GENE TECHNOLOGY

DEFINITIONS
• Gene Technology
refers to alteration in an
organism’s genotype which
alters protein synthesis during
translation.

• Genetic Engineering
refers to transfer of genetic
material from one organism to
another to produce
transformed organisms
 PROTEINS PRODUCED VIA
GENETIC ENGINEERING
• Examples of a few proteins produced industrially via genetic engineering
include:

1) Insulin
2) Growth hormone
3) Factor VIII
 PRODUCTION OF HUMAN INSULIN

• Before insulin from genetically modified

bacteria became available, people with
diabetes mellitus were treated with insulin
extracted from the pancreas of pigs or
cattle
• Steps to produce human insulin:

1. Extraction of the Human Insulin Gene

2. Insertion of the gene into the Vector
(plasmid)
3. Insertion of the Vector (plasmid) into the
bacteria
4. Selection of transformed Bacteria
5. Cloning
6. Isolation, Modification and Purification of
Insulin
 EXTRACTION OF THE HUMAN INSULIN
GENE

• The gene can be extracted or synthesised in different ways which are

summarised below:
1. Extraction of mRNA from β-cells of the Pancreas:
• Isolating mRNA is easier than extracting the human insulin gene from DNA
• The mRNA is devoid of introns and there are multiple copies of mRNA present
within the cytoplasm.
• This mRNA is then converted into single stranded complementary DNA
(cDNA) using the enzyme Reverse Transcriptase
• The single stranded cDNA is converted into double stranded complementary
DNA using the enzyme DNA Polymerase.
• Multiple copies of this gene can thereafter be formed using the Polymerase
Chain Reaction (PCR).
2. Isolating gene from the DNA:

The insulin gene can be extracted from the DNA within the β-cells of the
pancreas.

Introns are removed from the gene before multiple copies of the gene are
produced

3. Artificial Synthesis of the Gene:

Since the Amino Acid sequence of the Insulin protein is known, an artificial
gene can be synthesised. Multiple copies of the gene can be produced using
PCR.
 INSERTION OF GENE INTO THE VECTOR
• The human insulin gene is now modified by adding non-coding DNA segments which will
be cut by specific restriction endonucleases to produce sticky-ends.
• The same restriction endonuclease will serve to cut the vector plasmid at the same
palindromic site.
• These plasmids are extracted from the bacterium E. Coli, by exposing them to certain
enzymes which digests their cell wall.
• The plasmids are then mixed with the insulin gene to allow complementary base pairing to
occur between their sticky ends.
• The enzyme DNA ligase forms phosphodiester bonds between the ends of DNA fragments.
The resultant plasmid formed is known as the Recombinant DNA (rDNA).

Mixing the plasmid with Insulin gene can have 3 possible fates:
• Formation of Recombinant DNA.
• Reformation of the Plasmid.
• Formation of a circle of the wanted gene.
 INSERTION OF THE VECTOR INTO
THE BACTERIUM
• In order to allow uptake of the rDNA by E. Coli the bacterium is mixed with
the plasmids containing a high concentration of Ca2+ ions at 0◦C.
• These ions alter the permeability of the bacterial cell membrane to allow
uptake of the genetic material when given a heat shock by rapidly
increasing the temperature to 400C.
• Bacteria that take up rDNA are termed as transformed bacteria. Many
bacteria will however either take up circles of the wanted gene or the non-
recombinant plasmids.
• Less than 1% of the bacteria undergo transformation, thereby, making it
essential to select the transformed bacterium.
 SELECTION OF TRANSFORMED
BACTERIA
• Certain genes referred to as Marker genes are inserted along with the desired Insulin
gene to enable identification of the transformed microorganism.
• These marker genes could either be selectable markers or screening markers.

• Selectable markers:
are genes which enable identification of transformed organism by monitoring their
survival in the presence of a selective agent (for example, Antibiotics) – see replica
plating below.

• Screening markers:
are genes which enable identification of transformed organism by changes in their
visible appearances (eg GFP (green fluorescent protein) gene extracted from a
jellyfish)

• Due to the potential risk of spreading antibiotic resistant plasmids to pathogenic

