Biotechnology

principles and concepts

DR. PANKAJ KUMAR RAMTEKE

ASSISTANT PROFFESOR

DEPARTMENT OF ZOOLOGY

TGM RAMTEK

RTM NAGPUR UNIVERSITY

Introduction tobiotechnology

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”.

Oldest form of biotechnology

Application of

fermentation in

production of wine and

other alcoholic

beverages is also a

biotechnological

technique

Principles ofbiotechnology

• 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

• 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

• 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

• rDNA transferred into Escherichia coli- DNA replicate using

host’s 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

Steps to genetically modifyorganisms

Basic steps involved inprocess

Isolating Genomic DNA

Fragmenting DNA

ScreeningDNA

fragments

Insertion of DNA in vector

Introducing DNA in host

Culturing the cells

Transformation of host cell

Basic steps involved inprocess

Isolating genomic

DNA

Isolating

genomic DNA

from the donor.

Fragmenting this DNA

Fragmenting

this DNA using

molecular

scissors.

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’.

Basic steps involved in process

in Host

Culturingthe cells

Transformation of host cell

Introducing the recombinantIntroducing 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.

Biotechnology led to production of many products and

provides many services for human welfare.

Tools od recombinant dnatechnology

• Recombinant DNA technology is accomplished with tools:

1. Restriction Enzymes: Restriction endonuclease & Restriction

exonuclease

2. Polymerase enzymes

3. Ligase

4. Vectors

5. Host

Restrictionenzymes

• Enzymes which is used to make cut in DNAin

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

• 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

How restriction endonucleaseact??

• 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

• 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

Application of restrictionendonuclease

• 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

Diagrammatic representation of recombinant dnatechnology

Separation and isolation of dnafragments

• 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 DNAby

joining them with cloning vector

Electrophoresis- separation of

DNA fragments based on size

Staining- Ethidium bromide

Exposure to UV radiation

Cutting out DNAfragments

Cloningvectors

• 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

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

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 DNAbe

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

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

• 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

• 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 β- galactosidase- inactivation of enzyme-

insertional inactivation

• Presence of chromogenic substrate (β- galactosidase)- blue

coloured colonies (bacteria with plasmid with no insert)

• Presence of insert- inactivation of β- galactosidase- colonies do

not produce any colour- identify recombinant colony

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

Plant vector-Ti plasmid

Animalvector

Competent host (for transformation withrdna)

• 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 42ºC &

then back to ice

• Enable bacteria to take up rDNA

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

Techniques to introduce recombinantdna

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

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

Steps for dna extraction

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- agarosegel

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

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)

Polymerase chainreaction

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

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

• 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

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

bioreactors

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