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

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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

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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 micro-

organisms, especially bacteria, through a discussion of all facets of the technology.

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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

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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

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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.

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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

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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-

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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.

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Although conjugation in this exact form is not known to exist among the Gram-

positive 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

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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

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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.

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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.

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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

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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.

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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

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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.

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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.

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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

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

Bacillus subtilis Many proteins naturally exported into growth medium Non-pathogenic Easy to culture Some Bacillus enzymes excreted at high level (> 5 gl-1)

Still not much known about gene regulation Good, high-level expression vectors lacking High-level export of heterologous proteins not achieved

Saccharomyces cerevisiae 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

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

Filamentous fungi Large surface area to volume ratio should favour protein export Have been used in microbiology for over 40 years

Promoters/gene regulation poorly understood but may be similar to yeast Good expression systems lacking rheology of fermentations important

Actinomycetes Large surface area to volume ratio should favour protein export Widely used in industrial microbiology Good expression systems been developed

Promoters/gene regulation still poorly understood Rheology of fermentations important

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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

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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.

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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

A and B chains made separately as fusion proteins and joined invitro. Already on market.

Human Somatotrophin

191 amino acids E. coli Pituitary dwarfism

If used in treatment of osteoporosis then market size will be much larger. Has additional methione residue at N-terminus, but technology for removing this now Available already on market

IFN-α2 166 amino acids E. coli Hairy cell leukaemia Prophylaxis of common cold

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

IFN - Y 143 amino acids glycosylated

E. coli Treatment of cancers Treatment of viral diseases

In clinical trials

Tissue plasminogen activator

E.coli Yeast Animal cells

Thrombosis Animal cell culture most effective Way of producing active enzyme on the market

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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 self-assembles into a structure resembling virus particles Now on market

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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:

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• 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.

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• 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.

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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 host-

plasmid 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 (2,4,6-

trinitrotoluene) 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-to-

degrade 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 micro-

organisms may bring about spreading of resistance to the antibiotic.

It has been suggested that natural micro-organisms and genetically engineered micro-

organisms 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 micro-

organisms have died. Since some changes in single genes can convert benign micro-

organisms 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

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MICRO-ORGANISM

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 micro-

organisms 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 micro-

organism.

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 micro-

organisms 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

Lack of trytophan

Plac-hok Collapse of the membrane potential

IPTG

Plac-relF Collapse of the membrane potential

IPTG

xylS/Pm-lacI/Ptac-gef Collapse of the membrane potential

Lack of 3-methyl-benzoate

PR-hok/sok Collapse of the membrane potential

Temperature shift from 40oC to 30oC

PphoA-parB Collapse of the membrane potential

Phosphate limitation

nptI/sacR/b Cell lysis by levan accumulation

Sucrose

PL-nuc Decay of DNA and RNA

Temperature shift from 28 to 42 C

PphoA-T7Lys Cell lysis by T7-lysozyme

Phosphate limitation

fimB/fimE/PfimA-gef Collapse of the membrane potential

Stochastic switch on by invertion of the fimA-promoter

(IPTG = isopropyl-β-D-thiogalactoside)

4.2 Considerations when selecting a GEM

49

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 micro-

organisms with attributes which are necessary for survival and which allow

expression of the specific pathways required. Thus, it is an asset to have model

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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

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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.

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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.

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• 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.

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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.

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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

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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.

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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.

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