Chapter 12: Biotechnology :Principles and Processes

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Introduction

Overview

• Biotechnology uses living organisms or their enzymes to produce useful products and processes.

• European Federation of Biotechnology defines it as integration of natural science with organisms, cells, parts, and molecular analogues for products and services.

• Includes traditional and modern techniques like in vitro fertilisation, gene synthesis, DNA vaccines, and gene therapy.

• Recombinant DNA technology enables modification of microorganisms and other organisms to gain novel traits; also called genetic engineering.

• Herbert Boyer and Stanley Cohen pioneered genetic engineering by combining DNA segments and inserting them into bacteria to produce specific proteins.

• Paul Berg, awarded Nobel Prize in 1980, is considered the father of genetic engineering.

Memory Aids

• Biotechnology = Biology + Technology for useful products.

• Recombinant DNA = DNA recombined to create new traits.

• Boyer & Cohen = DNA cutters and inserters.

Quick Checkpoints

• What is the core definition of biotechnology?

• Who are the pioneers of genetic engineering?

• What is recombinant DNA technology?

Principles of Biotechnology

Core Techniques

• Genetic engineering: Altering DNA/RNA to change host organism's traits by gene introduction.

• Chemical engineering: Maintaining sterile, contamination-free conditions to grow desired microbes or cells for product manufacturing (antibiotics, vaccines, enzymes).

• Genetic engineering involves recombinant DNA creation, gene cloning, and gene transfer to isolate and introduce specific genes without unwanted ones.

• First recombinant DNA made in 1972 by Cohen and Boyer using plasmid DNA from Salmonella typhimurium carrying antibiotic resistance.

Memory Aids

• Genetic engineering = DNA editing + host modification.

• Chemical engineering = sterile growth for biotech products.

• Recombinant DNA = DNA + plasmid vector + ligase.

Quick Checkpoints

• What are the two main techniques in modern biotechnology?

• How is recombinant DNA constructed?

• What role do plasmids play in genetic engineering?

Genetically Modified Organisms

Creation Steps

• Identify DNA containing desired genes.

• Introduce identified DNA into host organism.

• Maintain introduced DNA in host and ensure its transfer to progeny.

• Alien DNA replicates only after integration into host chromosome.

• Origin of replication (ori) in DNA initiates replication; alien DNA linked to ori to enable cloning.

• Restriction enzymes cut DNA at specific sites; DNA ligase joins foreign DNA with plasmid vectors to form recombinant DNA.

Memory Aids

• GMO steps: Identify → Introduce → Maintain.

• Restriction enzymes = molecular scissors.

• DNA ligase = molecular glue.

• Plasmid vectors carry foreign DNA into host.

Quick Checkpoints

• What are the three basic steps to create GMOs?

• Why is origin of replication important?

• What enzymes are involved in recombinant DNA formation?

Tools of Recombinant DNA Technology

Restriction Enzymes

• Isolated first by W. Arber (1963); Nobel Prize awarded for discovery.

• Hind II: first restriction endonuclease; cuts at specific 6-base recognition sequence from Haemophilus influenzae.

• Enzyme names derive from source organism: genus (1st letter), species (next 2 letters, italics), strain (capital letter), and order of isolation (Roman numeral), e.g., EcoRI from Escherichia coli RY13.

• Restriction enzymes are nucleases: exonucleases remove nucleotides from DNA ends; endonucleases cut within DNA.

• Types based on properties:

  • Type I: 3 subunits, require ATP, Mg2+, S-adenosyl methionine; not used in rDNA.

  • Type II: require Mg2+; used in rDNA technology.

  • Type III: 2 subunits; not used in rDNA.

• EcoRI and HindIII produce sticky ends; EcoRII and HindII produce blunt ends.

• Other enzymes:

  • Alkaline phosphatase: removes 5' phosphate groups, modifies DNA ends.

  • DNA ligase: seals gaps in DNA fragments.

  • Reverse transcriptase: synthesizes cDNA from mRNA template.

