BIOTECHNOLOGY PRINCIPLES AND PROCESSES

Biotechnology

Bio-life, technology

When we use technology in living world and to

produce products for the welfare of mankind

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 Principles of biotechnology

Genetic engineering Bioprocessing

Sterile conditions are maintained to

Techniques that help to change the DNA
ensure the growth of only desired
or RNA of an organism
microbes

It ensures no contamination by any

The host becomes phenotypically
other microbes. Thus, protecting the
modified
product

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 OVERVIEW OF CHAPTER
1. Collection of sample [Genetic material]

2. Isolation of DNA

3. Fragments of DNA

(GENE)

‘Restriction enzyme’

‘Also called as called molecular scissor’

4. Separation of DNA fragments using "Gel electrophoresis”

5. Elution Extraction of DNA fragments from gel.

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 6. Desired gene

(Desired DNA)

7.

(Desired DNA) (Host cell)

8.Gene taxi Vector DNA

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 9. Transformation:- Introduction of rDNA into host cell

Non transformed Non-recombinant + Transformed Recombinant DNA + Transformed

CASE I CASE II CASE III

10.
Selection/screening
Selectable markers

Antibiotic
Chromogenic enzyme
resistant gena

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 11. Bioreactors/ fermenters.

Large Scale production

12. Downstream processing

Separation and purification of product.

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 1. Isolation of DNA
SPOOLING [DNA chilled ethanol precipitation of DNA strands]

Cell memb → lipid bilayer→ lipase

Plant cell Bacterial cell Fungi cell

Cell wall Cell wall Cell wall

Proteins→proteases
Cellulose enzyme Peptidoglycan enzyme Chitin enzyme

RNA →RNase cellulase lysozyme chitinase

NOTE : we do not use ‘DNase’

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 2. Fragmentation of DNA
WHY? Length of DNA- 2.2 m long
[Desired gene]
❏ History: 1963- hamilton Smith and his colleagues discovered isolated first
‘Restriction Enzyme’
● How and Where?: Hind-II was the first R.E. to be isolated from a bacteria
Causative agent for Pneumonia haemophilus influenzae
➢ Bacteriophage- Virus Infect bacteria

Virus will transfer its genetic material into bacteria

R.E. Restriction enzyme ‘present in bacterial cytoplasm,

will not restrict the growth of virus’
Two enzymes identified
DNA Methylase →adds methyl group to bacterial
genetic material such R.E. cannot act.
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 ➢ R.E. are always isolated from prokaryotic cells only.
➢ More than 900 R.E. from over 230 strand of bacteria

❏ Nomenclature: genus E CO R E order of isolation

Escherichia species strain first

coli RYI 3
❏ Class of enzyme:

Nucleases

Exonucleases Endonucleases

● 3’A TGCATG C–5’ ● 3’ATGCA TGC–5’

● Remove nucleotides from ● Break phosphodiester linkage within the
nucleic acids
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practice end of Nucleic
material acid
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 ❏ Properties of R.E

1.Specific enzyme → Breaks phosphodiester bond Slightly away from the restriction site.

2.ATP dependent enzyme

3.Palindromic sequence 4bp,6bp

(1) MADAM (2) NITIN

MADAM NITIN

Ex (1) 3’—GATG—5’ (2) 5′—GAATTC—3′

5’—CTAC—3’ 3′—CTTAAG—5′
→Not a palindromic seq. —5′
→Palindromic sequence
(3) 5'-GGG CCC-3' (4) 5'-TTTATA-3’
3'-CCC GGG-5' 3'-AAATAT-5’
→Palindromic seq. →Not a palindromic sequence
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 ❏ Mechanism of fragment production
Sticky ends/overhanging/ Staggering ends Blunt ends/flush ends

Eg. ECORV, Hind III, SmaI

Eg. ECORI, CIaI, Bam HI,

SalI, PvuI, Ps+I, Hind III

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 GEL ELECTROPHORESIS
Agarose Gel → Extracted from seaweed
→ Mucopolysaccharide
→ Producer sieve effect

❏ Principle
● DNA fragments move according to size
of the fragment. They move from
cathode (-) to Anode (+)
● smaller the fragment-farther It moves

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 ❏ ELUTION (Et Br)→Ethidium Bromide-‘dye’
UV radiations
DNA bands appear orange in color
Separate DNA fragment from the Gel- ‘Elution’

→Desired DNA fragment

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 4. Formation of rDNA

❏ VECTOR
● Is a sequence of DNA which is
autonomously tepliageng and acts as a
vehicle to carry desired DNA

