BIOTECHNOLOGY

PRINCIPLES AND PROCESSES

WHAT IS
BIOTECHNOLOGY?
Biotechnology is the use of living systems and organisms to develop or make
useful products, or "any technological application that uses biological
systems, living organisms or derivatives thereof, to make or modify products
or processes for specific use. Depending on the tools and applications, it
often overlaps with the (related) fields of bioengineering and biomedical
engineering.

The wide concept of "biotech" or "biotechnology" encompasses a wide range
of procedures for modifying living organisms according to human purposes,
going back to domestication of animals, cultivation of plants, and
"improvements" to these through breeding programs that employ artificial
selection and hybridization. Modern usage also includes genetic
engineering as well as cell and tissue culture technologies
STORY OF INSULIN AND
BIOTECH
The human body produces insulin to regulate blood sugar
levels. Diabetes occurs when the body does not produce
insulin or cannot produce enough insulin. People with diabetes
often need injections of insulin, which doctors first provided
patients through supplies taken from pigs and cows.
However, scientists did not know the long-term effects of
having animal insulin in your body. In 1978, Boyer was able to
take pieces of human DNA and isolate a gene for insulin using
biotechnology. He then inserted it into bacteria, which allowed
the gene to reproduce a larger quantity of insulin for diabetics.
This scientific advancement vastly improved quality of life for
many people living with diabetes and guaranteed their safety.

INSULIN CRYSTALS
PRINCIPLES
Two core techniques that enabled birth of modern biotechnology:
Genetic engineering: Techniques to alter the chemistry of genetic
material (DNA and RNA) to introduce into host organisms and
thus change the phenotype of the host organism.
Maintenance of sterile (microbial contamination-free) ambient
chemical engineering processes to enable growth of only the
desired microbe/eukaryotic cell in large quantities.
Conceptual development of the principle of genetic engineering:
Asexual reproduction preserves the genetic identity of species.
Sexual reproduction creates variation and creates unique
combinations of genetic makeup.

Traditional hybridization procedures used in plant and
animal breeding lead to inclusion of undesirable
genes along with desired genes.
The techniques of genetic engineering which includes
creation of recombinant DNA, use of gene
cloning and gene transfer, overcome this limitation and
allows us to isolate and introduce only one or a set of
desirable genes without introducing undesirable genes
into target organism
Three basic steps in genetically modifying an organism
Identification of DNA with desirable gene
Introduction of the identified DNA into the host.
Maintenance of introduced DNA in the host and
transfer of the DNA to its progeny.

TOOLS OF RECOMBINANT DNA
TECH
RESTRICTION ENZYMES
Restriction enzymes are involved in the 'breaking up' of DNA molecules.

Restriction enzymes occur naturally in certain bacteria and function as a defense mechanism against viral
DNA. When a virus attempts to inject its DNA or RNA, the bacteria releases restriction enzymes which slice
the viral DNA into short strands.

Restriction enzymes are an essential tool in recombinant DNA technology and are also known as DNA
scissors or cutting enzymes. Each restriction enzyme cuts at a specific location (recognition site) where a
particular sequence of nucleotides is found. This means that EcoRI will only cut between G and A in this
sequence. Restriction enzyme action using EcoRI is shown below.

Step 1: A section of DNA with the recognition site for EcoRI (underlined) is provided below:

Step 2: EcoRI cuts the DNA strand at the recognition site (between G and A).

Step 3: The DNA strand is fragmented.

This produces DNA fragments with exposed nucleotides or 'sticky ends'.

Other restriction enzymes, for example HindII, make a straight cut and produce DNA fragments with no
exposed nucleotides. These fragments are called 'blunt ends'.

Sticky ends and blunt ends can be joined together by another DNA enzyme, ligase, in a process called
ligation.

POLYMERASES
Polymerases are enzymes involved in the synthesis
of nucleic acids.
Polymerases can be used to:
produce copies of DNA during DNA replication and
repair. Polymerases 'read' an intact DNA strand as a
template to synthesise a new strand
produce copies of DNA from the fragments of DNA
from an extinct organism during a process called the
polymerase chain reaction (PCR)
produce copies of DNA from the fragments of DNA
from a crime scene using the PCR.

LIGASES
DNA ligase is involved in the repair and replication of
DNA. Ligases function to rejoin the DNA fragments in
a process called ligation. The DNA fragments join
together using the base pair ruling.
The exposed nucleotides of a fragment of DNA are
attracted by base pairing to another DNA fragment.
The fragments are linked by the enzyme DNA ligase,
which joins the fragments at their sugar-phosphate
backbones.
If the DNA fragments are from different sources
recombinant DNA is produced.

