Chapter 12: Biotechnology :Principles and Processes

Introduction

Overview

• Biotechnology uses live organisms or 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 create novel capabilities.
• Genetic engineering is the manipulation of genetic material; Berg is the "father of genetic engineering" (Nobel Prize 1980).
• Herbert Boyer and Stanley Cohen pioneered biotechnology by combining restriction enzymes and plasmids to produce proteins in bacteria.

Memory Aids

• Berg = Father of Genetic Engineering (Nobel 1980).
• Boyer + Cohen = Recombinant DNA pioneers.
• Biotechnology = Organisms + Science = Useful products.

Quick Checkpoints

• What is the EFB definition of biotechnology?
• Who are the founders of recombinant DNA technology?
• What is genetic engineering?

Principles of Biotechnology

Core Techniques

• Genetic engineering: Alters DNA/RNA to change host phenotype by gene introduction.
• Chemical engineering: Maintains sterile conditions for microbial or cell growth to produce antibiotics, vaccines, enzymes.
• Genetic engineering involves recombinant DNA creation, gene cloning, and gene transfer.
• First recombinant DNA made by Cohen and Boyer in 1972 using plasmid DNA from Salmonella typhimurium.
• Three steps to create GMOs/transgenic organisms:
  • Identify DNA with desired genes.
  • Introduce DNA into host.
  • Maintain DNA in host and transfer to progeny.
• Alien DNA replicates only when integrated into host chromosome with origin of replication (ori).
• Cloning = making multiple identical copies of DNA.
• Restriction enzymes cut DNA at specific sites; plasmids act as vectors to transfer DNA.
• DNA ligase joins cut DNA ends to form recombinant DNA (circular, autonomously replicating DNA).

Memory Aids

• GMO steps: Identify, Introduce, Maintain.
• Restriction enzymes = molecular scissors.
• DNA ligase = molecular glue.
• Plasmid = vector for gene transfer.

Quick Checkpoints

• What are the two core techniques of modern biotechnology?
• What is the role of restriction enzymes?
• How is recombinant DNA formed?
• What are the steps to create a transgenic organism?

Tools of Recombinant DNA Technology

Restriction Enzymes

• Isolated first by W. Arber (1963) in bacteria; Nobel Prize awarded to Arber, Smith, Nathan for discovery.
• Hind II was the first restriction endonuclease; cuts DNA at specific 6-base recognition sequence.
• Enzyme names derive from source organism: first letter genus, next two species (italicized), fourth letter strain (capital), Roman numeral order.
• Restriction enzymes are nucleases: exonucleases remove nucleotides from 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.
  • 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, modifies DNA ends.
  • DNA ligase seals gaps in DNA fragments.
  • Reverse transcriptase synthesizes cDNA from mRNA template.
• Restriction enzymes recognize palindromic sequences (same read 5'→3' on both strands).
• They cut DNA near center of palindrome, generating 3' or 5' protruding ends (sticky ends).
• Sticky ends have complementary sequences, enabling pairing and ligation by DNA ligase.
• Restriction fragments separated by gel electrophoresis using agarose gel and stained with ethidium bromide for visualization under UV light.

Memory Aids

• Restriction enzyme names = Genus (italic) + species (italic) + strain (capital) + order (Roman numeral).
• Sticky ends = EcoRI, HindIII; Blunt ends = EcoRII, HindII.
• Type II enzymes used in rDNA; Type I & III not used.
• Palindromic sequences read same 5'→3' on both strands.

Quick Checkpoints

• What defines a restriction enzyme's recognition site?
• Difference between sticky and blunt ends?
• Role of DNA ligase in recombinant DNA?
• How are DNA fragments separated after restriction digestion?

Cloning Vectors

Features and Types

• Vectors carry foreign DNA and replicate inside host cells; also called vehicle or carrier DNA.
• Common vectors: plasmids, bacteriophages, cosmids, YACs, BACs, transposons.
• Key features:
  • Origin of replication (ori): Start site for replication; controls copy number.
  • Selectable marker: Antibiotic resistance genes (ampicillin, tetracycline, kanamycin) to select transformants.
  • Cloning sites: Unique restriction sites (polylinker or MCS) for foreign DNA insertion.
• Insertional inactivation: foreign DNA insertion disrupts marker gene (e.g., B-galactosidase), enabling 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, tet^R, ori, rop, and multiple restriction sites.
• Shuttle vectors replicate in both eukaryotic and prokaryotic cells.

Memory Aids

• pBR322 = first cloning vector with amp^R and tet^R markers.
• Selectable markers help distinguish transformants by antibiotic resistance or color change.
• Polylinker = multiple cloning sites for easy DNA insertion.
• Agrobacterium Ti plasmid used for plant transformation.

Quick Checkpoints

• What is the role of origin of replication in vectors?
• How do selectable markers aid in recombinant selection?
• What is insertional inactivation?
• Name common vectors used in genetic engineering.

