after the genetically engineered microorganisms have died. Since some changes in single genes can convert benign microorganisms into serious pathogens, the potential effects of movements of genes from
one micro-organism to another can be very important. For example, gram-negative
and gram-positive bacteria, which can occur together in natural aquatic and terrestrial
environments, exchange plasmids exclusively with members of their own group;
many restrict exchange to their own species. However, some “promiscuous” plasmids
can transfer DNA between gram-negative and gram-positive bacteria and even from
bacteria to yeast cells and plants. Obviously, then, bacteria that carry promiscuous
plasmids would be poor choices for use outside the laboratory. Note, however, that
such an occurrence is considered very unlikely; pathogenesis is usually a
multifunctional state, so it is very unlikely that a benign micro-organism can be
switched into one that is harmful.
38
3.3 Possible Consequences of Using Genetically Engineered Micro-organisms
The potential harm associated with various genetically engineered micro-organisms is
shown in the table on the following page. Each letter (A through L) represents the
consequence of a particular combination of events and micro-organisms. For
example, the letters :
A,C represent the inadvertent release of micro-organisms known to be harmful to the
environment or to man e.g. in biological warfare or terrorism.
B,D represent the inadvertent release of micro-organisms known to be harmful to the
environment or to man e.g. in accidents at high-containment facilities where work is
being carried with dangerous micro-organisms.
E,I represent the intentional release of micro-organisms thought to be safe but which
prove harmful-when the safety of organisms have been misjudged.
F,J represent the intentional release of micro-organisms which prove safe as expected
e.g. in oil recovery, mining, agriculture and pollution control.
H,L represent the inadvertent release of micro-organisms which have no harmful
consequences e.g. in ordinary accidents with harmless micro-organisms.
G,K represent the inadvertent release of micro-organisms thought to be safe but
which prove harmful-the most unlikely possible consequence, because both an
accident must occur and a misjudgement about the safety must have been made.
Flow Chart of Possible Consequences of Using
Genetically Engineered Micro-Organisms
39
MICRO-ORGANISM
KNOWN HAZORDOUS
FOR ENVIRONMENT
FOR MAN
A. INTENTIONAL
RELEASE
C. INTENTIONAL
RELEASE
B. INADVERTENT
RELEASE
D. INADVERTENT
RELEASE
SUSPECTED SAFE
FOR ENVIRONMENT
INTENTIONAL
RELEASE
FOR MAN
INTENTIONAL
RELEASE
E. PROVES
HAZARDOUS
I. PROVES
HAZORDOUS
F. REMAINS SAFE
J. REMAINS SAFE
INADVERTENT
RELEASE
INADVERTENT
RELEASE
G. PROVES
HAZARDOUS
K. PROVES
HAZORDOUS
H. REMAINS SAFE
L. REMAINS SAFE
The biggest controversy surrounding genetically engineered micro-organisms has
centered around unforeseen harm - that micro-organisms thought safe might prove
harmful. Discussion of this kind of harm is hindered by the difficulty not only of
quantifying the probability of an occurrence but also of predicting the type of damage
that might occur. The different types of damage that can be conjured up are limited
40
only by imagination. The scenarios have included epidemics of cancer, the spread of
oil-eating bacteria, the uncontrolled proliferation of new plant life, and infection with
hormone-producing bacteria.
3.4 Possible Risks in the Laboratory
Other concerns raised due to the genetic engineering of micro-organisms involve the
hazards that might arise in the laboratory. However, it should be noted that a
consensus seems to have emerged among experts in biotechnology that genetic
engineering techniques present no special risks in themselves and, therefore, ought to
be governed by standard good laboratory practices. These practices are based on the
following :
1. A recognition that infectious micro-organisms can be classified according to the
risk they present to individuals in the laboratory and to the community at large;
2. Risk can be classified in various levels from low to high. The guidelines and
practices are geared to these increasing levels of risk;
3. Containment of the micro-organisms is the principal means of addressing the risks,
with recommended containment levels corresponding with each risk category; and
sound microbiological practices must be inculcated in the scientists, technicians
and other support staff.
