CL12_ Biotechnology_Principles and Process_Priya Jha.pdf

Biotechnology is the scientific technology which uses living organisms in the systems or processes for the manufacturing of useful products/services for human beings.
The term biotechnology was coined in 1917 by Karl Ereky to describe a process for large scale production of pigs.
The techniques of geneticengineering which include 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 the target organism.Among many, the two core techniques that enabled the birth of modern biotechnology are
(i) Alternation of constituents of genetic material (DNA or RNA) to change the phenotype of resultant organisms.
(ii) Production of the large number of microbes/eukaryotic cells in controlled environment to manufacture various products.

1. Biotechnology :
Principles and
Processes
‘The integration of natural science and
organisms, cells, parts thereof, and molecular
analogues for products and services’.
PRIYA JHA
PGT BIO

2. •Biotechnology is the scientific technology which uses
living organisms in the systems or processes for the
manufacturing of useful products/services for human
beings.
•The term biotechnology was coined in 1917 by Karl
Erekyto describe a process for large scale production
of pigs.
•The techniques of geneticengineeringwhich include
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 the
target organism.

3. (I)Principles of Biotechnology
•Among many, the two core techniques that
enabled the birth of modern biotechnology are
•(i) Alternation of constituents of genetic
material (DNA or RNA) to change the
phenotype of resultant organisms.
•(ii) Production of the large number of
microbes/eukaryotic cells in controlled
environment to manufacture various
products.

4. Two core techniques that enabled
birth of modern biotechnology:
1. 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.
2. Bioprocess engineering :Maintenance of
sterile (microbial contamination-free) ambient
chemical engineering processes to enable
growth of only the desired microbe/eukaryotic
cell in large quantities.

5. Three basic steps in genetically modifying
an organism
i)Identification of DNA with desirable gene
ii)Introduction of the identified DNA into the
host.
iii)Maintenance of introduced DNA in the host
and transfer of the DNA to its progeny.

7. (II)PROCESS OF RECOMBINANT TECHNOLOGY
(1)Isolating the DNA,
(2) Cutting the DNA at specific sites using restriction
enzymes,
(3) Amplifying the gene of interest (often using PCR),
(4) Inserting the desired DNA fragment into a vector (like
a plasmid) using ligase to create recombinant DNA,
(5) Transferring this recombinant DNA into a host cell (like
a bacterium), and finally,
(6) Selecting the host cells that have successfully
incorporated the new DNA to multiply and express
the desired product.

8. 1.Isolating the Genetic Material(DNA)

9. Gel electrophoresis
Gel electrophoresis involves
*preparing a gel slab
*loading DNA samples into wells
using a dye-containing buffer
*then running the gel by applying
an electric current to separate
fragments by size.
*Finally, the separated bands are
visualized under UV light after
staining the gel with a
fluorescent dye, such
asethidiumbromide .
The separated bands of DNA
are cut out from the agarose
gel and extracted from the
gel piece. This step is called
elution.

11. 2.The cutting of DNA at specific
locations
The cutting of DNA at specific locations
ismade possible by restriction endonucleases,
also known as restriction enzymes.These
enzymes act like molecular scissors,
recognizing and binding to specific DNA
sequences called restriction sites and cleaving
the DNA at or near these sites, which is a
fundamental process inrecombinant DNA
technology

12. (2) Cutting the DNA at specific sites using
restriction enzymes:
(III)TOOLS OF RECOMBINANT DNA
TECHNOLOGY
A.Restriction Endonucleases
B.Cloning Vector
C.Component Host(For
Transformation with Recombinant
DNA)

13. (A)Restriction Enzymes
•In the year 1963 two enzymes discovered from Escherichia coli which
restrict the growth of bacteriophagein it.
One of these added methyl groups to DNA.
Other cut the phage DNA. (restriction endonuclease)
•The first restriction endonucleasediscovered is HindII.
(Haemophilusinfluenza II –sequence osisolation)
• Hind II always cut DNA molecule at particular point by recognizing a
specific sequence of six base pairs. This is called recognition
sequence for Hind II.
• Till date around 900 restriction enzymes isolated from 200 strains of
bacteria each of which recognize different recognition sequences.
•Restriction enzyme belongs to nucleases
There are two kind of nucleases: (i) Exonuclease
(ii) Endonuclease

