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Lecture-3; genetic manipulations

Faculty of Engineering Science & Technology (ZUFEST),
Department of Biomedical Engineering
Genetic manipulation strategies in
environmental biotechnology
Genetic Manipulation
• Genetic engineering (GE), recombinant DNA
technology, genetic manipulation/modification
(GM) and gene splicing are the terms that apply to
the direct manipulation of an organism’s genes.
• GE uses the techniques of molecular cloning and
transformation to alter the structure and
characteristics of genes directly.
• GE aims at isolating DNA fragments and
recombining them.
Genetic Manipulation
Genes have been manipulated by man for a very long
time. It has been proposed that the exchange of genetic
information between organisms in nature is considerably
more commonplace than is generally imagined and could
explain the observed rates of evolution (Reanney 1976).
Genetic Manipulation
• Exchange involving a vector requires
compatibility between the organism
donating the genetic material, the vector
involved, and the recipient organism.
• Organisms which represent the ‘norm’,
frequently being the most abundant
members occurring in nature, are
described as ‘wild type’.
• Those with DNA which differs are described as ‘mutant’.
Alteration can be by the normal processes of evolution
which constantly produces mutants, a process which may
be accelerated artificially, or by deliberate reconstruction
of the genome.
Varied Applications
• Isolation of a particular gene, gene part or region of
a genome.
• Production of a particular RNA and protein
molecules in quantities.
• Improvement in the production of biochemicals and
commercially important organic chemicals.
• Production of varieties of plants having particular
desirable characteristics.
• Correction of genetic defects in higher organisms
• Creation of organisms with economically important
Training: Manipulation of Bacteria
Without Genetic Engineering
A general procedure is to take a sample of bacteria from the site of
contamination from which a pure culture is obtained in the
laboratory and identified, using standard microbiology techniques.
The ‘training’ may be required either to improve the bacterium’s
tolerance to the pollutant or to increase the capabilities of
pathways already existing in the bacterium to include the ability to
degrade the pollutant, or a combination of both.
Training: Manipulation of Bacteria
Without Genetic Engineering
Tolerance may be increased by culturing in growth
medium containing increasing concentrations of the
pollutant so that, over successive generations, the
microbe becomes more able to withstand the toxic
effects of the contaminant.
Improving the microbe’s ability to degrade a
contaminant, sometimes referred to as catabolic
Training: Manipulation of Bacteria
Without Genetic Engineering
Under laboratory conditions where cultures of
bacteria are isolated from each other to prevent crosscontamination, mutations are most likely to occur as a
result of an error in DNA replication.
An increased rate of error may be forced upon the
organism, speeding up the rate of mutation, by
including a mutagen in the growth medium.
Training: Manipulation of Bacteria
Without Genetic Engineering
A mutagen is a chemical which increases the rate of
error in DNA replication, often by causing a very limited
amount of damage to the DNA such that the DNA
polymerase, is unable to determine the correct base to
add in to the growing nucleotide chain.
If the error in the nascent strand cannot be recognized
and corrected, the fault becomes permanent and is
handed on through the generations.
Manipulation of Bacteria by Genetic
Genetic manipulation by the deliberate introduction of
defined genes into a specified organism is a very powerful
The techniques have produced some exciting hybrids in all
areas of research, both microscopic; bacteria and fungi,
usually described as recombinants, and macroscopic;
principally higher plants and animals, commonly described
as transgenics.
Basic Principles of Genetic Engineering
There are endless permutations of the basic cloning
procedures but they all share some fundamental
These are:
1. Enzymes, solutions and equipment necessary to
perform the procedures
2. Desired piece of DNA to be transferred
3. A cloning vector
4. Recipient cell which may be a whole organism.
5. Marker genes to ensure transfer
Enzymes, solutions and equipment
• Isolation of DNA and
purified from
contaminating material
• To insert the DNA into the
vector, ends must be
• Done by restriction
endonucleases and
staggered ends are
Enzymes, solutions and equipment
Preparation of the vector is dictated by the type of end
prepared for the insert DNA: flush or ‘sticky’.
