- 1. BIOTECHNOLOGY- principles and processes
2. Introduction to biotechnology Definition: Biotechnology is
the use of living systems and organisms to develop or make useful
products, or "any technological application that uses biological
systems, living organisms or derivatives thereof, to make or modify
products or processes for specific use European Federation of
Biotechnology (EFB) has defined biotechnology as The integration of
natural science and organisms, cells, parts thereof, and molecular
analogues for products and services. 3. Oldest form of
biotechnology 4. Application of fermentation in production of wine
and other alcoholic beverages is also a biotechnological technique
5. Principles of biotechnology Two important technique which enable
development of modern biotechnology: 1. Alteration of chemistry of
DNA & RNA to introduce into host organism to change phenotype
of host- Genetic engineering 2. Maintenance of sterile ambience to
enable growth of desired microbe/ eukaryotic cell in large
quantities for manufacture of biotechnological products like
vaccine, enzymes, beverages, drugs etc.- Chemical engineering 6.
Asexual reproduction in organism preserves genetic information
Sexual reproduction (hybridization) leads to variation- includes
undesirable gene with desirable gene Genetic engineering- isolate
& introduce only one or set of desirable genes without
introducing undesirable genes in target organism Techniques of
genetic engineering- creation of recombinant DNA, use of gene
cloning & gene transfer to host Recombinant DNA (rDNA)/ alien
DNA- cannot multiply itself until integrated in host genome When
inherited in host DNA- ability to replicate due to origin of
replication (host DNA)- initiates replication Alien DNA- linked
with host DNA replicates & multiply itself along with host DNA-
Cloning 7. Gene transfer to host require Vector Commonly used
vector- Plasmid (small, circular, double stranded, self replicating
extra chromosomal material of bacteria) First recombinant DNA was
constructed- Stanley Cohen & Herbert Boyer (1972) by linking
gene encoding for antibiotic resistance with plasmid of Salmonella
typhimurium Isolation of desirable gene (antibiotic resistant)-
cutting out piece of DNA from a plasmid responsible of antibiotic
resistance which involve molecular scissors- restriction enzymes
Desirable gene/ alien DNA linked with plasmid (vector) to transfer
into host organism Linking of DNA involves DNA ligase- acts on cut
DNA molecules & join their ends- new combination circular
autonomously replicating DNA created in vitro- Recombinant DNA 8.
rDNA transferred into Escherichia coli- DNA replicate using hosts
DNA polymerase- multiple copies of antibiotic resistant gene-
Cloning & E. coli will become antibiotic resistant Basic steps
in genetically modifying organism: 1. Identification of DNA with
desirable gene 2. Introduction of the identified DNA into host 3.
Maintenance of introduced DNA in the host and transfer of the DNA
into its progeny 9. Steps to genetically modify organisms 10. Basic
steps involved in process Isolating Genomic DNA Fragmenting DNA
Screening DNA fragments Insertion of DNA in vector Introducing DNA
in host Culturing the cells Transformation of host cell 11. Basic
steps involved in process Isolating genomic DNA Isolating genomic
DNA from the donor. Fragmenting this DNA Fragmenting this DNA using
molecular scissors. 12. Basic steps involved in process Insertion
of DNA in a vector Screening the fragments Screening the fragments
for a desired gene. Inserting the fragments with the desired gene
in a cloning vector. 13. Basic steps involved in process
Introducing in Host Culturing the cells Transformation of host cell
Introducing the recombinant vector into a competent host cell
Culturing these cells to obtain multiple copies or clones of
desired DNA fragments Using these copies to transform suitable host
cells so as to express the desired gene. 14. Biotechnology led to
production of many products and provides many services for human
welfare. 15. Tools od recombinant dna technology Recombinant DNA
technology is accomplished with tools: 1. Restriction Enzymes:
Restriction endonuclease & Restriction exonuclease 2.