16. Diagrammatic
representation
of recombinant
DNA
technology

17. The flow chart given below represents the process of
recombinant DNA technology. Identify A,B, C and D.
•A-Restriction endonuclease,
•B-Restriction endonuclease,
•C-DNA ligase,
•D-Transformation

18. •Exonucleaseremoves nucleotides from the free ends of the DNA.
• Endonucleasesmake cuts at specific positions within the DNA.
• Each restriction endonucleaserecognizes a specific palindromic
nucleotide sequences in the DNA.
( Palindromes are the group of letters that read same both forward
and backward, e.g. “MALAYALAM”)
•The palindrome in DNA is a sequence of base pairs that reads same
on the two strands when orientation of reading is kept same. Such
as 5'-GAATTC-3' and 3'-CTTAAG-5'.
•The restriction enzyme cut the strand of DNA little away from the
centre of the palindrome sites, but between the same two bases on
the opposite strand. This leaves single stranded portions at the
ends. There are overhanging stretches called sticky ends on each
strand.
• This stickiness of the ends facilitates the action of the enzyme DNA
ligases.
• The foreign DNA and the host DNA cut by the same restriction
endonuclease, the resultant DNA fragments have the same kind of
‘sticky-ends’ and these can be joined together using DNA ligases.

19. Steps in
formation of
recombinant
DNA by action
of restriction
endonuclease
enzyme -EcoRI

20. Nomenclature of Restriction Endonucleases
The name of the enzyme is derived from the name of organism
from which it is isolated.
(i) The first letter of the genus becomes the first letter of the
name (written in capital letter).
(ii) First two letters of the species make second and third letter of
the enzyme (written in small letters).
(iii) All these three letters are written in italics.
(iv) The fourth letter of the name of enzyme is the first letter of
strain (written in capital letter).
(v) The Roman number written at the end of the name indicates
the order of discovery of enzyme from that strain.

21. 3.Amplification of Gene of Interest
using Polymerase Chain Reaction
Polymerase chain reaction, orPCR, is a technique to make many
copies of a specific DNA regionin vitro(in a test tube rather than an
organism).
The basic steps are:
•Denaturation(96°C): Heat the reaction strongly to separate, or
denature, the DNA strands. This provides single-stranded template
for the next step.
•Annealing(55-65°C): Cool the reaction so the primers can bind to
their complementary sequences on the single-stranded template
DNA.
•Extension(72°C): Raise the reaction temperatures
soTaqpolymerase extends the primers, synthesizing new strands of
DNA.
Typically, the goal of PCR is to make enough of the target DNA region
that it can be analyzed or used in some other way. For instance,
DNA amplified by PCR may be sent forsequencing, visualized bygel
electrophoresis, orclonedinto a plasmid for further experiments.

22. Amplification of Gene:PCR

25. 4.Insertion of recombinant DNA in host cells
Mostly the host cell used is a bacterial cell. Yeast and
fungi can also be used as hosts. The process of transferring
recombinant DNA into the host cell is called transformation.
For transformation, bacterial cells are first made competent
by thermal treatment, electroporation, etc. to accept foreign
DNA molecules.
After the insertion process, a crucial step is to
identify which host cells have successfully taken up the
recombinant DNA.
•Selectable Markers:Recombinant DNA vectors often contain
genes for antibiotic resistance (like ampicillinresistance).
•Screening:When the transformed cells are spread on a
culture plate containing the corresponding antibiotic, only the
cells containing the resistance gene (and thus the
recombinant DNA) will survive and grow, while the
untransformed cells will die.

