Board exam focus — Biotechnology: Principles and Processes (CBSE & HBSE)

CBSE focuses on restriction enzymes, cloning vectors (plasmid features), recombinant DNA technology steps, gel electrophoresis, blue-white screening, PCR, and bioreactors. HBSE emphasises definitions, EcoRI, pBR322, transformation, PCR steps, and downstream processing.

Restriction Enzymes and Cloning Vectors

Biotechnology: Definition and Principles

Biotechnology is the use of biological systems (living organisms or their components) to develop or make products and processes for human benefit. Modern biotechnology specifically refers to recombinant DNA (rDNA) technology — the ability to combine DNA from two different organisms to create new genetic combinations.

Two core techniques enable modern biotechnology:

1. Genetic engineering (rDNA technology): ability to alter the chemistry of DNA and RNA; introduce resulting DNA into host organisms → change phenotype
2. Maintenance of sterile conditions (aseptic technique): enable growth of desirable organisms without contamination

Tools of Recombinant DNA Technology:

1. Restriction endonucleases (molecular scissors)
2. Polymerase Chain Reaction (PCR)
3. Cloning vectors (plasmids, phages, cosmids, BAC, YAC)
4. Host organisms (E. coli, Agrobacterium, yeast)
5. DNA ligase (molecular glue)
6. Gel electrophoresis (separation and analysis of DNA)

Restriction Endonucleases (Restriction Enzymes)

Restriction enzymes are bacterial endonucleases that cut double-stranded DNA at specific nucleotide sequences called recognition sites (typically 4-8 bp palindromes).

Nomenclature: Named after the bacterium they were isolated from:

• First letter: genus (capitalised, italicised)
• Second + third letters: species
• Fourth letter: strain
• Roman numeral: order of discovery

Examples:

• EcoRI: Escherichia coli strain RI — first enzyme discovered from this strain
• HindIII: Haemophilus influenzae strain d — third enzyme
• BamHI: Bacillus amyloliquefaciens H — first enzyme

Palindromic Recognition Sequences: Restriction enzymes recognise palindromic sequences — sequences that read the same on both strands in the 5' to 3' direction:

EcoRI recognition sequence:

• 5'–GAATTC–3'
• 3'–CTTAAG–5'

When EcoRI cuts, it creates staggered cuts:

• 5'–G AATTC–3'
• 3'–CTTAA G–5'

Result: sticky ends (cohesive ends) with single-stranded 5' overhangs (5'-AATT-3')

HindIII: 5'–A↓AGCTT–3' → 5' overhang (sticky ends: AGCT) SmaI: 5'–CCC↓GGG–3' → blunt ends (cuts in middle of palindrome)

Types of cuts:

• Sticky ends (cohesive ends): staggered cuts → overhanging single-stranded tails
• 5' overhang (EcoRI, HindIII) — more common; easier to ligate
• 3' overhang (KpnI, SphI)
• Blunt ends (PvuII, SmaI): flush cuts

Types of restriction enzymes:

• Type I: cut DNA far from recognition site; need ATP, SAM; not useful in rDNA technology
• Type II: cut within or near recognition site; no ATP needed; generate specific fragments; USED IN GENETIC ENGINEERING
• Type III: cut close to recognition site; need ATP

Applications:

• Creating compatible sticky ends for ligation of insert + vector
• DNA fingerprinting (RFLP — Restriction Fragment Length Polymorphism)
• Restriction mapping of genomes
• Southern blotting

Cloning Vectors

A cloning vector is a DNA molecule (carrier) into which a foreign DNA fragment is inserted and then introduced into a host organism where it will be replicated.