In sum, the following perspective suggests that when conducted according to
generally accepted practices, biotechnology offers no greater risk than other realms of
science :
The current monitoring mechanism of voluntary self-regulation in the form of
guidelines appears to be accurate for dealing with the risks presented to laboratory
workers by micro-organisms, whether genetically engineered or not. The guidelines
41
for good laboratory practices in the microbiological laboratory have been developed
over and are based upon several decades of experience. Even the newer guidelines
that are focused solely on recombinant DNA are the result of over 10 years of
experience with that technique in the laboratory. During this time, there have been no
reports of illnesses or injuries attributed to the recombinant DNA technique. Most
experts believe that laboratory work with recombinant DNA presents no risks beyond
those already inherent in the biological materials and systems being used.
3.5 Risk Assessment (RA)
As a result of the concerns previously mentioned an environmental risk assessment, as
specified in the Irish national law under the Genetically Modified Organisms
Regulations, 1994, must be carried out.
Risk Assessment (RA) is a process in which the probability or frequency of harm for
a given hazard (an event which has the potential to be harmful) is estimated. In all
cases of releases of genetically modified organisms (including micro-organisms) into
the environment, this assessment is mandatory. It must be carried out by the notifier,
i.e. the person or body seeking consent for a proposed release, and it must address
potential risks for human health and the environment. The assessment and its
evaluation by the competent authority are the core appraisal elements of a deliberate
release notification from a safety point of view.
The main elements of risk assessment and evaluation are as follows;
1. Identify hazards associated with the GMO,
2. Consider the environment in which GMO(s) will be released and intended
conditions of release; estimate extent of consequences for each hazard,
42
3. Consider environment in which GMO(s) will be released and intended conditions
of release; estimate likelihood that each hazard will occur,
4. Use results of steps 2 and 3 to estimate the risk for each hazard,
5. Consider those hazards which cause risk; if risk is not at an acceptable level, adjust
their impact by altering conditions of release or the GMO itself and repeat steps 2,
3 and 4 and
6. Consider risks from all hazards and evaluate overall risk of adverse effects to
human health and the environment.
Where a specific risk or a degree of uncertainty exists, appropriate risk management
techniques will be required to prevent adverse effects on people or the environment.
In the event that available management techniques are incapable of protecting human
health and the environment, consent may be refused by the competent authority.
3.6 Conclusion
It is evident that introducing new combinations of DNA through biotechnology is
equivalent to producing new variations in genetic material through the natural
processes of mutation and recombination. As such, the risks with each do not differ
markedly, except that biotechnology offers a means to produce these variations both
in a particular and tailored fashion and at a rate which far exceeds that found in
nature.
43
The problems that will arise from the use of biotechnology will likely be similar to
those faced in more traditional agricultural breeding programs. Ecological effects
will vary from one incident to the next, and may range from no effect to acute toxicity
in humans or other organisms to changes in growth rates for crop species. Although
some feel that prediction of the course of events following a planned release of a
biotechnology product is possible, there is a growing consensus that because of the
complexity of ecological relationships, unforeseen events will always happen with
some frequency. By extension, risks associated with biotechnology cannot be entirely
eliminated.
The lack of agreement concerning these risks is not surprising considering, among
other examples,
• The increasing experience in the manipulation of the environment exhibiting the
prospect for great success (e.g., the elimination of small pox) as well as failure
(introduction of harmful species, such as rabbits to Australia and African “killer
bees” to North America);
• The increasing understanding of the complexities and interlinkages in and among
ecosystems;
• The recent rapid advances in biotechnology including the emerging capabilities for
genetic control.
