Hari Om!

Chinmaya Vidyalaya,Kattakada
Botany notes
Biotechnology-Principles &processes
1. Two core techniques that enabled birth of modern biotechnology: (2m)
❖ Genetic engineering:
It helped to alter the chemistry of genetic material (DNA and RNA).
Then introduce it into host organisms, to change the phenotype of the host organism.
❖ Maintenance of sterile ambience(free from microbial contamination):
This enables growth of only the desired microbe in large quantities, for the
manufacture of products like antibiotics, vaccines, enzymes, etc.
2..(A) Advantage of asexual reproduction over sexual reproduction: (2m)
➢ Asexual reproduction: preserves genetic material
➢ Sexual reproduction: produces variations – genetic recombination’s,
which are useful to both organism and population
(B) Disadvantage of traditional hybridisation procedures used in plant and animal
breeding: (2 m)
It leads to inclusion and multiplication of undesirable genes along with the
desired genes. This limitation can be overcome by genetic engineering.
(C ) Recombinant DNA technology:
A desirable gene is isolated from source organism, then it is inserted into a vector to
form rDNA, later this rDNA is allowed to multiply in a host
3. First artificial recombinant DNA molecule (2m)
❖ Constructed by Stanley Cohen and Herbert Boyer in 1972
❖ They isolated antibiotic resistance gene from a plasmid using restriction
endonuclease
❖ Using DNA ligase, this gene was inserted into a plasmid of Salmonella typhii
(vector) to form rDNA molecule
❖ Placed rDNA molecule into a host like E.Coli and cloned it
4.Three Basic steps in genetically modifying an organism: (3m)
Identification of DNA with desirable genes
Introduction of the identified desirable DNA into the host
Maintenance of introduced DNA in the host and transfer of the DNA to its
progeny
5.Tools of Recombinant DNA Technology: (2m)
Enzymes – restriction endonucleases, DNA ligase, DNA polymerase.
Vectors- plasmids, bacteriophases, retrovirus, Agrobacterium tumifaciens
Host- bacteria, plant cell, animal cell
6.Nucleases: (2m)
- They cleave DNA or RNA molecule
- They are two types
❖ Exonucleases remove nucleotides from the ends of the DNA
❖ Endonucleases make cuts at specific positions within the DNA molecule.
7.Restriction endonucleases/restriction enzymes /Molecular Scissors: (2m)
a) What are R E and their action
❖ They are a type of endonucleases.
❖ They cleave DNA molecule at specific base sequence known as recognition
sequence.
❖ The recognition sequence possesses palindromic nucleotide sequence.
❖ Palindrome is a sequence of base pairs that reads same on the two strands in
opposite direction i.e. 5’ ->3’ and 3’ ->5’
3’ GAATTC 5’
5’ CTTAAG 3’
❖ They cut the strand of DNA a little away from the centre of the palindrome
sites, but between the same two bases on the opposite strands. This leaves
single stranded portions at the ends called Sticky ends. These ends facilitate
action of DNA ligase in joining foreign gene with vector DNA.
❖ Hind II and ECoRI are the examples of R E
b) Nomenclature of restriction endonucleases:(2m)
Eg: ECoRI is obtained from Escherichia coli RY 13 .
The first letter comes ‘E’ from the genus ‘Escherichia ‘
and the second two letters ‘Co ’from the species ‘coli’of the prokaryotic cell.
4th letter ‘R’is derived from the name of the strain-RY 13
Roman numbers indicate the order in which the enzymes were isolated from
the strains of the bacteria
Note: The first restriction endonuclease was HIND II obtained from Haemophilus
influenza.
It cuts DNA molecule at the recognition sequence having 6bp ( base pairs).
❖ c) Discovery of restriction endonucleases: (2m)
 ❖ -E.Coli restricted the growth of bacteriophage in its body, by producing two
enzymes.
- One enzyme added methyl groups to E. Coli DNA to protect from
restriction enzyme action.
- The other enzyme cut the DNA molecule of virus.
❖ This enzyme was Known as restriction endonuclease

*8.Formation of rDNA

9.RECOMBINANT DNA TECHNOLOGY -5 m/3m

Steps:
▪ Isolation of source DNA from the selected plant/animal/bacteria.
▪ Isolation of desirable gene from the source DNA. Using specific R E, the DNA
is cut and the desirable gene is obtained. This gene has sticky end
▪ Cut and open vector DNA (plasmid DNA) using the same R E
▪ Insertion of foreign gene into the plasmid with the help of DNA ligase to form
recombinant DNA( rDNA) or hybrid plasmid
▪ Insertion of rDNA into host cell: A suitable host cell like E. Coli is taken and
into this hybrid DNA is inserted. The bacteria with plasmid DNA is known as
transformant
 NOTE** Multiplication of gene : when the bacteria reproduce, the hybrid plasmid
present in it also get replicated . So, a large no. of copies of hybrid plasmid is raised.
Later using the R E the gene is isolated from the plasmid and it is used for gene
therapy
* expression of gene: In the bacteria the foreign gene is allowed to express by
transcription and translation. The product is collected from the bacteria and
used for therapeutic purposes.