Essential features of an ideal cloning vector:

1. Origin of Replication (ori): DNA sequence where replication starts; determines copy number (high copy = more plasmid per cell)
2. Selectable Marker: gene that allows identification of transformed cells (cells that took up the vector). Usually antibiotic resistance genes (ampR, tetR, kanR)
3. Cloning Sites (Multiple Cloning Site — MCS/Polylinker): restriction enzyme recognition sequences where insert DNA is ligated; ideally within a selectable marker (for blue-white screening)

Types of Cloning Vectors:

1. Plasmids:

• Small (1-30 kb), circular, extrachromosomal DNA; replicate independently
• pBR322 (first artificially constructed cloning plasmid, 1977):
• 4,361 bp; contains ampR (ampicillin resistance) and tetR (tetracycline resistance) genes
• Multiple restriction sites within tetR gene (EcoRI, ClaI, HindIII, SalI, BamHI, SphI, PvuI)
• Insertional inactivation: insert DNA into tetR → tetracycline sensitivity; cell still ampicillin resistant → screen on both antibiotics
• ori: ColE1 origin; ~15-20 copies per cell
• pUC vectors: contain lacZ (beta-galactosidase) gene with MCS → blue-white screening
• Ti plasmid of Agrobacterium tumefaciens: T-DNA (transferred DNA) integrates into plant genome → used as plant transformation vector

2. Bacteriophages (Phage Vectors):

• Lambda (λ) phage: can accommodate ~20-25 kb inserts (Charon phages, lambda gt series)
• M13 phage: filamentous phage; single-stranded DNA; useful for site-directed mutagenesis
• Advantage over plasmids: larger insert capacity; efficient infection

3. Cosmids:

• Hybrid of plasmid + lambda phage COS sites (cohesive ends)
• Accept inserts 35-45 kb
• Propagated as plasmid in bacteria

4. BAC (Bacterial Artificial Chromosome):

• Based on F factor (fertility plasmid) of E. coli
• Insert capacity: 100-300 kb
• Used in Human Genome Project

5. YAC (Yeast Artificial Chromosome):

• Contains yeast centromere, telomeres, autonomously replicating sequences (ARS), selectable markers
• Insert capacity: 200-2,000 kb (largest capacity)
• Used for cloning entire genes including introns; Human Genome Project
• Problem: chimeric clones (two different DNA pieces ligated together)

Steps of Recombinant DNA Technology

Steps of Recombinant DNA Technology

Recombinant DNA (rDNA) technology involves several coordinated steps to produce an organism with a desired foreign gene.

Step 1: Isolation of DNA (from source organism)

Step 2: Cutting DNA with Restriction Enzymes

• Source DNA (insert) and vector DNA are digested with the SAME restriction enzyme (or compatible enzyme producing compatible sticky ends)
• This ensures that both have compatible sticky ends → can be ligated together
• Restriction digestion in appropriate buffer, temperature (usually 37°C for most Type II enzymes)

Step 3: Gel Electrophoresis (Analysis and Separation)

• DNA fragments separated by agarose gel electrophoresis based on size
• DNA is negatively charged → migrates toward anode (+) in electric field
• Smaller fragments migrate faster → further from wells
• Gel concentration: 0.8-2.0% agarose; higher % = better separation of small fragments
• DNA visualised by staining with ethidium bromide (EtBr) — intercalates between DNA bases → fluoresces under UV light (orange-pink bands)
• DNA ladder (size marker): known-size fragments run alongside; used to estimate unknown fragment sizes
• Desired band cut from gel → DNA eluted (recovered) — called elution

Step 4: Ligation

• Purified insert + vector (both cut with compatible restriction enzyme) are incubated with DNA ligase (T4 DNA ligase)
• DNA ligase forms phosphodiester bonds between compatible sticky ends (or blunt ends)
• Products: recombinant plasmid (insert + vector), re-ligated vector (self-ligation), unligated insert
• Self-ligation of vector is reduced by treating vector with alkaline phosphatase (removes 5'-phosphate → cannot be re-ligated without insert)

Step 5: Transformation (Introduction into Host)

Methods of introducing recombinant DNA into host cells:

a) Chemical competence (Heat shock method):