Polymerase enzymes 3. Ligase 4. Vectors 5. Host 16. Restriction
enzymes Enzymes which is used to make cut in DNA in recombinant DNA
technology 1963 two enzymes were isolated from Escherichia coli
which restricts the growth of bacteriophage One enzyme added methyl
group to DNA & other cut DNA and was named Restriction
endonuclease Function of Restriction endonuclease depend on
specific DNA nucleotide sequence First endonuclease isolated was
Hind II; studies proved that Hind II always cut DNA at particular
point by recognizing a specific sequence of six base pairs-
recognition sequence of enzyme 900 restriction enzymes isolated
from 230 strains of bacteria 17. Restriction enzymes belongs to
nuclease class of enzymes. Its of two types: 1. Restriction
exonuclease removes nucleotides from the end of DNA 2. Restriction
endonuclease cut at specific positions within DNA While naming
restriction enzymes- first letter represent genomic name &
second two letter represent species name of the prokaryotic cell
from which the enzyme was isolated Eg.- EcoRI- isolated from
Escherichia coli RY 13; R- name of strain, I order in which the
enzyme were isolated from that strain of bacteria 18. How
restriction endonuclease act?? R.E inspects DNA sequence to find
its specific recognition site to bind & cut specifically each
of the two strand of the double helix at their sugar phosphate
backbone R.E recognizes specific palindromic nucleotide sequence in
DNA Palindrome in DNA- sequence of base pairs that reads the same
on two strands when orientation of reading is kept the same, i.e.,
5 3 in both strands 19. R. E cut the strand away from the center of
palindrome sites but between same two bases on the opposite strand
leading to formation of single stranded portion at the ends- Sticky
ends Sticky end- hydrogen bond with their complementary cut
counterparts- DNA ligase 20. Application of restriction
endonuclease Used in the field of genetic engineering to form
recombinant molecules of DNA- composition of DNA from different
sources Same R.E (vector & source DNA)- same kind of sticky
ends which can be joined by DNA ligase 21. Diagrammatic
representation of recombinant dna technology 22. Separation and
isolation of dna fragments Action of R. E result fragments of DNA-
separated by Gel Electrophoresis Depends on negative nature of DNA
External electrical field is applied to the medium- DNA move to
anode Medium/ matrix used is agarose gel- natural polymer,
extracted from sea weed Medium- acts as sieve & separate DNA
according to their sizes; smaller fragments- farther Separated DNA-
stained with Ethidium bromide & exposed to UV radiations-
bright orange coloured bands of DNA in gel Separated bands of DNA-
cut out from agarose gel & extracted from gel piece- Elution
Purified DNA fragments- constructing recombinant DNA by joining
them with cloning vector 23. Electrophoresis- separation of DNA
fragments based on size Staining- Ethidium bromide Exposure to UV
radiation Cutting out DNA fragments 24. Cloning vectors A cloning
vector is a small piece of DNA, taken from a virus or a plasmid of
bacteria, that can be stably maintained in an organism, and into
which a foreign DNA fragment can be inserted for cloning purposes
Common vectors- Plasmids & Bacteriophage Salient features of
cloning vectors: 1) Ability to replicate within bacterial cell
independent of chromosomal DNA 2) High copy number per cell;
Plasmids- 15- 100 copies per cell 3) Alien DNA when integrated with
vector can multiply equal to copy number of vector 4) Vectors can
be engineered for easy linking of foreign DNA & selection of
recombinant from non- recombinants 25. Features to facilitate
cloning into vectors Features required to facilitate cloning into
vectors: 1. Origin of replication (ori) 2. Selectable marker 3.
Cloning sites 4. Vectors for cloning genes in plants & animals
26. 1. Origin of replication (ori): Sequence on DNA where
replication starts/ initiates Alien DNA when linked to it-
replicate within host cell Regulates & control copy number of
linked DNA/ alien DNA/ target DNA If multiple copies of target DNA
required, then target DNA be cloned in a vector whose origin of
replication supports high number 2. Selectable marker: Identify
& eliminate non- transformants from transformants &
selectively permit growth of transformants Transformation-
procedure through which rRNA introduced into host bacterium- change
in phenotype Eg.- E. coil- resistance against ampicillin,
chloramphenicol, tetracycline or kanamycin- selectable markers
which is lacked in normal E. coli 27. 3. Cloning sites Region in
the vector where ligation of target DNA takes place Created by
commonly used specific R. E at the recognition sites/ restriction
sites within a vector where ligation of alien DNA takes place
pBR322- genetically engineered plasmid; widely used E. coli cloning
vectors; the ampR gene- encodes for ampicillin resistance protein,
and the tetR gene- encodes for tetracycline resistance protein;
have specific restriction sites for different Restriction
endonucleases (BamH I, Sal I, Hind III, Cla I, EcoR I, Pvu I Pvu
II, Pst I) Presence of more than one recognition site within vector
will complicate gene cloning Ligation of alien DNA- restriction
site present in one of two antibiotic resistance genes Inactivate
the gene- enable to choose transformants from non transformants 28.
