PRINCIPLES OF

BIOTECHNOLOGY
(i)Genetic engineering
(ii)Bioprocess engineering

GENETIC ENGINEERING:-
Techniques to alter the chemistry of genetic
material (DNA and RNA) to introduce these into host
organisms and thus change the phenotype of the host
organism
BIOPROCESS ENGINEERING:-
Maintenance of sterile (microbial contamination-
free) ambience in chemical engineering processes to
enable growth of only the desired microbe/eukaryotic
cell in large quantities for the manufacture of
biotechnological products like antibiotics, vaccines,
enzymes, etc
 RECOMBINANT DNA
TECHNOLOGY-TOOLS

I. Restriction
Nuclease
II. Cloning Vectors

RESTRICTION NUCLEASE:-

They are used to cut a DNA molecule at specific and

desired site. They are also called as Molecular Scissors
CLONING VECTORS:-

A Cloning Vector is a small piece of DNA that can be

stably maintained in an organism, and into which a
foreign DNA fragment can be inserted
for cloning purposes
 FEATURES OF
CLONING VECTOR
I. Origin Of replication
II. Selectable marker
III. Cloning vector

ORIGIN OF REPLICATION:
This the sequence of DNA where the
replication starts called the Ori gene.
SELECTABLE MARKER:
It is required to identify recombinant from
non-recombinant vectors.
CLONING SITES:
In order to link the alien DNA, the vector
needs to have very few preferable single,
recognition sites for commonly used restriction
endonuclease.
 SAD

pBR 322

pBR322 was one of

the first widely used E.
coli cloning vectors.

pBR322 is 4361 base pairs in length and

has two antibiotic resistance genes the gene bla
encoding the ampicillin resistance (AmpR) protein, and
the gene tetA encoding the tetracycline resistance
(TetR) protein. It contains the origin of replication of
pMB1, and the rop gene, which encodes a restrictor of
plasmid copy number. The plasmid has unique
restriction sites for more than forty restriction
enzymes. If a foreign DNA ligated or inserted at the
Bam H I site of tetracycline resistance gene in the
vector pBR322,the recombinant plasmid will lose
tetracycline resistance.(insertional inactivation)
 RECOMBINANT DNA
TECHNOLOGY

PROCESSES:

▪ Isolation of DNA
▪ Fragmentation of DNA by restriction endonuclease
▪ Isolation of desired DNA fragment by gel
electrophoresis
▪ Ligation of DNA fragments with a vector by DNA
ligase
▪ Transferring the recombinant DNA into the host
▪ Culturing the host cells in a medium at large scale
in a bioreactor.
▪ Extraction of desired products by downstream
processing
 AMPLIFICATION OF
GENE- PCR
-DENATURATION
-ANNEALING
-EXTENSION

DENATURATION:-
Double stranded DNA is made single
stranded by heating DNA at 94o c.
ANNEALING:-
Two sets of primer are added to the
medium at around 50oc.
EXTENTION:-
Deoxyribonucleotides triphosphates
are added in the medium. Taq polymerase catalyses the
polymerization reaction using nucleotides extending
from the primer to the 5’ end of the template.
 DOWNSTREAM
PROCESSING
Obtaining the foreign
gene product or
recombinant product

Downstream processing:
After biosynthesis inside the bioreactor, the product
has to be subjected through a series of processes
before it is ready for marketing.
▪ The process includes separation and purification,
which are collectively referred as downstream
processing.
▪ The product has to be formulated in suitable
preservatives.
▪ Such formulation has to undergo through clinical
trials as in case of drugs.
 INDEX
PAGE NO TOPIC

1 GENE

2 HOW GENES WORK?

3 BIOTECHNOLOGY

4 PRINCIPLES OF BIOTECHNOLOGY

5 RECOMBINANT DNA TECHNOLOGY-TOOLS

6 FEATURES OF CLONING VECTOR

7 pBR 322

8 RECOMBINANT DNA TECHNNOLOGY

9 AMPLIFICATION OF GENE - PCR

10 DOWNSTREAM PROCESSING

11 GENETIC ENGINEERING

12 DRAWBACKS

13 CONCLUSION