10.Diagram

(text book diagram must be drawn )

11.Separation and Isolation of DNA fragments by Gel electrophoresis: 3m
Technique of separating DNA fragments based on their lengths by Agarose Gel
Electrophoresis,
STEPS
❖ Cut DNA by restriction endonuclease. It results in formation of DNA fragments
of varied lengths
❖ Load the wells of agarose gel with DNA solution ( containing DNA fragments)
and apply electric field
❖ DNA being –vely charged moves towards +ve electrode( anode).
❖ During this movement the shorter fragments move faster through the pores of
the gel and settle at the bottom. Whereas larger fragments settle at the top of
the gel Stain the gel with Ethidium bromide. Later expose it to UV radiation
❖ Now DNA fragments appear as bright orange coloured bands.
❖ Cut and separate DNA fragments from the agarose gel. This step is known as
Elution.

12.Cloning Vectors (Vehicles for Cloning):

Cloning Vector is DNA or RNA molecule, into which a foreign gene can be ligated
and cloned in a suitable host .
Types of cloning vectors:
a)plasmid 2m
❖ It is extra chromosomal, double stranded circular DNA molecule
❖ It can independently replicate
❖ It provides antibiotic resistance to the bacteria
❖ there can be several plasmids in a bacteria. Therefore they have high copy
no. per cell
b) Bacteriophage 2 m
❖ -Viruses that infect bacteria are called bacteriophases.
❖ A bacteria may possess several bacteriophases. Therefore they have high
copy No per cell.
Both plasmids and bacteriophases are used to clone genes in E coli
c) Agrobacteriumtumifaciens: 2m/3m
➢ It is a vector for cloning genes in plants
 ➢ Present in the soil and infects several dicot plants and causes a
disease called crown gall tumor.
➢ Possess Ti plasmid( tumor inducing plasmid) which delivers a
piece of DNA known as T- DNA into the plant cells when the
bacteria infects them.
➢ T- DNA transforms plant cells into a tumor. So that more
chemicals required for the growth of the pathogen can be
synthesized.
➢ Ti plasmid can be modified into a cloning vector by disarming it
(unabling it to cause disease). The disarmed bacteria is used for
delivering desirable genes into plant cells
d) Retrovirus:
❖ They contain RNA as the genetic material
❖ They have the ability to transform normal cells into tumor cells.
❖ They can be disarmed and used to deliver genes into animal cells.
Copy number: No of copies of vectors present in a cell
12. Salient features of a Vector:
-Ori
-Selectable markers
-cloning sites
a)origin of replication (ori). 2m
➢ It is a base sequence on vector from where replication starts.
So foreign gene linked to this sequence gets replicated.
➢ It also controls the copy No of linked DNA/ foreign gene
b) selectable marker : 2m
❖ Antibiotic resistance genes such as tetR and ampR act as selectable markers
for E coli.
❖ These help in identifying and eliminating non-transformants and selectively
permitting the growth of transformants.
❖ Transformation is a process by which rDNA is introduced into the host cells
for cloning a foreign gene.
❖ Host cell with rDNA molecule is called transformant
c) Cloning sites: 2m
❖ It is a recognition site/ restriction site present on vector, at which a specific R
E can cut open the plasmid for inserting a foreign gene
❖ Single recognition site is preferred. If there are more than one recognition
sites, the RE will generate several fragments which will complicate the gene
cloning.
 ❖ When foreign DNA is introduced into the coding sequence of the antibiotic
resistant gene , there is insertional inactivation of the gene.So the transformed
bacteria loses antibiotic resistance .This helps in selecting recombinants
selected non-recombinants.
13. Structure of vector pBR322: 3m 5m

➢ It is a E coli cloning vector.It has ORI, selectable markers and cloning

sites/restriction sites
➢ Ori – sequence from where replication starts . Any foreign gene linked to it
can be made to replicate within the host (E coli).It also controls the copy
No of foreign gene
➢ Selectable markers- tetR and ampR are the genes encoding for antibiotic
resistance to tetracycline and ampicillin respectively . If a foreign gene is
inserted at Bam HI site of tetR, the plasmid will loose the tetracycline
resistance. The markers help in identifying and eliminating non-
transformants and selectively permitting the growth of transformants.
➢ Rop- codes for proteins involved in the replication of the plasmid
➢ Restriction site:. These are the sites on the plasmid at which a specific R E
can cut open the plasmid for inserting a foreign gene.
➢ Hind III , Eco RI , Bam HI , Sal I, Pvu II , Cla I. are restriction enzymes,
they can cut and open vector at specific sites
14. Selection of transformants using antibiotic resistance gene as cloning site
3m
vector pBR322 has two antibiotic resistance genes- tetR and ampR. They provide
resistance to antibiotics tetracycline and ampicillin respectively.
a) If a foreign gene is inserted at Bam HI site of tetR, the plasmid will lose the
tetracycline resistance due to insertional inactivation. But they still have ampicillin
resistance
b) The bacteria possessing this recombinant plasmid are called transformants/
recombinants. They can grow in ampicillin containing medium but not on tetracycline
containing medium .
c) Whereas Non-transformants will grow on the medium containing both the
antibiotics
d) In this method one of the antibiotic resitance genes helped in identifying and
eliminating non-transformants and selectively permitting the growth of transformants
by a plating method.
Dis advantage of this method:
Selection of transformants from non-transformants using antibiotic resistance gene is
cumbersome procedure because it requires simultaneous plating on two plates
having different antibiotics