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 4. Formation of rDNA ➢ Properties of vector
● Ori [origin of replication]:-
a.Allows vector DNA to start self
replication
b.Copy Number: No of copies made by
vector DNA inside a host cell [more the
copy number better is vector DNA]

● Selectable markers:-
This is a sequence of vector DNA which allows
to select Non-transformants from transformants.
e.g., 1.Antibiotic resistant genes
→TetR
→AmpR
→KanR - kanamyan resistant gene
2.chromogenic enzyme →colour by product
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 ● Size of vector:-

● Smaller the vector better is the

transformation efficiency

● Cloning site:-
● This refers to the site of restriction
enzyme present in the vector DNA

● Preferably, for each R.E one site

should be present in vector DNA

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 ➢ Examples of vector DNA
a.PLASMID:-

→Extra-chromosomal, self-replicating, circular, dsDNA, without histone proteins.

→Found in prokaryotic cells.
→Has sequences of virulence/Resistance
b.PBR322:-
P-plasmid
B-boliver
R-rodriguez
322-attempt

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 c.PUC18 d.Ti plasmid [Tumour inducing plasmid]

[only for reference]

T-DNA [Transfer DNA
P-plasmid

UC-university of california

18- attempt (order of development)

Vir genes
Multiple cloning site
(MCS)

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 ● Tumour inducing plasmid
➔ T-DNA/ Transfer DNA
Producer rapid
cytokinin cell
Vir genes to help
+ auxin division
T-DNA transfer Wounded plant
into plant genome
Crown gall
disease Agrobacterium
T-DNA seq. tumefaciens
Gets transferred into plant enters into
genome enters Ti plasmid plant
into plant cell
● Recombinant DNA
Stanley cohen & herbert boyer, 1972

Salmonella typhi bacteria was used to make first rDNA

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 5. Transformation
→ Is a process in which we transfer rDNA into host cell

Steps:- 1) competency 2) transfer

a. Competency
Treats with divalent cation
(ca2+, Mg2+) Solution

No. of pores increases

within the membrane.

↑se probability of
transformation
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 b. Methods for transformation

i. Bacterial cell → heat shock method ● Electroporation

Divalent cation
42℃
Ice

Host cell 42℃

Transformation occurs
1.Divalent Ion solution
2.High voltage electrical impulse of tex
fixed interval of time
3.Transformation occurs

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 ii. Plant cell
Gene gun/Biolistic

Bullet

Tungsten [Non- reactive

Gold element

Gene Gun Plant cell

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 iii. Animal cell

Desired DNA

Microinjection

Directly inject rDNA

into host cell.

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 6. Selection/ Screening

No change in structure of vector DNA

Bacterial cell

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 7. Bioreactors/ fermenters

→100-1000 litre Capacity

→for large scale production of desired product.

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 Conditions:-
Sterile condition →contamination free
Inlet port →we add sample into bioreactor
Outlet port →we take out our Product
Optimum nutrition →for growth of host cell
Optimum temperature →for max growth of host cell
Aerators →for proper supply of O2 as to avoid
anaerobic respiration
Antifoam agents →to avoid formation of foam.
Coolants →which keep temperature down.

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 Types of bioreactors

Simple stirred Spargered

Agitator

To allow o2 and nutrients

to reach every cell

Sparger
Breaks the air into
small bubble of o2
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 ● Types of Batches
1. Closed batch

• Nutrition Added one time

• Host cells
• Product – Extracted once

2. Open batch/continuous
• Continuously nutrition is added
• Continuously sample is added
• Continuously product is extracted
• In this host cells are maintained in exponential / log phase
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 3. Fed batch
• Nutrition and sample are added at once
• products are extracted on a regular Interval of time

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 8. Downstream processing

• Separation and purification of

product from bioreactor
• Centrifugation
• Chromatography

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 9. Upstream processing

• Expression of the product

PCR-POLYMERASE CHAIN REACTION
• Discoverer:- KARY MULLIS
• Principle:- In-vitro amplification of desired
DNA sequence.
Outside the cell

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 • process:-

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 • Material required:-
1) dNTPs-deoxynucleoside triphosphate
2) Primers/Annealers
Oligonucleotide sequences
3) Taq polymerase = 100-110OC
Thermostable
Thermus aquaticus [Bacteria]
4) Mg² + →co-factor for Taq polymerase

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 → Formula:-
n = no. of PCR cycles
Number of DNA copies = 2n
2n = 24 = 16
16 x 2 = 32

•Application of PCR :-

1. Forensic
2. Diagnosis
3. Evolutionary study
4. Detect mutation
5. DNA fingerprinting

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