CHAPTER: BIOTECHNOLOGY: PRINCIPLES AND PROCESSES

Biotechnology deals with microorganisms, plant or animal cells or their enzymes to produce
products and processes useful to humans.
The term biotechnology was given by KARL EREKY(1919)
According to European federaton of biotechnology ( EFB ), BIOTECHNOLOGY IS THE
integraton of natural science and organisms, cells , parts and molecular analogues for
products and services.
In 1972, Stanley Cohen and Herbert Boyer constructed the frst recombinant DNA.
Steps carried out in constructng frst recombinant DNA.
i. A gene encoding antbiotc resistance in the natve plasmid of Salmonella
typhimurium V. was identfed. PLASMID is an autonomously replicatng circular
extra chromosomal DNA.
ii. The desired DNA was cut at specifc locatons by restricton enzymes.
iii. The cut DNA was linked to plasmid DNA and transferred to E. coli for gene
multplicaton.

TOOLS OF RECOMBINANT DNA TECHNOLOGY

The key tools required for the recombinant DNA technology are:
i. Restricton enzymes ii. Polymerase enzymes iii. Ligases iv. Vectors v.
host organism/ cell

PROCESS OF RECOMBINANT DNA TECHNOLOGY

It involves the following steps:
I. ISOLATION OF DNA
The bacterial / plant/ animal cell is broken down by enzymes to release DNA, along
with RNA, proteins, polysaccharides and lipids.
Bacterial c ell is treated with enzyme lysozyme
Plant cell is treated with enzyme cellulase
 Fungal cell is treated with enzyme chitnase
DNA is removed by treatment with DNAase , RNA is removed by treatment with
ribonuclease and proteins are removed with protease. Afer all treatments, the purifed DNA is
precipitated by adding chilled ethanol.

II. FRAGMENTATION OF DNA BY RESTRICTION

ENDONUCLEASES.
The DNA is cut using restricton enzymes.
RESTRICTION ENZYMES are called molecular scissors and are responsible for cutng
DNA. They are present in bacteria to provide a type of defence mechanism called
restricton modifcaton system.
The frst restricton endonuclease, Hind II, was isolated by Smith, Wilcox and Kelley 1968) from
Haemophilus infuenza bacterium. It was used to cut DNA molecules at a partcular point by
recognizing a specifc sequence of six base pairs read the same on both the DNA strands, when
orientaton of reading is kept same, known as the recogniton sequence or palindromic
sequence .
eg. 5’ ----- G A A T T C --------3’
3’ ----- C T T A A G --------5’
ENZYMES NAMING OF RESTRICTION
The frst leter is derived from the genus name and the next two leters from the species name
of the prokaryotc cell from where the enzymes are extracted. The roman numbers, following
the name, show the order in which the enzymes were isolated from the bacterial stain.
Eg. EcoRI is derived from Escherichia coli RY 13, HindII from Haemophilus infuenza Rd, BamHI
from Bacillus amyloliquefaciens h, EcoRII from E. coli R 245, etc.
Restricton enzymes belong to a class of enzymes called nucleases and are of two types:
i. Exonucleases- cut DNA at the ends.
ii. Endonucleases- cut at specifc positons within the DNA.
 MECHANISM OF ACTION OF ENDONUCLEASES
Every nuclease inspects the entre DNA sequence for the palindromic sequence. On
fnding the palindromic sequence , the endonuclease binds to the DNA. IT CUTS THE
OPPOSITE strands of DNA in the sugar – phosphate backbone, a litle away from the
centre of the palindromic sites but between the bases on both strands.
This results in the formaton of single stranded overhanging stretches at the end of
each strand is called stcky ends.
The stcky ends facilitate the acton of the enzyme DNA ligase by readily forming
hydrogen bonds with complementary strands
III. ISOLATION OF A DESIRED DNA FRAGMENT
Using agarose gel electrophoresis, the actvity of the restricton enzymes can be checked. Since
the DNA is negatvely charged, it moves towards the positve electrode or anode and in the
process, DNA fragments separate out based on their sizes because of the sieving property of
agarose gel. Hence, smaller the fragment size, the farther it will move.
The separated DNA fragments are visualized afer staining the DNA with ethidium bromide
followed by exposure to UV radiaton.
The DNA fragments are seen as coloured bands. The separated bands of DNA are cut out and
extracted from the gel piece. This step is called eluton.
The purifed DNA fragments are used to form recombinant DNA which can be joined with
cloning vectors.
IV. AMPLIFICATION OF GENE OF INTEREST USING PCR
The polymerase chain reacton is a reacton in which amplifcaton of specifc DNA sequences is
carried out in vitro.
This technique was developed by Kary Mullis in 1985.
PCR carried out in the following three steps:
a. Denaturaton: the double stranded DNA is denatured by subjectng it to high
temperature of 950 C for 15 seconds. Each separated single strand acts as template for
DNA synthesis.
b. Annealing: two sets of primers are added which anneal to the 3’ end of each separated
strand. Primers act as initators of replicaton.
c. Extension: DNA polymerase extends the primers by adding nucleotdes complementary
to the template provided in the reacton.
 A thermostable DNA polymerase ( Taq polymerase ) is used in the reacton which can
tolerate the high temperature of the reacton.
All these steps are repeated many tmes to obtain several copies of desired DNA.
V. LIGATION OF DNA FRAGMENT INTO A VECTOR
The vector DNA and source DNA are cut with the same endonuclease to obtain stcky ends.
These are then ligated by mixing vector DNA , gene of interest and enzyme DNA ligase to form
recombinant DNA
VECTORS are the DNA molecules that can carry a foreign DNA segment into the host cell.
Vectors may be:
a. Plasmid b. bacteriophage
pBR 322 is the frst artfcial cloning vector developed in 1977 by Boliver and Rodriguez from E.
coli plasmid.
The following features are required to facilitate cloning into a vector.
A.Origin of replicaton ( ori )
This is a DNA sequence that is responsible for initatng replicaton. Any piece of DNA linked to
this sequence can replicate within the host cells.
Ori also control the copy numbers of the linked DNA.
B.Selectable marker
It helps to select the host cells which contain the vector( transformants) and eliminate the non
transformants.
Transformaton is defned as the procedure by which a piece of DNA is introduced into a
bacterial host.
Genes encoding resistance to antbiotcs like ampicillin, tetracycline are useful selectable
markers for E. coli.
The normal E.coli do not carry resistance against any of these antbiotcs.
C. Cloning sites
To link the alien DNA vectors require any few recogniton sites for the restricton enzymes.
More than one recogniton sites within the vector can complicate the gene cloning so ligaton of
alien DNA can be carried out a restricton site present in one of the two antbiotc resistance
genes.
 VI. INSERTION OF RECOMBINANT DNA INTO THE HOST CELL/
ORGANISM
Introducton of ligated DNA into recipient cells occurs by several methods before which the
recipient cells are made competent to receive the DNA.
DNA is a hydrophilic molecule, cannot pass through cell membrane. So bacteria should be made
competent to accept the DNA molecules.
Competency is the ability of a cell to take up foreign DNA.
The cell is made competent by the following methods:
a. Chemical method