In general, the accuracy of predictions of ecological, economic and social effects of
releasing a genetically engineered micro-organism depends on the specific organism,
the type of genetic information introduced, the particular environment into which it is
released, and the availability of detailed ecological information. Even so, the
complexity of ecology is such that prediction is likely to remain problematic.
44
45
4.0 Release and monitoring of Genetically engineered microorganisms in the environment.
It is not always easy to identify what is meant by "releasing" a genetically modified
organism into the environment. For example a genetically engineered sheep in a field
might not be considered to have been released, so long as the fences are strong
enough, but a genetically modified bacterium placed in the same field would be
considered to have been released. In order to determine if an organism is "released" it
has to be determined whether it can escape.
4.1 Containment of genetically engineered micro-organisms.
As micro-organisms are not contained in the environment by physical barriers, i.e. a
fence or wall, it will be deemed to be released when introduced into the natural
environment, whether introduced intentionally or by accidental means.
There are two strategies of biological containment of genetically engineered bacteria
in use. These are passive and active biological containment.
The concept of biological containment.
In the past, there was a consensus between molecular biologists and ecologists that
every disturbance of the ecological steady-state is an undesirable event. Furthermore,
there was a fear that some GEMs could be harmful for humans. Because of the lack
of predictable behaviour of GEMs the decision was made to use only the strains
which cannot establish or persist in different environments. The concept of the
biological containment was thus born. This concept demands that foreign genes must
be introduced only into so-called safety strains. Such safety strains should not be able
46
to transfer their foreign DNA to other organisms. Their ability of survival,
propagation and spreading have to be restricted only to laboratory conditions.
To fulfil safety guidelines, any introduced genetic material of commercially used
GEMs must be:
• Limited in size to consist only of the gene/genes of interest
• Well characterised in the function of all the gene products
• Free of certain sequences, e.g. gene products which are potentially toxic to other
organisms
The vectors used in gene technology should have features which fulfil the criteria
described above. An important additional characteristic is to prevent the transfer of
recombinant DNA by making the plasmids poorly mobilizable.
For the biological containment of GEMs there are two strategies. The initial strategy
was to use chromosomal mutations which altered the bacteria so that they would
poorly survive outside the laboratory. The mechanism can be regarded as a passive
containment strategy. The second one is an active strategy which based on the
construction of a suicide system. A simple suicide system consists of two parts. One
part is the control sequence, which usually consists of a promoter and, if necessary,
contains additional sequences involved in its regulation. The second part is a gene
which codes for a product that is toxic for the cell. The choice of the promoter and
the induction mechanisms strongly depends on the use of the appropriate microorganism.
Passive containment:
47
In simple form this is the addition of genes that cause an organism to require
particular nutrients that are not normally found. When you deliberately stop
supplying this nutrient, the organism dies.
An example of passive containment of GEMs is the best known safety strain of E.coli
: K12. This strain is absolutely not viable outside the laboratory, because in addition
to other limitations this strain is not able to synthesise D-amino pimelic acid, an
essential constituent of the bacterial cell wall not naturally occurring in the
environment. This kind of mutation could prevent the prolonged persistence of
intentionally released bacteria in soil or groundwater, and yet allow sufficient time for
the recombinant micro-organisms to fulfil its engineered purpose. This guarantees an
easy culturing of the GEMs in the laboratory, however, this approach does not
guarantee quick killing of unintentionally released GEMs.
Active containment:
GEMs must be able to compete successfully for a time with indigenous microorganisms to perform their special tasks in soil or groundwater. Therefore, the idea
was conceived to use so-called conditionally lethal biological containment systems,
which are induced under defined environmental conditions. Conditional suicide
systems can be expected to produce a predictable killing of GEMs.
The theory of this system is as follows: (For example) a bacterium could be designed
to destroy a particular pollutant, such as crude oil, and a gene could be inserted that
kills the bacterium if there is no crude oil around. Therefore the bacteria can be
sprayed onto an oil-spill, they will destroy the oil, and when all of the oil is gone they
will kill themselves.