• Restriction enzymes recognize palindromic sequences: same sequence 5'→3' on both strands.

• They cut DNA near the center of palindrome, producing 3' or 5' overhangs (sticky ends) that facilitate recombination.

Memory Aids

• Enzyme name = Genus + species + strain + order (e.g., EcoRI).

• Type II enzymes are the rDNA workhorses.

• Sticky ends = cohesive ends = facilitate ligation.

• Palindromic sequences read same 5'→3' on both strands.

Quick Checkpoints

• What determines restriction enzyme specificity?

• Difference between sticky and blunt ends?

• Role of DNA ligase in recombinant DNA?

• Types of nucleases and their functions?

Gel Electrophoresis

Principle and Procedure

• Separates charged molecules by size under electric field.

• DNA is negatively charged; moves towards positive electrode (anode).

• Agarose gel (from seaweed) is common matrix; smaller fragments move faster through pores.

• DNA fragments stained with ethidium bromide (EtBr) and visualized under UV light as orange bands.

• Separated bands can be excised and purified (elution) for further use.

Memory Aids

• Smaller DNA fragments travel farther in gel.

• EtBr + UV = DNA visible as orange bands.

• Gel wells made by comb in agarose mold.

• Elution = cutting and purifying DNA from gel.

Quick Checkpoints

• Why does DNA move towards anode?

• How does fragment size affect migration?

• Purpose of ethidium bromide staining?

• What is elution in gel electrophoresis?

Cloning Vectors

Features and Types

• Vectors carry foreign DNA and replicate in host cells; also called carrier DNA.

• Common vectors: plasmids, bacteriophages, cosmids, YACs, BACs, transposons.

• Essential features:

  • Origin of replication (ori): initiates replication; controls copy number.

  • Selectable marker: antibiotic resistance genes (ampicillin, tetracycline, etc.) to select transformants.

  • Unique cloning sites: restriction sites for inserting foreign DNA; often in polylinker/multiple cloning site (MCS).

• Insertional inactivation: foreign DNA insertion disrupts marker gene (e.g., B-galactosidase), allowing color-based selection of recombinants.

• Vectors for plants and animals:

  • Agrobacterium tumefaciens Ti plasmid modified for plant gene delivery.

  • Disarmed retroviruses used for animal gene delivery.

• pBR322: first artificial cloning vector (1977); contains amp^R and tet^R genes, ori, and multiple restriction sites.

• Shuttle vectors replicate in both eukaryotic and prokaryotic cells.

Memory Aids

• ori = start replication; high copy number for more DNA.

• Selectable markers = antibiotic resistance for easy screening.

• Polylinker = multiple unique restriction sites for cloning.

• Insertional inactivation = loss of color indicates recombinant.

Quick Checkpoints

• What is the role of ori in vectors?

• How do selectable markers help in cloning?

• What is insertional inactivation?

• Examples of vectors used in plants and animals?

Competent Host

Preparation and Methods

• Competent hosts can uptake recombinant DNA; includes E. coli, yeast, animal, and plant cells.

• DNA is hydrophilic; cannot cross membranes without treatment.

• Bacterial competence induced by divalent cations (e.g., Ca2+) to increase membrane permeability.

• Transformation steps: incubate cells with DNA on ice, heat shock, then back on ice.

• In eukaryotes, transformation is called transfection.

• Vectorless gene transfer methods:

  • Biolistic/gene gun: DNA-coated gold/tungsten particles shot into plant cells.

  • Microinjection: direct injection of DNA into animal cell nucleus.

  • Electroporation: electric pulses create temporary membrane pores.

  • Chemical methods: polyethylene glycol (PEG) facilitates DNA entry.

Memory Aids

• Ca2+ treatment makes bacteria competent.

• Heat shock helps DNA uptake in bacteria.

• Gene gun for plants; microinjection for animals.

• Electroporation = electric pore formation.

Quick Checkpoints

• How is bacterial competence induced?

• Difference between transformation and transfection?

• Methods to introduce DNA into eukaryotic cells?

• Role of heat shock in transformation?