Competent Host (For Transformation with Recombinant DNA)

Host Preparation and Methods

• Competent hosts (E. coli, yeast, animal, plant cells) are required to take up recombinant DNA.
• DNA is hydrophilic; cannot cross membranes without treatment.
• Bacterial cells made competent by treating with divalent cations (e.g., Ca2+) to increase DNA uptake through cell wall pores.
• 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 bombarded into plant cells; used in crops like wheat, rice, maize.
  • Microinjection: Direct injection of DNA into animal cell nucleus using micropipette.
• Electroporation creates temporary pores in membranes using lysozyme or calcium chloride.
• Chemicals like polyethylene glycol (PEG) facilitate DNA entry into host cells.

Memory Aids

• Competency increased by Ca2+ treatment and heat shock.
• Biolistic method for plants; microinjection for animals.
• Electroporation and PEG aid DNA uptake.

Quick Checkpoints

• Why must bacterial cells be made competent?
• Difference between transformation and transfection?
• Methods to introduce DNA into eukaryotic cells?
• Role of divalent cations in transformation?

Processes of Recombinant DNA Technology

Steps Involved

• Isolation of genetic material (DNA) from cells.
• Cutting DNA at specific sites using restriction enzymes.
• Amplification of gene of interest by PCR.
• Insertion of recombinant DNA into host cells.
• Production of foreign gene product.
• Downstream processing for product purification.

Memory Aids

• Remember the sequence: Isolate, Cut, Amplify, Insert, Produce, Process.

Quick Checkpoints

• What is the first step in rDNA technology?
• Which enzyme cuts DNA at specific locations?
• How is the gene of interest amplified?
• What is the purpose of downstream processing?

Isolation of Genetic Material

Key Points

• Isolation means removing plasmid or genomic DNA from cells in pure form.
• Cell membranes and walls are broken using enzymes like lysozyme (bacteria), cellulase (plants), chitinase (fungi).
• RNA removed by ribonuclease; proteins removed by protease.
• Purified DNA precipitates with chilled ethanol.

Memory Aids

• Use specific enzymes to break cell walls and remove contaminants.
• Precipitate DNA with cold ethanol for purity.

Quick Checkpoints

• Which enzymes help break cell walls during DNA isolation?
• How are proteins and RNA removed from DNA samples?
• What is the role of chilled ethanol in DNA isolation?

Cutting of DNA at Specific Locations

Key Points

• Restriction enzymes cut purified DNA at specific sequences.
• Digestion progress checked by agarose gel electrophoresis.
• Cut gene of interest and vector DNA are joined using DNA ligase to form recombinant DNA.

Memory Aids

• Restriction enzymes act like molecular scissors at specific sites.
• Gel electrophoresis confirms DNA cutting.

Quick Checkpoints

• What enzyme cuts DNA at specific sites?
• How is the success of DNA cutting verified?
• Which enzyme joins DNA fragments to form recombinant DNA?

Amplification of Gene of Interest using PCR

Key Points

• PCR replicates DNA in vitro using primers and DNA polymerase.
• Thermostable Taq polymerase allows repeated cycles at high temperatures.
• Three steps per cycle: denaturation (90°-98°C), primer annealing (40°-60°C), extension (72°C).
• Amplifies DNA up to a billion times.
• Applications: pathogen diagnosis, DNA fingerprinting, prenatal diagnosis, gene therapy.

Memory Aids

• Denature, Anneal, Extend = PCR cycle steps.
• Taq polymerase works at high heat without denaturing.

Quick Checkpoints

• What enzyme is used in PCR?
• What are the three steps of a PCR cycle?
• How many times can PCR amplify DNA?

Insertion of Recombinant DNA into Host Cell

Key Points

• Recipient cells are made competent to take up recombinant DNA.
• Vector and alien DNA cut by same restriction enzyme to produce complementary sticky ends.
• DNA ligase joins sticky ends forming recombinant DNA.
• Selectable markers like antibiotic resistance genes help identify transformed cells.
• Example: Ampicillin resistance gene allows selection of transformed E. coli on ampicillin plates.

Memory Aids

• Competent cells take up DNA from surroundings.
• Selectable markers identify successful transformants.

Quick Checkpoints

• What makes cells competent?
• How are recombinant DNA fragments joined?
• What is a selectable marker?

Obtaining the Foreign Gene Product

Key Points

• Foreign gene expresses protein called recombinant protein in host cells.
• Cells grown in continuous culture to maintain active growth phase for high protein yield.
• Bioreactors provide optimal conditions: temperature, pH, oxygen, nutrients.
• Two types of stirred-tank bioreactors: simple and sparged (with air bubbles).
• Bioreactors have systems for mixing, oxygen delivery, foam control, temperature and pH control, and sampling.

Memory Aids

• Bioreactors optimize growth and protein production.
• Sparged bioreactors improve oxygen supply with air bubbles.

Quick Checkpoints

• What is a recombinant protein?
• Why use continuous culture systems?
• What conditions do bioreactors control?

Downstream Processing

Key Points

• Involves separation and purification of the product after biosynthesis.
• Product formulation with preservatives and clinical trials for drugs.
• Strict quality control testing is essential.
• Processes vary depending on the product.

Memory Aids

• Downstream = purification and quality control.
• Formulation and testing ensure product safety.

Quick Checkpoints

• What is the purpose of downstream processing?
• Why is quality control important?
• Does downstream processing vary by product?