Biotechnology-Textbook-v-3-2016-2017
CHAPTER 1: INTRODUCTION TO BIOTECHNOLOGY
1.1 What is Biotechnology?
Biotechnology is the use of a living organism for one’s own benefit; its roots date back to early civilization through agriculture (cultivation of crops, domestication of animals).
In modern biotechnology, gene splicing and recombinant organisms take center stage, enabling genetic engineering.
The biotechnology industry has grown to affect daily life via healthcare products, vaccines, and industrial processes.
1.2 Biotechnology: Healing, Fueling & Feeding the World
Biotech is technology based on biology, harnessing cellular and biomolecular processes to develop technologies and products.
Historically, microorganisms have been used for >6,000 years to make food (bread, cheese) and preserve dairy products.
Modern biotech aims to combat diseases, reduce environmental footprint, improve food security, generate cleaner energy, and make manufacturing processes safer and cleaner.
Current impact indicators:
>250 biotechnology healthcare products and vaccines available to patients (for previously untreatable diseases).
>13.3 million farmers worldwide using agricultural biotechnology to increase yields and reduce crop damage.
>50 bio-refineries across North America to convert renewable biomass into biofuels and chemicals.
Healing (examples): reducing infectious disease rates, saving millions of lives, tailoring treatments to individuals, improving disease detection, etc.
Fueling (examples): fermentation-based production, enzymes and biocatalysts as microscopic manufacturing plants, improving process efficiencies and reducing petrochemical reliance.
Feeding (examples): biotech crops with insect resistance, herbicide tolerance, enhanced nutrition, reduced pesticide use, and improved oil content for cardiovascular health.
1.3 What is Biotechnology Used for?
Four main subfields: green (plants), blue (aquatic), white (industrial/bioprocessing), red (medical).
Green biotech is the oldest and most widely used; blue biotech is relatively rare.
White biotech focuses on using biological organisms to produce or process chemicals for industry; can include bioremediation (e.g., bacteria degrading environmental pollutants).
Red biotech involves medical applications: drug production via modified organisms, gene therapy, and genome manipulation.
Debate: GMOs and environmental/health concerns versus potential for environmental friendliness and food security.
1.4 Safety in a Biotechnology Lab
Lab safety is a shared responsibility; following rules protects individuals and the lab community.
Lab security is emphasized due to hazardous materials and potential misuse; post-9/11 regulations increased security measures.
1.5 Proper Handling & Storage of Chemicals & Reagents
MSDS (Material Safety Data Sheets): each chemical labeled with chemical identity, hazard ingredients, physical/chemical properties, fire/explosion data, reactivity, health hazards, handling precautions, and control measures.
NFPA ratings: color-coded blue (health), red (flammability), yellow (reactivity); numbers indicate hazard level.
8 sections of MSDS standard; details include TLV (threshold limit value) exposure levels and emergency contacts.
1.6 General Safety Precautions in Handling Hazardous Chemicals in the Lab
Routes of exposure: inhalation, skin/eye contact, ingestion, injection; use PPE and fume hoods.
Safety rules by chemical type (flammables, corrosives, reactives, toxic chemicals) with practical handling guidance.
Common hazardous chemicals and hazards (carcinogens, mutagens, neurotoxins, teratogens, nephrotoxins, hepatotoxins, corrosives).
1.7 Biological Safety: Containment
Live organisms and recombinant DNA are handled as biosafety hazards; treat all living materials and recombinant DNA as potential hazards.
Routes of exposure mirror chemical hazards; maximize containment and training.
Practices include restricting access, PPE, hand washing, no mouth pipetting, disinfection, autoclaving regulated waste, insect/rodent control, and using a laminar flow cabinet where available.
1.8 Disposal of Hazardous Chemicals & Biological Materials
Hazardous chemical disposal subject to state/federal regulations; autoclave bags for biohazard waste; autoclave to kill organisms.
Clean benches with bleach after messy sessions; turn off equipment when leaving.
1.9 Micropipettors & Tip Boxes
Each station has a dedicated micropipettor set; tips labeled to workstation for tracking.
1.10 Glassware
Proper washing/storage areas; do not leave glassware in sinks; labeling conventions; use label tape for labeling; broken glass disposed in designated box; clean-up rules.
1.11 Biotechnology Student Checkout Duties
Lab etiquette and workspace cleanliness; label solutions and prep forms; return equipment; wipe benches with bleach; wash glassware; assist others; ensure hazardous materials stored properly; turn off balances, hot plates, spectrophotometers, and other equipment.
CHAPTER 2: MATH SKILLS & BASIC TOOLS IN A BIOTECH LAB
2.1 Mass & Volume with SI Units
Mass measured directly, by difference, or by subtraction; units: mass in grams (g); volume in milliliters (mL); 1 mL = 1 cm^3.
Volume measurements typically with graduated cylinders; micropipettes for small volumes.
Volume concepts: cuboid volume V = L × W × H.
Volume by displacement for irregular solids: Solid Volume = Total Volume − Liquid Volume.