CLONING VECTORS
A cloning vector is a small piece of DNA into which a
foreign DNA fragment can be inserted. The insertion
of the fragment into the cloning vector is carried out
by treating the vehicle and the foreign DNA with a
restriction enzyme that creates the same overhang,
then ligating the fragments together. There are many
types of cloning vectors. Genetically engineered
plasmids and bacteriophages (such as phage ) are
perhaps most commonly used for this purpose.
Other types of cloning vectors include bacterial
artificial chromosomes (BACs) and yeast artificial
chromosomes (YACs).


Cloning vectors based
on E.coli plasmids

Simplest cloning vectors based on small bacterial plasmids are the
most widespread in gene cloning experiment. A large number of
different plasmid vectors are available for use with E.coli and these
vectors combine ease of purification with desirable properties such
as high transformation efficiency, convenient selectable markers for
transformants and recombinants, and the ability to clone reasonably
large pieces of DNA. One of the most popular vector used in gene
cloning is pBR322, and it's nomenclature is shown below:

pBR322 Plasmid

p- A plasmid

BR- The laboratory in which the vector was originally
constructed(Bolivar and Rodriguez, the two researchers who
developed pBR322).

322- This number distinguishes the plasmid from other plasmids
developed in the same laboratory.

COSMID
A cosmid is a plasmid that carries a cos site the
sequence yielding cohesive ends (Figure-8).
Cosmids are hybrids between a phage DNA
molecule and a bacterial plasmid, and are designed
in such a way that the enzymes that package the
DNA molecule into the phage protein coat need only
the cos sites in order to fuction. A typical cosmid has
replication origin, unique restriction sites and
selectable markers from the plasmid; therefore
selection strategy for obtaining the recombinant
vectors is based on that for the contributing plasmid.
Cosmid vectors are constructed using recombinant
DNA techniques.


Plant viruses as cloning
vectors

Most plants are subjected to viral infection and vast
majority of plant viruses have genomes of RNA. RNA
viruses are not so useful as potential cloning vectors
because manipulations with RNA are rather more difficult
to carry out. Two classes of DNA virus are known to infect
higher plants, the caulimoviruses and geminiviruses, but
neither is identically suited for gene cloning. Caulimovirus
vector could only be used to clone very short pieces of
DNA. Geminiviruses at first appear more promising as
they naturally infect important crops such as maize and
wheat. But during the infection cycle the geminivirus
genome undergoes rearrangements and deletions, which
could scramble up any additional DNA that had been
inserted, an obvious disadvantage for a cloning vector.
Animal viruses as cloning
vectors

Retroviruses, though have single-stranded RNA genomes but provides perhaps the
most promising vector system of all. During the process of reverse transcription,
sequences from the termini of viral RNA are duplicated to generate long terminal
repeats(LTRs). These long terminal repeats contain both the promoter and the
polyadenylation signal for the transcription of viral mRNAs. The specificity of proviral
DNA integration is also determined by the long terminal repeats. Although retroviruses
can integrate at many sites within the cellular genome, integrative recombination
always occurs at particular sites at the ends of the LTRs. The sequences appropriately
inserted between the two LTRs will be integrated intact which contrasts sharply with the
integration of papovavirus or adenovirus DNA, during which extensive rearrangements
of the integrated viral sequences are commonplace. A further great advantage of
retroviruses is that they are natural transducing viruses.

Baculoviruses, enable large amounts of proteins to be obtained from genes cloned in
insect cells. One of the major proteins encoded by the virus genome is polyhedrin,
which accumulates in very large quantities in the nuclei of infected cells, since the gene
has an extremely active promoter. The same promoter can be used to drive the
over expression of a foreign gene engineered into the baculovirus genome, and large
quantities of protein can be produced in infected insect cells in culture. This method is
being used increasingly for large-scale culture of proteins of animal origin, since the
insect cells can produce many of the post-translational modifications of animal proteins
which a bacterial expression system.

PLAY GENE CLONING

PROCESSES
1. ISOLATION OF GENETIC MATERIAL
2. CUTTING OF DNA AT SPECIFIC
LOCATIONS
3. GEL
ELECTROPHORESIS
PLAY VIDEO
4.
AMPLIFICATIO
N OF GENE OF
INTEREST
USING PCR
PLAY VIDEO
5. INSERTION
OF
RECOMBINA
NT DNA INTO
THE HOST
CELL
6. OBTAINING FOREIGN
GENE PRODUCT