If alien DNA is ligated at Bam HI site in tetracycline resistance
gene- recombinant DNA loose its resistance against Tetracycline
(transformants/ recombinants) Transformants can be selected from
non transformants- plating bacteria on ampicillin containing
medium- bacteria grows Transformants growing in ampicillin
transferred to tetracycline medium- transformants cannot grow &
non recombinants grow- due to gene gets inactivated- insertion 29.
Selection of recombinants due inactivation of antibiotics-
cumbersome process due to simultaneous plating on two different
antibiotics Alternative selectable marker developed- differentiates
to produce colour in the presence of chromogenic substance
Recombinant DNA sequence is inserted within coding sequence of
enzyme - galactosidase- inactivation of enzyme- insertional
inactivation Presence of chromogenic substrate (- galactosidase)-
blue coloured colonies (bacteria with plasmid with no insert)
Presence of insert- inactivation of - galactosidase- colonies do
not produce any colour- identify recombinant colony 30. 4. Vectors
for cloning genes in plants & animals: Bacteria & virus-
transform eukaryotic cell by delivering genes Agrobacterium
tumifaciens (pathogen of dicot plants)- deliver a DNA piece- T- DNA
& transform normal cell to tumor which produces chemicals
required by the pathogen Retrovirus- transforms normal animal cell
to cancerous cell An understanding of mechanism of delivering genes
by pathogens to their host can help to understand the tools of
pathogen & can be used to deliver genes of interest in human
Tumor inducing plasmid (Ti) of Agrobacterium tumifaciens- modified
into cloning vector- non pathogenic to plants & have ability to
deliver genes of interest to variety of plants Retrovirus-
disarmed; delivers genes of interest in animals When gene of
interest is ligated into a suitable vector- transferred into
suitable host (bacteria, plant or animal)- multiply 31. Plant
vector- Ti plasmid 32. Animal vector 33. Competent host (for
transformation with rdna) rDNA should enter cell to transform cells
But DNA cannot enter cell in a medium without treatment-
hydrophilic Host should be made competent to take up DNA (vector)
Different techniques to make host competent: 1.Chemical Treatment:
Treating hosts (bacteria) with specific concentration of a divalent
cation (Ca)- increases efficiency & make DNA enter through cell
wall of host Heat shock (sudden change in temperature)- mixture of
rDNA & host cells Incubation of host & rDNA on ice &
then placing them at 42C & then back to ice Enable bacteria to
take up rDNA 34. 2. Microinjection: rDNA directly injected into
nucleus of host cell Common for animal cell 3. Biolistics &
gene gun: Host cell bombarded with high velocity micro- particles
of gold or tungsten Micro- particles are coated with recombinant
DNA Used for plant cell 4. Disarming pathogen: Pathogenicity of
vector is removed- disarmed pathogens Disarmed pathogen vector-
allowed to infect host & transfer r DNA 35. Techniques to
introduce recombinant dna 36. Process of recombinant DNA technology
Recombinant DNA technology involves following steps: 1. Isolation
of genetic material DNA 2. Fragmentation of DNA by restriction
endonuclease 3. Isolation of desired DNA fragments 4. Ligation of
the DNA fragments into a vector 5. Transferring rDNA into host 6.