The first most crucial step is to find a suitably controlled promoter. It should have no
or very low basal activity under permissive conditions but should be highly induced
48
by a distinct signal, such as by temperature changes, metabolites, chemical inducers
or nutrient limitation. The second part of a suicide system is a promoterless gene
which codes for a host toxic protein.
Examples of model suicide systems of E.coli (P=promoter)
System
Killing By
Induction By
Ptrp-hok
Collapse of the
membrane potential
Collapse of the
membrane potential
Collapse of the
membrane potential
Collapse of the
membrane potential
Collapse of the
membrane potential
Collapse of the
membrane potential
Cell lysis by levan
accumulation
Decay of DNA and
RNA
Cell lysis by T7lysozyme
Collapse of the
membrane potential
Lack of trytophan
Plac-hok
Plac-relF
xylS/Pm-lacI/Ptac-gef
PR-hok/sok
PphoA-parB
nptI/sacR/b
PL-nuc
PphoA-T7Lys
fimB/fimE/PfimA-gef
(IPTG = isopropyl-β-D-thiogalactoside)
4.2 Considerations when selecting a GEM
49
IPTG
IPTG
Lack of 3-methylbenzoate
Temperature shift from
40oC to 30oC
Phosphate limitation
Sucrose
Temperature shift from
28 to 42 C
Phosphate limitation
Stochastic switch on by
invertion of the fimApromoter
When selecting and designing a GEM with suitable attributes for use as an agent for
in-situ bioremediation, those features which are crucial for it to function effectively
and safely in the environment include:
•
Its ability to survive and multiply in the ecosystem into which it is introduced.
•
The ability of the GEM to effectively function in the role for which it was
designed under the conditions prevailing in the ecosystem.
•
The stability of new genetic material and the potential for this material to transfer
laterally to indigenous organisms
•
The effects, if any, of the GEM on the structure and function of the ecosystem into
which it is introduced.
However, despite many containment efforts, one cannot prevent an unintentional
release of GEMs outside the laboratory with absolute security.
4.3 Monitoring of GEMs in the environment.
The large-scale application of genetically modified micro-organisms in the
environment has raised concerns about potential environmental impacts. Assessment
of potential risks associated with the environmental release of GEM requires adequate
methods of monitoring the fate of the GEM in the environment. The major challenge
for the development of suitable monitoring techniques is the fact that only a minor
fraction of the total bacterial community in the environment is accessible to
cultivation techniques.
When considering the use of GEMs in the environment for bioremediation, the
ecology of the ecosystem should be understood in order to select and design microorganisms with attributes which are necessary for survival and which allow
expression of the specific pathways required. Thus, it is an asset to have model
50
systems in which to assess the ability of the GEMs to degrade target pollutants in situ
conditions prior to actual field use. .Microcosms are used for this purpose, as they
contain the components necessary for expression of ecosystem processes, e.g. the
flow of energy, carbon and nutrients. It is these processes which affect the fate and
activity of introduced GEMs and can be reciprocally altered by their presence in the
ecosystem. Microcosms allow the experimenter to maintain GEMs in contained
systems and to control and monitor selected ecosystem parameters.
Factors affecting survival
Several biotic and abiotic factors affect the survival and establishment of introduced
GEMs. These factors may also affect the degree of interaction of the introduced
GEMs or the DNA with the environment.
•
Biotic factors
Host micro-organism (ability to compete with indigenous micro-organisms, survival
under field conditions, distribution in the field)
Predators / parasites
Vectors of microbial transport (for example earthworms)
Type and variability of vegetation
•
Abiotic factors
Physical factors
Temperature
Humidity
Oxygen
Proportion of organic substances
Soil types ( proportion of sand, clay, silt)
Proportion of humic substances
51
Pore size distribution
Chemical factors
pH
Nutrient availability
Conductivity
Chemical contamination
Cation exchange capacity
Fungicide application
4.4 Detection Techniques
The detection and enumeration of previously released GEMs presents many
challenges. Traits such as antibiotic resistances, bioluminescence or other enzymes
encoded by the genetic construct can be used for the detection of the GEM in the
presence of the indigenous microbial population. The availability of information on
the genetically engineered micro-organism and the genetic modification ( marker
genes, promoter sequences) is a prerequisite for the development of specific detection
techniques.