• If it is flush, it does not much matter how that was
achieved so long as the vector receiving it is also flush.
• If it is sticky, the appropriate sticky end must be prepared
on the vector by a suitable restriction endonuclease.
Enzymes, solutions and equipment
The prepared insert, or ‘foreign’
DNA is incubated with the
prepared vector in an aqueous
solution containing various salts
Ligase is an enzyme, the
function of which is to make the
bond between the free phosphate
on a nucleotide base and the
neighboring ribose sugar, thus
‘repairing’ the DNA to make a
complete covalently linked chain.
DNA for transfer
• A piece of double-stranded DNA
which contains the coding sequence
for a gene.
• Obtained from a number of sources,
for example, genomic DNA, a cDNA
library, a product of a polymerase
chain reaction (PCR) or a piece of
DNA chemically produced on a DNA
synthesizer machine.
• Another source is DNA copy of an
RNA virus as in the replicative form
of RNA viruses.
Genomic Libraries
Genomic DNA, in this context, is material which has been
isolated directly from an organism, purified and cut up into
pieces of a size suitable to be inserted into a cloning vector.
These pieces may either be ligated in total mixture, into a
suitable vector to produce a genomic library,
Genomic libraries are very
useful, as they may be amplified,
and accessed almost limitlessly,
to look for a specific DNA
sequence thus reducing the
amount of work involved in any
one experiment.
cDNA libraries
In eukaryotes, the first product of transcription from DNA is not
messenger RNA (mRNA) but heterogeneous nuclear RNA
This is mRNA prior to the removal of all the noncoding sections,
or introns, which are discarded during the processing to produce
the mature mRNA.
Complementary DNA (cDNA) is DNA which has been artificially
made using the mature mRNA as a template, which is then used
as the template for the second strand.
Thus the synthetic DNA product is simply a DNA version of the
mRNA and so should overcome the problems of expression
outlined above
Polymerase chain reaction
• The polymerase chain reaction
(PCR) is a powerful technique
which amplifies a piece of DNA of
which only a very few copies are
• The process is repeated by a
constant cycling of denaturation of
double-stranded DNA at elevated
temperature to approximately 95
◦C, followed by cooling to
approximately 60 ◦C to allow
annealing of the probe and
complementary strand synthesis.
Cloning vectors
• A cloning vector is frequently a plasmid or a bacteriophage
(bacterial virus) which must be fairly small and fully sequenced,
able to replicate itself when reintroduced into a host cell, thus
producing large amounts of the recombinant DNA for further
• It must carry on it ‘selector marker’ genes. These are different
from the reporter genes which are indicators of genomic integrity
and activity.
• A common design of a cloning vector is one which carries two
genes coding for antibiotic resistance. The ‘foreign’ gene is
inserted within one of the genes so that it is no longer functional
therefore it is possible to discriminate
Cloning vectors
Standard cloning vectors normally carry only
selector marker genes required for plasmid
construction. To make the manipulations
easier, these genes normally contain a
multicloning site (MCS) which is a cluster of
sites for restriction enzymes constructed in
such a way to preserve the function of the
Cloning vectors
This is pGEM (Promega 1996) which
has a MCS in the s-gal gene. This codes
for s-galactosidase from the E. coli lac
operon, which has the capacity to
hydrolyse x-gal, a colourless liquid, to
produce free galactose and ‘x’ which
results in a blue pigment to the colony.
Thus the screening for successful
insertion into the MCS is a simple
scoring of blue (negative) or white
(possibly positive) colonies. The
success of the experiment can be
determined quickly as this cloning
vector also has sequences at either
side of the MCS which allows for rapid
DNA sequencing.
Expression vectors
These are similar to the vectors described above but in
addition have the required signals located before and
after the ‘foreign’ gene which direct the host cell to
translate the product of transcription into a protein.