Culturing host cells in a medium at large scale 7. Extraction of
desired product 37. 1. Isolation of Genetic material (DNA) Most of
eukaryotic cells have DNA as genetic material cells To form rDNA,
the DNA should be pure, i.e., free from all biomolecules DNA-
located inside nucleus enclosed by plasma membrane, genes
interwined with histone protein Cell have to break open to release
DNA & other macro molecules (RNA, proteins, polysaccharides
& lipids) Cells are lysed by lytic enzymes: lysozyme
(bacteria), cellulase (plant cell), chitinase (fungus)
Macromolecules- treated with specific enzymes to remove; RNA-
ribonuclease, Proteins- Protease, Purified DNA precipitates with
addition of Ethanol- appear as collection of fine threads in
suspension 38. Steps for dna extraction 39. 2. Cutting of DNA at
specific location Purified DNA (source DNA/ desirable gene DNA
& vector DNA) incubated with specified restriction enzymes for
the process- Digestion at optimal condition Progression of
restriction enzyme digestion is checked- agarose gel
electrophoresis (source DNA & vector DNA) DNA- negatively
charged so move towards positive electrode (anode) Once gene of
interest & cut vector with space for gene of interest is
isolated, DNA ligase is added This step is a preparation of
recombinant DNA 40. 3. Amplification of Gene of Interest using PCR
PCR (Polymerase Chain Reaction)- technology used to amplify a
single or a few copies of a piece of DNA to generate thousands to
millions of copies of a particular DNA sequence in vitro.
Components required- Primers (small chemically synthesized
oligonucleotides that are complementary to the regions of DNA),
Nucleotides & DNA polymerase- incubated together Steps in PCR
1. Denaturation of double stranded DNA by heating- single stranded
DNA 2. Primer is added- binds to complementary region- Annealing 3.
DNA polymerase- polymerization with nucleotides in medium; genomic
DNA act as template 4. Replication repeated many times- 1 billion
copies of desirable DNA- Amplification & involve thermostable
DNA polymerase- Thermus aquaticans- to facilitate replication even
at high temperature (denaturation) 41. Polymerase chain reaction
42. 4. Insertion of Recombinant DNA into Host cell/ organism
Insertion of rDNA possible by making host competent Achieved by-
Chemical treatment, microinjection, biolistic & gene gun method
or disarming pathogen Phenotype of organism alters when rDNA is
transferred to host Eg.- rDNA with gene resistance against
antibiotic Ampicillin transferred to E. coli (host)- phenotype
alters; resistant against ampicillin- transformants When plated on
agar plate with ampicillin- transformed will grow (change in
phenotype) & non- transformed- die Presence of ampicillin
resistance gene- select transformed cells & considered as
Selectable marker 43. 5. Obtaining the Foreign gene product Alien
DNA binds with cloning vector- rDNA, transferred to suitable host
(bacteria, plant or animal cell) rDNA- expresses itself (target
protein) under appropriate optimal conditions & after cloning
of rDNA to particular number of copies Target protein- produce in
large scale In heterologous host if protein encoding gene is
expressed- recombinant protein Heterologous host- gene or gene
fragment that does not naturally present in host & the gene
expresses itself (recombinant protein) Cells which harbor cloned
genes of interest- grown in laboratory in small scale Culture of
dividing host- used to extract desired protein & then purified
by different separation techniques 44. In large scale cells can be
cultured in continuous culture system Here the used medium is
drained out form one side & fresh medium added from other to
maintain the cells- physiologically most active log/ exponential
phase This culture method produce a larger biomass leading to
higher yields of desired protein Biomass- biological material
derived from living, or recently living organisms To produce large
quantities of products- Bioreactors are employed (100- 1000
liters)- large volumes of culture is processed Bioreactors- vessels
in which raw materials (recombinant organism like microbial plant,
animal or human cell & culture medium with ambient condition)-
biologically converted into specific products (protein/ enzymes)
Optimal growth is achieved with growth conditions- temperature, pH,
substrate, salts, vitamins & oxygen 45. Structure of
Bioreactors: Commonly used- stirring type Cylindrical & with
curved base to facilitate mixing contents Equipped with stirrer-
even mixing & oxygen availability throughout reactor Oxygen
delivery system- bubble air through reactor Agitator- thorough
mixing of contents Also have foam control system, temperature
control system, pH control system & sampling port Sampling
port- withdraw small volumes of culture periodically to check the
progression 46. bioreactors 47. 6. Downstream Processing Downstream
processing refers to the recovery and purification of biosynthetic
products, particularly pharmaceuticals, from natural sources such
as animal or plant tissue or fermentation broth Products obtained
from bioreactors- subjected to series of process before marketing
as a finished product Process include separation & purification
of biological products Products are formulated with suitable
preservatives & then should undergo thorough clinical trials
like drugs Strict quality control testing is required for the final
biological products
