BIOTECHNOLOGY

Biotechnology deals with techniques of using organisms or enzymes from organisms to

produce products and processes useful to humans.

Two core techniques of Biotechnology are,

1. Genetic engineering, to alter the chemistry of genetic material to introduce them into host
organisms & thus change the phenotype of host. It allows to isolate and introduce only one or a set of
desirable genes into target organism. Presence of origin of replication along with the alien DNA allows
self replication of rDNA producing a clone of desired gene.

2. Maintenance of sterile environment to enable growth of only desired microbe/eukaryotic

cell in large quantities for the products like antibiotics, vaccines etc.

Stanley Cohen and Herbert Boyer first started biotech process in 1972. They isolated
antibiotic resistance gene from a plasmid of Salmonella typhimurium, using restriction enzyme. This
piece of DNA was then linked with another plasmid, using DNA ligase. This rDNA was transferred to
[Link], where it produces multiple copies. Thus there are three basic steps in genetic modification of
organisms,

1. Identification of DNA with desirable genes.

2. Introduction of identified DNA into host.

3. Maintenance of introduced DNA in the host and transfer of the DNA to its progeny.

Tools of rDNA technology

1. Enzymes. 2. Vectors 3. Host organisms. 4. Bioreactors

Enzymes:
Enzymes to cut DNA of different organisms and to joining them together.

[Link] enzymes 2. DNA ligase.

1. Restriction enzymes:
These are DNA cutting enzymes found in bacteria which restrict viral infection by cutting DNA of virus.
Restriction enzymes are of two types as Exonucleases and Endonucleases . Exonuclease remove
nucleotides from the ends of DNA. Endonucleases make cut at specific position within DNA.

Restriction endonucleases (REN): Recognize restriction site having specific recognition sequence.
It consists an inverted repeat of nucleotides called Palindromes of 4-8 base pairs. It reads same way in
both strands, but in opposite direction. 51 –G A A T T C- 31

31—C T T A A G -51

REN are considered as molecular scissors or knives . Hind II was the first REN identified. Now
more than 900 REN are isolated , which recognize different restriction sites. Majority of REN produces
staggered or sticky end cuts, where two strands of DNA are cut, a few bases apart. This creates single
stranded tails in each end of the fragment, which are complementary to each other. When same REN
are used, the resulting DNA fragments have same kind of sticky ends which can be joined together
easily.

Eg: EcoR I– Escherichia coli RY 13.

In REN names, first letter (E) refers to the genus genus name and next two letters (co) represent
the species of prokaryotic cell from which they are isolated. Next letter (R) represent the name of the
strain and the roman number (I) indicates the order in which the enzyme isolated from that bacteria.
2. DNA ligases: DNA fragments resulted due to REN action can be joined to any other DNA
fragments treated with same enzyme to form recombinant DNA. This is called as Annealing. DNA
ligases join sugar phosphate backbone of two DNA fragments by forming phosphor-diester bond
between them. So are called as molecular stitchers.

CLONING VECTORS:
Vectors are gene carriers or gene vehicles. They are used to transport desired genes into a recipient cell
or organism. They have the ability to replicate in an appropriate host cell. Plasmids and Bacteriophages
are common vectors.

Plasmids are small circular, autonomously replicating dsDNA present in some bacteria, in addition to
chromosomal DNA. It has genes for antibiotic resistance. In a bacteria one or two copies plasmids are
present, but its number can go up to 100 or more. Plasmids are ideal vectors because they can be cut at
specific site and desired gene can be joined to produce rDNA. They can self replicate to produce a clone
of desired gene.

Eg: 1. Ti plasmid of Agrobacterium tumefaciens.

2. Ri plasmid of Agrobacterium rhizogenes.

Now artificially engineered plasmids like pBR-322, pUC-18 etc. are available. They have many cloning
sites and recombinants formed can be easily selected by marker technique.

Important features of pBR 322 are,

1. Origin of replication( ori):

This is a sequence from where replication starts and any piece of DNA linked to this can be made to
replicate within host cell. It also control the number of copies of linked DNA. Close to it ‘rop’ site, coding
for protein involved in replication of plasmid.

2. Selectable marker:

Marker helps in identifying and eliminating non transformants and selectively permitting the growth
of transformants. The genes encoding resistance to antibiotics such as, ampicillin, tetracycline,
Kanamycin etc. are selectable markers for [Link]. Normal [Link] do not carry resistance against any
antibiotics.

3. Cloning sites:

The vector should have one or a few cloning sites to insert alien DNA. The alien DNA is ligated to a
restriction site present within the marker gene. So the foreign DNA is ligated either to BamHI or Sal I
site of tetracycline resistance marker gene or Pst I and Pvu I are sites of ampicillin resistance gene.
When a gene is inserted into TetR gene, the recombinant plasmid loose tetracycline resistance. Bacteria
having such plasmid will not grow in a medium containing tetracycline, but can grow in a medium with
ampicillin. Non recombinants will grow on medium containing both antibiotics. Here one antibiotic
resistance gene helps in selecting transformant and the other antibiotic resistance gene gets
‘inactvated due to insertion’ of alien DNA, and helps in selection of recombinants.

Alternative selective markers based on ability to produce color in the presence of chromogenic
X-gal compound in the medium. A specific gene is inserted within the coding sequence of β-
galactosidase enzyme of plasmid. This inactivate the enzyme. The process of inactivation of the
expression of selectable marker gene by inserting a gene of interest is called insertional inactvation.
Plasmid without an insert produce blue coloured colonies. Plasmid with insert do not produce any
colored colonies and are considered as recombinants.
Vectors for cloning genes in plants and animals:
Agrobacterium tumifaciens, a pathogen of several dicot plants, causing crown gall disease, is able to
deliver a piece of DNA known as T-DNA to transform a normal cell into a tumor cell. It also directs the
tumor cells to produce chemicals required for the pathogen. The plasmid of Agrobacterium is called
Ti plasmid. This plasmid is modified and is no more pathogenic, but can deliver a gene of our interest
into a variety of plants.