Two general approaches are used for detecting GEMs:
Cultivation based methods and direct methods not using cultivation, such as total
DNA extraction followed by analysis or immunofluoresence microscopy.
•
Cultivation based detection of GEMs
Selective cultivation
This often takes advantage of antibiotic, heavy metal or herbicide resistances encoded
by the GEM. The application of selective cultivation techniques improves the limit of
detection since the natural background is reduced.
52
The most frequently used antibiotic resistant marker gene is the nptII gene conferring
a kanamycin and neomycin resistance to its host. Deliberate release of GEMs marked
with antibiotic resistance genes is not desirable when the respective antibiotics are of
medical importance.
Reporter Genes
Reporter genes are defined as genes conferring distinctive phenotypic properties
which allow the marked organism to be tracked in the presence of the indigenous
microbiota. Detection of GEMs containing reporter genes such as luc, lux, xylE or
gusA by plating onto selective media is highly sensitive and specific.
An example of this type of detection system is the lacZY genes from E.coli coding for
a -galactosidase and a lactose permease as marker genes for fluorescent
pseudomonads. The expression of the marker gene can be detected on an X-Gal
resulting in blue colonies.
Immunological detection
Prerequisite for immunological approaches is the availability of specific antibodies
(specific for the bacterial host or a gene product encoded by the recombinant DNA).
Antibodies coupled to magnetic beads have been applied for selective recovery of
Pseudomonas putida cells.
Gene probes and PCR
Most GEMs carry unique DNA stretches which makes their specific detection
possible by means of gene probes or the polymerase chain reaction. The specific and
unequivocal detection of recombinant DNA is made possible by an appropriate
selection of adequate primer systems, even in the presence of naturally occurring
genes.
53
•
Direct detection methods
Microscopic methods
In situ hybridisation of whole cells using fluorescently labelled oligonucleotides
targeted to the16S rRNA or 23S rRNA allows for the detection of micro-organisms in
their natural microhabitat. The microscopic identification of individual cells provides
information on the cell morphology, the spatial distribution, and the growth rate,
independantly of their culturability.
Direct DNA extraction from soil samples
This allows the detection of the construct(i) in GMOs which became nonculturable
due to environmental stress; (ii) in bacteria which are not accessible to cultivation
techniques; (iii) persisting as free DNA adsorbed to soil particles. Methods of nucleic
acid extraction from environmental samples have two approaches: (i) The cells are
lysed directly within the environmental sample; and (ii) the cells are lysed after
recovery of the bacterial fraction from soil or sediment particles.
Direct lysis
Soil is directly subjected to cell lysis conditions using, for instance, freezing/thawing,
ultrasonication, microwave, bead beater and/or lysozyme treatment steps followed by
alkaline SDS treatment. Thus direct extraction of DNA from soil.
54
5.0 LEGISLATION REGARDING GENETICALLY
ENGINEERED ORGANISMS.
5.1 Eu Legislation.
The potential environmental impact of products containing or consisting of live
GMOs which are deliberately released into the environment is controlled under EU
Directive 90/220/EEC. Commonly known as the Deliberate Release Directive, the
Minister for the Environment and Local Government has overall responsibility for its
implementation in Ireland. A proposal from the European commission to amend the
directive, in the light of scientific and technical advances and operational experience,
is currently before the EU Council of Environment Ministers and the European
Parliament.
Under the Deliberate Release Directive, the term “Deliberate Release” covers
intended releases of GMOs for research and development purposes, and the placing of
products containing or consisting of GMOs on the EU market.