The cell is treated with specifc concentraton of a divalent caton such as calcium to
increase pore size in cell wall.
The cells are incubated with recombinant DNA on ice, followed by placing them briefy
at 42o C and putng it back on ice. This is called heat shock treatment. The bacteria now
take up the recombinant DNA
b. Physical methods

Micro injecton method: recombinant DNA is directly injected into the nucleus of an
animal cell.
Biolistcs or gene gun method: cells are bombarded with high velocity micro partcles of
gold or tungsten coated with DNA in plants.

VII. CULTURING THE HOST CELLS

The vtransformed host cells are grown in appropriate nutrient medium at optmal conditons.
The DNA gets multplied and expresses itself to form desired product.
Bioreactors are vessels of large volumes ( 100- 1000 litres) in which raw materials are
biologically converted into specifc products.
It provides all the optmal conditons like temperature, p h , substrate, salt , vitamins and
oxygen.
Strred tank bioreactors are commonly used bioreactors. It has following components:
a. An agitator system

b. An oxygen delivery system

c. Foam control system

 d. Temperature control system

e. Ph control system

f. Sampling ports to withdraw cultures periodically.

Sparged strred tank reactor is a strred type reactor in
which air is bubbled.
VIII. EXTRACTION OF DESIRED GENE PRODUCT
When a protein encoding gene is expressed in a heterologous host, it is called a recombinant
protein.
On small scale, the cells are grown on cultures in a laboratory and then the expressed protein is
extracted and purifed by diferent separaton techniques.
On large scale, the cells are grown in a contnuous culture system in which fresh medium is
added from one side to maintain cells in exponental growth phase and the desired protein is
collected from the other side.

IX. DOWNSTREAM PROCESSING

All the processes to which a product is subjected to before being marketed as a fnished
product are called downstream processing.
It includes:
a. Separaton of product from the reactor b. purifcaton of
the product c. formulaton of products with
preservatves d. quality control testng and clinical trials
in case of drugs.
 WORKSHEET
BIOLOGY
GRADE 12
CHAPTER: BIOTECHNOLOGY- PRINCIPLES AND PROCESSES
LEVEL 1
1. What is recombinant DNA?

2. What is the host called that produces a foreign gene product? What is this product
called?

3. Write the two components of the frst artfcial recombinant DNA molecule constructed
by Cohen and Boyer.

4. Why is it essental to have a selectable marker in a cloning vector?

5. Why does DNA move towards the anode in gel electrophoresis?

6. How is the acton of exonuclease diferent from endonuclease?

7. Write the name of the enzymes that are used for isolaton of DNA from bacterial and
fungal cells respectvely.

LEVEL 2

8. Why is origin of replicaton required to facilitate cloning into a vector?

9. DNA being hydrophilic cannot pass through the cell membrane of a host cell. Explain

how does recombinant DNA get introduced into the host cell to transform the later.
10. Write the use of the following in biotechnology:

a. Chilled ethanol b. micro injecton c. bioreactor d. plasmid

11. Write the diference between r DNA and r protein.

12. Explain with the help of a suitable example the naming of a restricton endonuclease.

13. Write the role of Ori and restricton site in a cloning vector p BR322.

14. Explain palindromic sequence with the help of a suitable example

LEVEL 3

I. APPLICATION BASED QUESTIONS:

15. Write any three ways used to introduce a desired DNA segment into a bacterial cell in

recombinant technology.

16. A recombinant DNA is formed when stcky ends of vector DNA and foreign DNA join.

Explain how the stcky ends are formed and joined.

17. How and why is the bacterium Thermus aquatcus employed in recombinant DNA

technology

II. CRITICAL THINKING QUESTION:

18. What would happen when you grow a recombinant in a bioreactor but forget to add

antbiotc to the medium in which the recombinant is growing?

19. Would you like to choose an exonuclease enzyme while producing a recombinant DNA

molecule?

20. You have created a recombinant DNA molecule by ligatng a gene to a plasmid vector.

By mistake, your friend adds exonuclease to the tube containing recombinant DNA. How

will your experiment get afected as you plan to go for transformaton now?