• E. coli cells treated with cold CaCl2 (calcium chloride) → makes cell membrane permeable (creates competent cells)
• Mix with recombinant DNA → heat shock (42°C for 90 seconds) → cold → DNA enters cells

b) Electroporation:

• Short, high-voltage electric pulses → transient pores in membrane → DNA enters
• Used for bacteria, yeast, mammalian cells

c) Agrobacterium-mediated transformation (for plants):

• T-DNA from Ti plasmid of Agrobacterium tumefaciens transfers to plant genome
• Foreign gene cloned into T-DNA → integrates into plant chromosomes
• Most widely used method for dicot plants

d) Biolistics (Gene gun / microprojectile bombardment):

• DNA coated onto gold or tungsten microparticles (0.4-1.2 μm)
• Fired at high velocity (using compressed gas — helium; or electric discharge) into plant tissue/cells
• Used for monocots (maize, wheat, rice — resistant to Agrobacterium) and organelle transformation

e) Microinjection:

• Directly inject DNA into nucleus using fine glass needle
• Used in animal cells, oocytes, pronuclear stage embryos

f) Liposome-mediated (Lipofection):

• DNA encapsulated in lipid vesicles (liposomes) → fuse with cell membrane → deliver DNA

Step 6: Selection of Transformants

• Plating on selective medium with antibiotic (ampicillin, tetracycline, kanamycin)
• Only cells that took up the vector (and its antibiotic resistance gene) survive
• Colony PCR: individual colonies screened by PCR to confirm insert

Step 7: Identification of Recombinants Among the transformants, need to identify which cells have recombinant plasmid (with insert) vs non-recombinant (vector without insert).

Insertional Inactivation (pBR322):

• Insert DNA cloned into BamHI/SalI site within tetR gene
• Transformants grown on ampicillin plates (all survive) and then replica plated onto tetracycline plates
• Recombinants: ampR but tetS (grow on Amp, NOT on Tet) — tetR gene disrupted
• Non-recombinants: ampR and tetR (grow on both)

Blue-White Screening (pUC vectors, pBluescript):

• lacZ gene (codes for beta-galactosidase) present in vector; MCS is WITHIN lacZ
• Plated on medium with IPTG (inducer) + X-gal (chromogenic substrate for beta-galactosidase)
• Non-recombinant (no insert): lacZ intact → produces beta-galactosidase → cleaves X-gal → blue colour
• Recombinant (insert disrupts lacZ): no functional beta-galactosidase → X-gal not cleaved → white colonies
• Blue = non-recombinant; White = recombinant

Step 8: Screening and Expression

• Screen library by colony hybridisation (using labelled probe complementary to gene of interest)
• Or PCR screening
• Positive clones grown in large culture → protein expression
• Optimise expression: strong promoter, codon optimisation, fusion tags, protease inhibitors

PCR, Bioreactors and Downstream Processing

PCR (Polymerase Chain Reaction)

PCR is an in vitro technique for amplifying specific DNA sequences exponentially from a small starting template. Developed by Kary Mullis (1983); Nobel Prize in Chemistry 1993.

Components needed for PCR:

1. DNA template: contains the target sequence to be amplified; can be genomic DNA, cDNA, or any DNA
2. Primers (forward and reverse): short synthetic oligonucleotides (~18-25 nt) complementary to the sequences flanking the target region on each strand; define the boundaries of amplification
3. Taq DNA Polymerase: thermostable DNA polymerase from Thermus aquaticus (hot spring bacterium); active at 72°C; stable up to 95°C; extends from 3'-OH end of primers
4. dNTPs (deoxyribonucleoside triphosphates): building blocks (dATP, dCTP, dGTP, dTTP)
5. Buffer with Mg2+: MgCl2 is essential cofactor for Taq polymerase

PCR Cycle (Three Steps, repeated 25-40 times in a thermocycler):

1. Denaturation: 94–96°C for 30 seconds

• Double-stranded DNA is heated → hydrogen bonds break → two single strands separate
• All enzyme activities inactive at this temperature