The directive provides that separate notification and consent procedures should apply
to proposals to undertake releases for research and development and marketing
purposes. Proposed releases are examined individually, and development must
proceed on a gradual basis, i.e. subject to satisfactory evaluation of each step in terms
of safety for human health and the environment.
In addition to ensuring that GMO releases will not have an adverse effect on the
environment, the Deliberate Release Directive is also intended to harmonise the
relevant laws, regulations and administrative procedures in the individual Member
States of the EU. To meet this internal market requirement, the provisions of the
Directive apply uniformly throughout the Community.
55
5.2 National Legislation.
The Deliberate Release Directive has been given effect in Irish law under the
Genetically Modified Organisms ( GMO ) Regulations, 1994 and the Environmental
Protection Agency is the competent authority.
The Regulations,
• express relevant provisions of the Directive in Irish law; in general these concern
regulatory procedures,
• designate the EPA as national competent authority,
• introduce a control system for research and development releases of GMOs,
• introduce procedures for processing notifications for consent to place genetically
modified products on the EU market,
• specify fees and other charges payable to the EPA,
• provide for maintenance by the EPA of a public register of release notifications,
• provide for enforcement action by the EPA, including powers to prosecute
offences, and
• enable the EPA to appoint an Advisory Committee on GMOs.
Under the provisions of the EU and national legislation already in place, a person or
body cannot proceed with the deliberate release of a GMO in Ireland unless prior
consent has been granted by the competent authority. The current requirements are as
follows;
• in the case of proposed research and development releases in this country consent
must be obtained from the EPA under the GMO Regulations, 1994,
• in the case of proposals to place products ( other than food products ) containing
live GMOs on the open market, EU wide consent must be obtained under the
56
Deliberate Release Directive. In such cases, a notification seeking consent may be
submitted to the competent authority of any Member State of the EU.
Under the Deliberate Release Directive, consent to place a product containing or
consisting of GMOs on the EU market can only be granted provided the product has
satisfactorily completed the research and development stage or undergone an
environmental risk assessment similar to the one provided for in the Directive.
Procedures for the marketing of products operate at Community as well as national
level, since a consent granted by the competent authority of any Member State is
valid for the whole Community. In these circumstances, a consent by a competent
authority must have the agreement of the competent authorities in all Member States.
Where agreement is not reached at competent authority level, Member State
Procedures, which may involve reference to the EU Council of Ministers, apply.
57
6.0 Conclusion
The technology and techniques of genetic engineering came to the fore in the 1970’s
as expertise in the area underwent unprecedented expansion.
Micro-organisms used in this technology are varied and while no one perfect
organism has emerged the importance of E. coli is undeniable due to its ease of
manipulation and its extensively studied biochemical process.
Areas into which this technology has expanded include :
health care, agriculture, food and drink, environment, waste management, energy and
biological warfare.
Coupled with growth in this technology is the increasing public concern and debate
over the merits and defects of the whole process of genetic engineering. Concerns are
centred around the fear that altered microbes might run amok or that their genes
would hop unpredictably to other organisms.
As a result of these concerns an environmental risk assessment as specified in the
Irish national law under the Genetically Modified Organisms Regulations, 1994, must
be carried out. This regulation is in place to ensure the safe application of modern
biotechnology throughout the community. The collected studies of bacteria in their
native habitats suggests the GEMs can be put into the environment safely. It is
however essential that environmental biotechnologists gain all the information needed
to reduce the riskss to the barest minimum.
58
References
1) Genetically Modified Organisms and the Environment. A consultation Paper
(summary) by The Department of the Environment and Local Government.
2) Recombinant DNA Technology - the Biochemistry Society England by Peter
Moore Bsc. PhD. Applications of recombinant DNA technology and the ethical
and safety implications.