Processes of Recombinant DNA Technology

Overview

• Sequence of steps: Isolation of DNA → Cutting DNA → Amplification by PCR → Insertion into host → Foreign gene expression → Downstream processing

• Goal: Produce desired gene product using recombinant DNA

Memory Aids

• \"I Cut And Insert Obtain Downstream\" (Isolation, Cutting, Amplification, Insertion, Obtaining product, Downstream)

Quick Checkpoints

• What are the main steps in recombinant DNA technology?

• Why is each step important?

Isolation of Genetic Material

Key Points

• Isolation means extracting pure plasmid or genomic DNA from cells

• Cells are broken by enzymes: lysozyme (bacteria), cellulase (plants), chitinase (fungi)

• RNA removed by ribonuclease; proteins by protease

• Purified DNA precipitated using chilled ethanol

• DNA spooling: DNA strands wound on glass rod to separate from solution

Memory Aids

• Enzymes for cell wall: Lysozyme (bacteria), Cellulase (plants), Chitinase (fungi)

• Remove RNA with ribonuclease, proteins with protease

Quick Checkpoints

• Which enzymes help break different cell types?

• How is DNA purified after cell lysis?

Cutting of DNA at Specific Locations

Key Points

• Restriction enzymes cut DNA at specific sequences

• Digestion checked by agarose gel electrophoresis

• Source DNA and vector cut with same enzyme to create compatible ends

• DNA ligase joins gene of interest and vector to form recombinant DNA

Memory Aids

• Restriction enzymes = molecular scissors

• Ligase = molecular glue

Quick Checkpoints

• How do restriction enzymes recognize cut sites?

• Why use same enzyme for vector and insert?

Amplification of Gene of Interest using PCR

Key Points

• PCR replicates DNA in vitro to produce billions of copies

• Uses two primers, DNA polymerase (Taq), nucleotides, and template DNA

• Three steps per cycle: Denaturation (90-98°C), Annealing (40-60°C), Extension (72°C)

• Taq polymerase is thermostable, enabling repeated cycles

• Applications: pathogen detection, mutation analysis, DNA fingerprinting, gene therapy

Memory Aids

• PCR cycle: Denature → Anneal → Extend

• Taq polymerase from Thermus aquaticus survives high heat

Quick Checkpoints

• What are the three steps of a PCR cycle?

• Why is Taq polymerase essential for PCR?

Insertion of Recombinant DNA into the Host Cell/Organism

Key Points

• Recipient cells made competent to take up recombinant DNA

• Vector and insert DNA have complementary sticky ends joined by ligase

• Transformed cells express selectable marker (e.g., antibiotic resistance)

• Selection on antibiotic plates allows growth of only transformed cells

Memory Aids

• Selectable marker = gene allowing identification of transformed cells

• Competent cells = cells able to uptake foreign DNA

Quick Checkpoints

• How are cells made competent?

• What is the role of selectable markers?

Obtaining the Foreign Gene Product

Key Points

• Foreign gene expressed under optimal conditions to produce recombinant protein

• Continuous culture maintains cells in log phase for high protein yield

• Bioreactors provide controlled environment: temperature, pH, oxygen, nutrients

• Types of bioreactors: simple stirred-tank and sparged stirred-tank

• Stirred-tank bioreactor mixes culture and supplies oxygen evenly

• Sparged bioreactor bubbles air to increase oxygen transfer efficiency

Memory Aids

• Recombinant protein = protein from foreign gene in host

• Bioreactor = vessel for controlled cell growth and product formation

Quick Checkpoints

• What conditions are controlled in a bioreactor?

• Difference between simple and sparged stirred-tank bioreactors?

Downstream Processing

Key Points

• After biosynthesis, product undergoes separation and purification

• Formulation with preservatives and clinical trials required for drugs

• Strict quality control testing ensures product safety and efficacy

• Processes vary depending on product type

Memory Aids

• Downstream = purification and finishing steps after production

Quick Checkpoints

• Why is downstream processing essential?

• What quality controls are necessary?

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