Reporter genes
• There are many such genes in common use and these
usually code for an enzyme.
• The most common is β-galactosidase. This enzyme,
supplied with the appropriate reagents, may also
catalyze a color change by its activity.
• Other reporter genes produce enzymes which can
cause the emission of light such as the luciferase
isolated from fireflies, or whose activity is easy and
quick to assay like the bacterial β glucuronidase
(GUS), which is probably the most frequently used
reporter gene in transgenic plants.
Analysis of Recombinants
I. The design of the plasmid was such that insertion of
‘foreign’ DNA allows for a colour test, or causes a change
in antibiotic sensitivity, either to resistance (positive
selection) or sensitivity (negative selection). This
constitutes the first step in screening.
II. The second stage is usually to probe for the desired
gene using molecules which will recognise it and to
which is attached some sort of tag, usually radioactive
or one able to produce a colour change.
III. The next stage is normally to analyse the DNA isolated
from possible recombinants, firstly by checking the size
of the molecule or pieces thereof, or by sequencing the
Analysis of Recombinants
DNA sequencing has become a standard part
of recombinant analysis procedure. However,
if a large number of samples are to be
analysed it is usually quicker and cheaper to
scan them by a procedure described as a
Southern blot.
Recombinant Bacteria
• Genetic engineering of micro-organisms for use in environmental
biotechnology has tended to focus on the expansion of metabolic
pathways either to modify the existent metabolic capability or to
introduce new pathways.
• This has various applications, from the improved degradation of
contaminants, to the production of enzymes for industry, thus
making a process less damaging to the environment.
• One such experimental example taken from ‘clean technology’
with potential for the manufacturing industry, is a strain of
Eschericia coli into which was engineered some 15 genes
originating from Pseudomonas. These were introduced to
construct a pathway able to produce indigo for the dyeing of
Recombinant Yeast
• Yeast, being unicellular eukaryotes, has become popular for
cloning and expressing eukaryotic genes.
• These are fairly simple to propagate, some species being
amenable to culture in much the same way as bacteria.
• There are several types of plasmid vector available for genetic
engineering, some of which have been constructed to allow
replication in both bacteria and yeast
Recombinant Virus
• The insect virus, Baculovirus, has been shown to be the
method of choice for the overexpression of genes in
many applications of molecular biology
• The viral genome is large relative to bacterial plasmids
and so DNA manipulations are normally carried out on
a plasmid maintained in Eschericia coli.
• One example of interest to environmental
biotechnology is the replacement of p10, one of the
two major Baculovirus proteins, polyhedrin being the
other, by the gene for a scorpion neurotoxin, with a
view to improving the insecticidal qualities of the virus
Transgenic Plants
• Currently, genetic engineering in
agribiotechnology is focusing on genetic
modifications to improve crop plants with
respect to quality, nutritional value, and
resistance to damage by pests and
• Other avenues aim to increase tolerance
to extreme environmental conditions, to
make plants better suited for their role in
pollutant assimilation, degradation or
dispersion by phytoremediation, or to
modify plants to produce materials which
lead to the reduction of environmental
Transformation of plants
There are two practical problems associated with genetic
engineering of plants which make them more difficult to
manipulate than bacteria.
Firstly they have rigid cell walls and secondly they lack the
plasmids which simplify so much of genetic engineering in
The first problem is overcome by the use of specialised
techniques for transformation, and the second by
performing all the manipulations in bacteria and then
transferring the final product into the plant.
Transformation of plants
• The most popular method of transforming plants is by the Ti
plasmid but there are at least two other methods also in use.
• The first is a direct method where DNA is affixed to
microscopic bullets which are fired directly into plant tissue.
An example of this technology is the introduction into
sugarcane, of genes able to inactivate toxins produced by the
bacterium, Xanthomonas albilineans, causing leaf scald
• The second is by protoplast fusion which is a process whereby
the plant cell wall is removed leaving the cell surrounded only
by the much more fragile membrane. This is made permeable
to small fragments of DNA and then the cells allowed to
recover and grow into plants.