3) Principles of Gene Engineering (Fourth Edition) - An Introduction to Genetic
Engineering by R.W. OLD and S.B. Primrose Blackwell Scientific Publications
ISBN O-632-02608-1
4) Advances in Life Sciences - Transgenic Organisms Biological and Social
Implications Edited by J. Tomiuk, K. Wohrmann, A. Sentker, Birkhauser ISBN O
3-7643-5262-0
5) Monitoring Genetically Manipulated Micro-organisms in the Environment, Edited
by Cliff Edwards (Wiley Biotechnology Series) ISBN O 471-93795-9
6) Release of Genetically Engineered and other Micro-organisms, Edited by John C.
Fry and Martin J. Day. (Plant and Microbial Biotechnology) ISBN O-521-417562
7) Understanding DNA and Gene Cloning by Karl Druca
8) Environmental Gene Release by M.J. Bazin and J.M. Lynch
9) Genetics by Weaver Hedrick, ISBN 0-697-10021-9
10) Genetic Technology, A New Frontier, Office of Technology Assessment (1982)
by Westview Press/Croom Helm
11) Introductory Microbiology By F.C. Ross
12) A Guide to Modern Biology-Genetics, Cells and Systems By E. Lawrence
ISBN 0582-44272-9
59
13) Molecular Biology of the Cell By B Alberts, D Bray, J Lewis, M Raff, K Roberts,
J.D. Watson. ISBN 0-8153-1620-8
14) Gene Cloning-An Introduction By T.A. Brown ISBN 0-412-62240-8
15) Biotechnology-A Guide to Genetic Engineering By P. Peters ISBN 0-697-120635
16) Recombinant DNA By J.D. Watson, M Gilman, J. Witkowski and M. Zoller
ISBN 0-7167-2282-8
17) Encyclopedia of Microbiology, Volume 2, (By Various Authors)
18) Journal of Industrial Microbiology by STOCKTON
19) Pollution and Toxicology 7R 1991 Volume 25 Part 8 (979-984), Feasibility of
Wastewater treatment using genetically engineered micro-organisms by Fujita-M;
Ike-M;
20) Journal of Industrial Microbiology : STOCKTON, Volume 16 NO. 1 Jan. 1996
(42-45), Ethanol production from hemicullulose hydrolysates of agricultural
residues using genetically engineered E.coli strain KO11 by A. Asghari, RJ
Bothast, JB Doran and LO Ingram
21) Journal of Industrial Microbiology : STOCKTON, Volume NO. 16 March 1996
(145-155), Review optimising inducert culture conditions for expression of
foreign proteins under the control of the lac promoter by RS Donovan, CW
Robinson and BR Glick
22) Journal of Industrial Microbiology : STOCKTON, Volume NO. 16 March 1996
(189), Biosorption of Cadmium cobalt, nickel and strontium by a Bacillus simplex
strain isolated from the Vadose zone by NB Valentine, H. Bolton Jr., MT
Kingsley, GR Drake, DL Balkkwill and AE Plymale
60
23) The Ecologist Volume 28 NO.3 May/June 1998 (146-153), The Biotechnology
Bubble General Genetic Engineering
24) Applied and Environmental Microbiology May 1998 Volume 64 NO5 (18521859), Genetically engineered Saccharomyces yeast capable of effective cofermentation of glucose and xylose
25) Techniques in applied microbiology Volume 31 1995, Progress in industrial
microbiology by B. Sikyta
26) Scientific America Jan. 1998, Bacterial Gene Swapping in Nature
27) Biological Warfare, http://www.calpoly.edu/ddrjones/biowar-e3.html
28) Transgenic animals, http://www.synapse. ndirect.co.uk/science/clone.html
29) General genetic engineering, http://webzone/.co.uk/www/srtproject/genengl.htm
30) The role of micro-organisms in genetic engineering, http://wwwmicro.msb.le.ac.uk/109/GeneticEngineering.html
31) Biotechnology in Waste Management, http://www.aba.asn.au/leaf8.html
32) http://home.cord.edu/faculty/moewes/environment/320x/mestery.html
61