2. Annealing: 50–65°C for 30 seconds (temperature depends on primer Tm)

• Temperature lowered → primers anneal (bind by complementary base pairing) to their specific target sequences on single-stranded template
• Forward primer binds template strand (3'→5'); Reverse primer binds coding strand (5'→3')
• Annealing temperature typically ~5°C below primer Tm

3. Extension (Elongation): 72°C for 30 seconds–1 minute (depends on amplicon size; ~1 kb/min for Taq)

• Taq polymerase extends from 3'-OH of each primer in 5'→3' direction
• Synthesises new complementary strand using dNTPs
• Product: new double-stranded DNA copy of target region

Product accumulation:

• After n cycles: 2^n copies theoretically (actually ~10^9 from single molecule after 30 cycles)
• Exponential amplification (doubles each cycle)

Applications of PCR:

1. Forensics: DNA fingerprinting from tiny samples (hair, blood drop, saliva)
2. Medical diagnostics: detect HIV (early infection), drug-resistant bacteria, genetic mutations, HPV, SARS-CoV-2
3. Prenatal diagnosis: detection of genetic disorders in fetal DNA (from amniocentesis/CVS)
4. Phylogenetics: amplify ancient DNA (museum specimens, mummies)
5. Cloning: amplify genes of interest with restriction sites incorporated in primers
6. Sequencing: prepare templates for DNA sequencing

Variants: RT-PCR (reverse transcription PCR — detect RNA; convert to cDNA first), Real-time PCR (qPCR — quantitative; detect amplification in real time using fluorescent dyes), Multiplex PCR, Digital PCR.

Bioreactors

A bioreactor is a vessel in which large-scale microbial or cell culture is carried out to produce a desired product (enzyme, antibiotic, recombinant protein).

Simple stirred-tank bioreactor:

• Large cylindrical vessel (100 L to 100,000 L scale)
• Stirrer (impeller): maintains homogeneous culture; prevents settling
• Baffles: prevent vortex formation
• Sparger: introduces air/O2 for aerobic organisms
• Temperature control: water jacket or heating coil
• pH monitoring and control: pH probes + acid/base addition
• Dissolved oxygen monitoring: O2 probe
• Foam control: antifoam agent (silicone oil) or mechanical foam breaker
• Sampling port: for monitoring culture progress (cell density, metabolite concentration)
• Inoculation port: for adding inoculum
• Outlets for products

Types of bioreactors:

• Batch bioreactor: closed system; all nutrients added at start; culture grows, accumulates product, then harvested; simplest; not optimal for maximum yield
• Fed-batch bioreactor: nutrients added in portions during culture; prevents substrate inhibition; increases yield
• Continuous bioreactor (chemostat): fresh nutrients continuously pumped in; culture fluid + products continuously removed; steady state maintained; maximum productivity per unit time

Other designs: airlift bioreactor (gentle agitation by air), hollow fibre bioreactor (mammalian cells), fluidised bed bioreactor (immobilised cells)

Downstream Processing

After fermentation, the product must be isolated and purified from the complex culture medium — this is called downstream processing.

Steps:

1. Cell disruption (for intracellular products): sonication, French press, bead beating, detergents, enzymatic lysis
2. Separation of biomass (centrifugation): low-speed centrifugation removes cells and debris
3. Concentration: ultrafiltration, evaporation, precipitation (ammonium sulphate salting out)
4. Chromatography (Purification):

• Affinity chromatography: protein binds specific ligand on column; eluted with competing compound; highest specificity (e.g., His-tag protein on Ni-NTA resin)
• Ion-exchange chromatography: separates by charge
• Size-exclusion (gel filtration) chromatography: separates by molecular weight
• Hydrophobic interaction chromatography: separates by surface hydrophobicity

1. Formulation: addition of stabilisers, pH adjustment, sterilisation, lyophilisation (freeze-drying), quality testing
2. Quality control and validation: especially critical for pharmaceutical proteins (sterility testing, potency, purity, safety)

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