The cancer causing retrovirus disarmed and are used to deliver genes into animal cells.

COMPETENT HOST:
Suitable host cell is necessory for the expression of transformed gene. Escherichia coli (E. coli) is most
popular host. It can be easily cultured, nonpathogenic and multiply for every 20 min. Any vector
plasmid with alien DNA will replicate and express in [Link].

The bacteria are made competent to take up DNA by treating with specific concentration of Ca+2, which
causes pores in its cell wall. These cells are incubated in ice along with rDNA, followed by heat shock
briefly at 42oc and then putting back to ice. This enables the bacteria to take up rDNA.

Alien DNA can also be introduced by microinjection, biolistic and by using disarmed pathogens .
In microinjection rDNA is directly introduced into nucleus of animal cell by micro-syringe. In biolistic
(gene gun) method, plant cells are bombarded with high velocity micro-particles of gold or tungsten
coated with rDNA. Disarmed pathogen vectors when allowed to infect the cell, transfer the
recombinant DNA into the host.

BIOREACTORS:
The host cell with rDNA can be multiplied in a continuous culture system, where in the used medium is
drained out from one side while fresh medium is added from the other side to maintain the cells in
active exponential phase. This type of culture leads to higher yield of desired proteins. For this
bioreactors are used.

Bioreactors are steel tanks of 100-1000 litres capacity in which raw materials are biologically converted
into specific products using microbes, plant, animal or human cells. It provide optimal growth conditions
like temperature, pH, substrate, salts, vitamins, O2, etc.

Stirred-tank reactors are cylindrical or with curved base to facilitate the mixing of contents. It may be
1. Simple stirred tank bioreactor. 2. Sparged stirred tank bioreactor.

The stirrer facilitates mixing and oxygen availability throughout the bioreactor. Alternatively air can be
bubbled through the reactor. The bioreactor has an agitator system, an oxygen delivery system, a foam
control system, temperature and pH control system and sampling parts to test the sample periodically.
Separation and isolation of DNA fragments:

DNA fragments formed due to REN action can be separated by Agarose gel electrophoresis. In agarose
gel DNA fragments moved towards anode, due to their –ve charge. DNA fragments separate according
to their size through sieving effect provided by agarose gel. Smaller fragments moves farther away
compared to larger fragments.
 Separated fragments are stained with Ethidium bromide and then exposed to UV radiations. DNA
fragments appears as bright orange colored bands. The separated bands are cut out from gel and DNA
are extracted. This is called as elution. This DNA can be used for rDNA technology.

Polymerase chain reaction (PCR):

Discovered by Kary Mullis in 1985. Here multiple copies of gene or DNA is synthesized in vitro
using two sets of primers and DNA polymerase. It is done in thermal cycler machine.

The technique is based on the principle that DNA gets separated into two single strands at high
temperature ( 940 C).The single stranded molecules are then converted into double stranded by DNA
polymerase. If the process is repeated many times, billions of copies of DNA can be produced. It is
achieved by the use of thermo stable DNA polymerase (Taq polymerase – Thermus aquaticus), which
remain active in high temperature. The amplified fragment can be used to ligate with a vector for
further cloning.
Processes of rDNA technology:
1. Isolation of DNA:
 Bacteria, plant or animal tissue is treated with enzymes like lysozyme(bacteria), cellulose(plant)
or chitinase (fungus) to break open cell membrane.

 Histone proteins are removed by treating with proteases, RNA can be removed by treating with
ribonucleases. Other molecules can be removed by appropriate enzymes.

 Purified DNA is ultimately precipitates after the addition of chilled ethanol, which looks like
acollection of fine threads in the suspension.

[Link] of DNA at specific location:

 DNA are cut at specific site by REN. Agarose gel electrophoresis is employed to check the
progression of REN action. DNA is a –vely charged molecule, hence it moves towards +ve
electrode.

 Suitable vector also treated with same REN to have complementary sticky ends.

3. Amplification of gene of interest using PCR:

 Multiple copies of a desired gene can be obtained by PCR , using thermal cycler machine. Here
DNA replication is repeated many times to get millions of copies of DNA.

4. Ligation:
The gene and vectors are mixed together ,in the presence of DNA ligase. Fragments having
complementary ends are first joined by H2 bonds and then by Phospho-diester bonds to form
rDNA molecule.

5. Insertion of rDNA into host cell / organism:

 The host cell like [Link] are made ‘competent’ to receive rDNA.

 Host cell absorb rDNA and are selected by marker technique. If rDNA contain antbiotic
(eg: ampicillin) resistant gene, the host cell become ampicillin resistant cell. It can grow in a
media having ampicillin, but the non- transformed cells will die in ampicillin.

 When host cell multiply, the rDNA passed on to progeny cells. So a large population of host cell
with a copy of rDNA is produced.

6. Obtaining the foreign gene product:

 The alien DNA transferred into a bacteria, plant or animal cell must express to produce
recombinant protein. On large scale it is done by continuous culture systems, using bioreactor.

7. Downstream processing:
 Product formed in a bioreactor has to be subjected through a series of processes, before it is
ready for marketing as a finished product.

 It includes separation and purification, collectively referred as downstream processing.

 The product has to be formulated with suitable preservatives. Such formulations has to
undergo clinical trials, in case of drugs.
 Strict quality control testing for each product is also required. Down stream processing
and quality control testing vary from product to product.

Recombinant DNA technology

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