26. 5.Obtaining the Foreign Gene product or Recombinant
product
•The protein encoding gene is expressed in a
heterogeneous host is called a recombinant
protein.
• The host is cultured in a continuous culture
system provided in bioreactor.
• A bioreactor provides optimum growth conditions
(temperature, pH, substrate, salts, vitamins,
oxygen)
• Bioreactor covert the raw materials into specific
product, specific enzyme

27. Bioreactor

28. Obtaining the Foreign Gene Products
•Scale-Up Production in a
Bioreactor:
•Culture the recombinant
cells on a large scale in a
bioreactor, which
provides and maintains
optimal growth
conditions (temperature,
pH, nutrients) for mass
production of the foreign
product.

29. (B)Cloning Vector:pBR322
•Cloning vectors:“A cloning
vector is a small piece of DNA
into which a foreign DNA can
be inserted for cloning
purposes.”
• The plasmid and
bacteriophageshave the
ability to replicate within
bacterial cells independent of
the control of chromosomal
DNA.
• Alien DNA linked with the
vector multiply its number
equal to the copy number of
the plasmid or bacteriophage.

30. Characteristics of a Cloning Vector
The following features are essential to facilitate
cloning into a vector
(i) A vector should contain a repliconthat
enables replication in the host cells.
(ii) It should have several marker genes.
(iii) It should have a unique cleavage site within
one of the marker gene.
(iv) For the expression of cloned DNA, the vector
DNA should contain suitable control elements
such as promoter, terminators and ribosome
binding sites.

31. Features of cloning vector
(i)Origin of replication:Theori, or origin of replication, is the sequence at
which replication of DNA begins. This sequence is also linked to the copy
number of the vector, and so controls how many times your gene of
interest will be produced in the host cell.
(ii)Selectable markers:Selectablemarkers are genes that help identify
bacteria that have successfully transformed, or taken up the recombinant
plasmid. Antibiotic resistance genes are commonly used markers.
(iii)Cloning sites:Allcloning vectors need sites that will allow the insertion of
foreign DNA into it. Most commonly, this involves the use of restriction
enzyme recognition sites. Ideally, the enzyme should have1, or a
maximum of2recognition sites, preferably within the selectable marker
sequences
Cutting the vector within the selectable marker will
interrupt that gene, and allow for identification of recombinants. For
example, using a cloning site within an ampicillinresistance gene will
result in recombinants that are no longer resistant to that antibiotic.

32. (iv).Vectors for cloning genes in plants and
animals:
Vectors (Vehicle DNA):It is defined as a DNA molecule that can be used to
carry a DNA segment (gene) to be cloned.
• Agrobacteriumtumefaciens, a pathogenic bacterium of several dicot
plants.
• This bacterium contains a plasmid called Ti-plasmid.(tumor inducing)
• In natural condition the A.tumifacienstransfer the T-DNA into the plant
which transform normal plant cells into a tumor and direct these
tumor cells to produce the chemical required by the pathogen.
• Retroviruses in animals have the ability to transform normal cells into
cancerous cells.
• The dis-armed retroviruses are being used to transfer gene into
animals.
• In Ti-plasmid the T-DNA is replaced by the gene of interest, still
A.tumifaciensable to transfer the gene into the plant without causing
tumor in plants.

33. (C)Competent Host (for transformation with
recombinant DNA)
• DNA is a hydrophilic molecule; it cannot pass through cell
membranes. • In order to force bacteria to take-up the plasmid, the
bacterial cells must first be made ‘competent’ to take up DNA.
• The bacterial cell is treated with divalent cationssuch as calcium,
which increases the efficiency of DNA up take by the bacteria.
• Recombinant DNA and the bacterial cells are incubated in ice,
followed by placing them briefly at 42oC (heat shock) and then
putting them back in ice.
• By microinjection the recombinant DNA directly injected into the
nucleus of the animal cell.
• Plant cells are bombarded with high velocity micro-particles of gold
or tungsten coated with DNA in a method known as biolisticsor
gene gun.
• The disarmed pathogen vectors which when allowed infecting the
cell transfer the recombinant DNA into the host.

35. 6.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.