Examples of developments in plant GE
The purpose of these examples is to illustrate the potential plant
genetic engineering could bring to future practical applications in
the field of environmental biotechnology.
Broad range protection
A general strategy to protect plants from various viruses,
fungi and oxidative damage is by a range of agents, has
been proposed using tobacco plants as a model.
The transgenics express the iron-binding protein, ferritin,
in their cells which appears to afford them far-ranging
Examples of developments in plant GE
Resistance to herbicides
• ‘Glyphosate’, one of the most widely used herbicides, is an
analogue of phosphoenol pyruvate and shows herbicidal
activity because it inhibits the enzyme 5-enolpyruvylshikimate3-phosphate synthase.
• The gene coding for this enzyme has been identified, isolated
and inserted into a number of plants including petunias. In this
case, the gene was expressed behind a CaMV promoter and
introduced using A. tumefaciens, leading to very high levels of
enzyme expression.
• As a consequence, the recombinant plants showed significant
resistance to the effects of glyphosate
Examples of developments in plant GE
Improved resistance to pests
• Plants have an inbuilt defence mechanism protecting them
from attack by insects but the damage caused by the pests
may still be sufficient to reduce the commercial potential of
the crop.
• Plants are being engineered to have an increased self-defence
against pests.
• With a view to increasing resistance to sustained attack, the
genes coding for the δ-endotoxin of the bacterium, Bacillus
thuringiensis (Bt).
• Examples are of synthetic B. thuringiensis δ-endotoxin genes
transferred, in the first case, by A. tumefaciens into Chinese
cabbage (Cho et al. 2001) and in the second, by biolistic
bombardment into maize
Examples of developments in plant GE
Improved resistance to disease
• Bacteria communicate with each other by way of small diffusible
molecules such as the N-acylhomoserine lactones (AHLs) of Gram
negative organisms described as ‘quorum sensing’, they are able
to detect when a critical minimum number of organisms is
present, before reacting.
• These responses are diverse and include the exchange of plasmids
and production of antibiotics and other biologically active
• Plants are susceptible to bacterial pathogens such as Erwinia
carotovora, which produces enzymes capable of degrading its cell
walls. The synthesis of these enzymes is under the control of
• The rationale behind using AHLs for plant protection is to make
transgenic plants, tobacco in this case, which express this signal
Examples of developments in plant GE
Improved tolerance
• The example is of bacterial rather than plant modification but
impinges on interaction between the two. Pseudomonas syringae
produces a protein which promotes the formation of ice crystals
just below 0 ◦C thus increasing the risk of frost damage.
• They transferred it to the bacterium Eschericia coli to simplify the
genetic manipulations. the mutants were able to compete with
the wild type and protect this particularly susceptible crop against
frost damage.
• Salt tolerance in tomatoes has been established by introducing
genes involved in Na+/H+ antiport, the transport of sodium and
hydrogen ions in opposite directions across a membrane.
• Improved tolerance to drought, salt and freezing in Arabidopsis
has been achieved by overexpressing a protein which induces the
stress response genes.
Examples of developments in plant GE
Improved plants for phytoremediation
• The genetic modification of a poplar to enable
mercury to be removed from the soil and converted to
a form able to be released to the atmosphere. This
process is termed ‘phytovolatilisation’
• A bacterial gene encoding pentaerythritol tetranitrate
reductase, an enzyme involved in the degradation of
explosives, has been transferred into tobacco plants.
The transgenics have been shown to express the
correct enzyme to determine their ability to degrade
Examples of developments in plant GE
New products from plants
• The rape plant, Arabidopsis thalia has
become a popular choice for the production
of recombinant species.
• One such recombinant is a rape plant, the
fatty acid composition in the seed of which
has been modified. It now produces
triacylglycerols containing elevated levels of
trierucinic acid suitable for use in the
polymer industry
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