bacteria, screening markers are usually preferred over selectable markers.
REPLICA PLATING
 CLONING
• Genetically Modified Organisms (GMO) identified through marker genes are
selected and grown in large fermenters.
• This will produce a large population of bacteria which contains the gene
coding for human Insulin.
• Rapid division of these bacteria via binary fission will, therefore, enable
production of large volumes of insulin.
• Insulin is a quaternary protein containing two polypeptide chains A & B. The
genes for these chains are usually inserted in different plasmids.
• The polypeptide chains are isolated from the culture medium and treated
with chemicals to produce the quaternary protein Insulin.
• Insulin is then purified for human consumption.
• Using Eukaryotic yeast cells rather than Prokaryotic E. Coli will enable
formation of the quaternary protein Insulin within the yeast cells due to the
presence of rER and Golgi apparatus.
• The Insulin gene is usually inserted along with its promoter to ensure that the
gene gets transcribed.
• Promoters are short segments of DNA which are essential for gene
transcription due to the following reasons:
1. Promoters serve as the site to which the enzyme RNA polymerase binds to
initiate transcription.
2. Promoters also help identify which strand of DNA serves as the template for
transcription.
 BENEFITS OF PRODUCING
GENETICALLY ENGINEERED HUMAN
INSULIN
• Less likely to cause allergic reactions.
• Production of genetically engineered human Insulin is relatively
cheaper, if Insulin is produced on large scale.
• Is acceptable to religious groups who are against the usage of Insulin
derived from pigs or cattle.
• Likely to be more effective in causing reduction in the plasma glucose
because of its better binding to the Insulin receptor.
• Human Insulin has a more rapid onset of action and is less likely to cause
tolerance.
 TOOLS FOR GENETIC
ENGINEERING
• Recombinant DNA (rDNA):
The DNA that has been altered by genetic engineering and which now contains
lengths of nucleotides from two different organisms
• Reverse Transcriptase:
is responsible for producing single stranded cDNA using the mRNA.
• DNA Polymerase:
is responsible for producing double stranded cDNA using single stranded cDNA via
rules of complimentary base pairing.
• Restriction Endonucleases:
is an enzyme that cleaves DNA into fragments at or near specific recognition sites
within the molecule
• Plasmids:
are small circles of DNA in a bacterial cell that is physically separated from a
chromosomal DNA and can replicate independently
• Vector:
is a DNA molecule used as a vehicle to artificially carry foreign genetic material into
another cell, where it can be replicated and/or expressed
 MARKER GENES
• Markers are genes that are added to DNA segment to help identify
transformed organisms. There are two types of markers:

1. Selectable Markers

2. Screening Markers.
 SELECTABLE MARKERS
• Selectable Markers are genes which enable identification of transformed organisms
by monitoring their survival in the presence of a selective agent, such as an
antibiotic.

• Antibiotic resistance genes are usually added to the plasmids.

• Replica Plating is a commonly used technique for identification of transformed

bacteria that have taken up the recombinant DNA.

• The antibiotic resistance genes used in selectable markers may spread to

pathogenic bacteria which can thereby increase the incidence of antibiotic
resistance.
 REPLICA PLATING
• Consider a plasmid which contains antibiotic resistance genes for Ampicillin
and Tetracycline.
• The desired gene (for example human insulin gene) is inserted within the
Tetracycline gene which makes the rDNA contain a functional copy of
ampicillin resistance gene but a non-functional copy of the tetracycline
resistance gene.
• Bacteria that take up the rDNA containing Insulin gene will, therefore, survive
in the presence of ampicillin but will be susceptible to the drug tetracycline.
• Bacteria are cultured onto an agar plate containing the antibiotic ampicillin.
The plate is left for a day and observed for the formation of bacterial
colonies after a given amount of time.
• The position of the bacterial colonies on the agar plate is marked. This plate
is labelled as the Master Agar Plate.
• A velvet cloth is pressed against the agar which enables the bacterial
colonies to stick to the cloth
• The velvet coth is now pressed against a second agar plate rich in antibiotic
Tetracycline
• Bacteria that contain the tetracycline resistance gene will survive while
others will be susceptible
• The second agar plate is referred to as the Replica Plate
• The missing colonies on the replica plate when compared with the master
agar plate will enable identification of transformed microorganisms.
• Master Agar Plate enables growth of both transformed and non-transformed
organisms which contain the ampicillin resistance gene
• Replica Plate, however, only allows growth of non-transformed organisms
that do not contain the desired gene. Transformed organisms containing the
desired gene die due to susceptibility to tetracycline
REPLICA PLATING
 SCREENING MARKERS
• Screening markers are genes that enable identification of transformed
organisms based on the changes in their visible appearance.
Commonly used screening markers include:
• GFP gene (Green Fluorescent Protein gene):
the GFP gene is extracted from the Jellyfish. The protein produced
by this gene fluoresces green under UV light.

• β-GUS gene (coding for β-Glucuronidase Enzyme):

β-GUS gene which codes for the enzyme β-Glucuronidase enables identification of transformed
organisms by breaking down non-blue glucuronides to produce a toxic blue product.

• The Blue White Method:

which involves use of a marker gene known as the Z gene. Z gene codes for the enzyme β-
Galactosidase, which splits a colourless galactoside to produce harmless blue product.

• The marker gene for GFP codes for a non-enzymatic protein, whereas, the other two screening
markers codes for enzymes. These enzymes can produce a large number of product molecules
since they can be reused.