15. Selection of recombinants from non-recombinants on the basis of

colouring reaction. (Blue-White screening) 3m
In this method lac Z gene producing beta galactosidase is used as a marker, to
select transformants from non-transformants.
a) In non- transformants: (blue in colour)
lac Z gene present in plasmid vector produces enzyme beta-galactosidase.
This enzyme cleaves a chromogenic substrate into a blue coloured product,
Blue colour bacterial colonies appear
b) In Transformants: ( white in colour)
▪ Insert a foreign DNA into the coding sequence of lac Z
▪ Insertional inactivation of the gene occurs . So, lac Z gene does not produce
the enzyme beta-galactosidase .
▪ In the absence of the enzyme the bacteria do not produce blue colour
▪ They appear as white coloured colonies.
c) By this way, we can differentiate transformants (white colour) from
nontransformants (blue colour) colonies
NOTE: Chromogenic substrate: X-gal is hydrolyzed to form 5-bromo-4-chloro-
indoxyl, in presence of beta galactosidase .
16. Methods to introduce rDNA into host cells:
a) Chemical method in bacterial cells: 2m
❖ DNA being a hydrophilic molecule, cannot pass through cell membranes.
Hence the bacteria
❖ DNA being a hydrophilic molecule, cannot pass through cell membranes.
Hence the bacteria should be made competent to accept the DNA molecule.
 ❖ Bacterial cell is treated with divalent cation such as calcium ions to increase
pore size in the cell wall.
❖ The bacterial cells are then incubated with rDNA on ice followed by placing
them briefly at 42oC and then putting them back on ice. This is called Heat
Shock treatme
❖ This enables the bacteria to take up the recombinant DNA
b) Physical methods- for animal and plant cells
i) microinjection method in animal cells: 2 m
rDNA is directly injected into the nucleus of the animal cell through micro
pippetes
ii) Biolistics / Gene gun method in plants: 1m
In this method, microscopic particles of gold / tungsten are coated with the
DNA of interest and bombarded onto cells.
c) Disarmed Pathogen Vectors:
such as Agrobacterium tumefaciens , Retro virus, which when allowed to infect the
cell, transfer the recombinant DNA into the host.
17. Isolation of genetic material (DNA): 2m
DNA should be isolated in pure form, from bactrerial/fungal/plant cells.
❖ Using the enzymes Lysozyme (bacteria), cellulase (plant cells), chitinase
(fungus), the cells are broken and nuclear contents are separated.
❖ Later RNA and proteins are removed by ribonuclease and proteases.
❖ Using chilled ethanol the pure DNA is obtained
18. Amplification of Gene of Interest using PCR: 3m/5m
➢ PCR stands for Polymerase Chain Reaction. In this reaction, multiple
copies of the gene of interest are synthesized in vitro using two sets of
primers and the enzyme DNA polymerase.

There are 3 steps in PCR cycle

a) Denaturation:
Double stranded DNA is denatured by breaking H-bonds to get two DNA templates
b) Annealing:
- Primers are chemically synthesized oligo-nucleotides and they are
complementary to DNA templates
- Primers are added on the templates in 5’ 3’
c) Extension:
 • To the primer deoxyribonucleotides are added at 3’ end in presence of
Taq polymerase
• In this way we get 2 DNA molecules
• All these steps are repeated many times to get several copies of the
desired DNA segment.

➢ Taq DNA Polymerase : 2m

➢ The DNA polymerase used in PCR reaction is Taq polymerase.
➢ It is isolated from a bacterium, Thermus aquaticus.
➢ It is thermostable and can remain active at high temperature induced
during denaturation of DNA.
19. a) simple stirred-tank bioreactor:
o Bioreactor is a large cylindrical vessel in which raw materials are
converted into useful products and enzymes, with the help of microbes.
o The reactor provides optimal growth conditions of pH, temp, substrate
and other micronutrients
o Stirrer facilitates even mixing and oxygen availability throughout the
bioreactor. Alternatively air can be bubbled through the reactor.
o It has an agitator system, an oxygen delivery system and a foam
control system, as well as temperature and pH control systems
o Through the sampling ports, small volumes of the culture can be
withdrawn periodically.
b) Sparged stirred tank bioreactor: sterile air bubbles are sparged , to increase the
surface area for oxygen transfer
20.Downstream Processing: 2 m
The raw product producedin the bioreactor is separatedand purified first and
thereafter, if required preservatives are added and marketed
Recombinant proteins: 2m
Foreign gene present in rDNA molecule expresses in heterologous host ( transgenic
organism) to produce desired proteins. These proteins are known as recombinant
protein