Density d = m/V; density is an intensive property, enabling interconversion between mass and volume.
2.2 Metric Units
Metric prefixes and conversions (based on powers of ten); importance of scientific notation for small measurements.
Example conversion process using the staircase concept to shift decimal places.
2.3 Difference Between Accuracy & Precision
Accuracy: closeness to a known/accepted value.
Precision: reproducibility or consistency of measurements.
Analogy: hitting bullseye vs. clustering around a point.
2.4 Common Biotechnology Lab Equipment
Volume measurement tools:
Erlenmeyer flasks (not calibrated for accurate volumes).
Beakers (not reliable for precise volume).
Graduated cylinders (calibrated for reasonable accuracy).
Volumetric flasks (high accuracy for standard solutions).
Pipets including Pasteur, Beral, serological, Mohr, volumetric, automatic micropipettes, and multichannel pipettes.
Mass measurement: balances (mechanical vs electronic); analytical electronic balances with high sensitivity.
pH measurement: pH meters (calibration with buffers); pH readouts to 0.1 pH unit.
Light measurement: spectrophotometers (VIS, UV/VIS, scanning, NanoDrop, microplate readers).
Solution preparation devices: magnetic stirrers, vortex mixers.
Microbiology tools: autoclaves, biosafety hoods, fermentors.
Microscopy: brightfield/light, stereoscope, SEM, TEM; magnifications and usage tips.
2.5 Using the Spectrophotometer
Spectrophotometer measures transmittance or absorbance of light; relationship: absorbance increases with sample density.
Blank cuvette used to subtract cuvette/solvent contributions; auto-zero procedure.
Wavelength selection guided by absorption spectrum of the analyte; peak absorption gives best measurement precision.
2.6 Microscopes
Types: Light microscope (40x, 100x, 400x), Stereoscope, Scanning Electron Microscope (SEMs) and Transmission Electron Microscope (TEM).
Magnification calculations: Total magnification = magnification of ocular × objective.
Focusing sequence: start with scanning, switch to medium, then high; adjust diaphragms and lighting; use fine adjustment for high power; avoid damage.
Drawing and sample prep practices for microscopy (wet mounts, staining, oil immersion).
CHAPTER 3: MICROBIOLOGY AND BACTERIA
3.1 Origin of Life
Life originated ~4 billion years ago; earliest cells were prokaryotic (no nucleus or true organelles).
Oldest prokaryotic fossils ~3.5 billion years old; lithotrophic and fermentative metabolisms among early prokaryotes.
Eukaryotes with nuclei appeared ~1.5–2 billion years ago.
3.2 Prokaryotic Life
Domains: Bacteria, Archaea; prokaryotic cells lack a defined nucleus.
Archaea (formerly archaebacteria) are a separate domain; endosymbiotic theory links mitochondria and chloroplasts to bacteria.
3.3 Size, Distribution & Ecology
Typical bacterial cells ~1 μm in diameter; overwhelmingly abundant in soil and oceans; 10^9–10^9 cells per gram of soil; bacteria surpass eukaryotes in number and biomass in many habitats.
Endosymbiosis and mutualistic relationships between bacteria and eukaryotes; nitrogen fixation and methanogenesis as prokaryotic-specific metabolisms.
3.4 Structure & Function of Prokaryotic Cells
Three architectural regions: appendages (flagella, pili), cell envelope (capsule, cell wall, plasma membrane), cytoplasmic region (DNA, ribosomes, inclusions).
Flagella: motility; patterns include polar and peritrichous; chemotaxis response to stimuli.
3.5 Coverings of the Prokaryotic Cell
Cell wall composition differs by Gram staining:
Gram-positive: thick peptidoglycan layer with teichoic acids; retains crystal violet.
Gram-negative: thin peptidoglycan; outer membrane contains LPS (endotoxin); counterstain reveals pink.
Capsule, slime layer, glycocalyx: adherence, protection from phagocytosis, desiccation prevention; biofilms (e.g., dental plaque by Streptococcus mutans).
Fimbriae and pili: attachment and conjugation (sex pilus).
3.6 Cytoplasmic Constituents
Prokaryotic chromosome typically one circular DNA molecule; plasmids may be present.
Ribosomes (70S) responsible for protein synthesis; inclusions act as nutrient reserves.
3.7 Identification of Bacteria
Microscopy: cell shape (bacillus, coccus, spirilla), motility, Gram staining.
Use Bergey’s Manual as a field guide; follow with biochemical tests for definitive ID.
3.8 Applications of Bacteria in Industry & Biotechnology
Fermentation, antibiotics production, vaccines, enzymes; industrial biotech uses bacteria for biosynthesis and gene engineering foundations.
3.9 Growing Bacteria in Culture
Culture media types: broth (liquid), solid media with agar; defined vs undefined; selective vs differential media.
Aseptic technique and streaking for isolation to obtain pure cultures from mixed populations.
Inoculation tools: loop and stab inoculators; sterile technique; streaking pattern to isolate colonies.
3.10 Bacterial Colony Characteristics
Observing colony size, shape, margin, surface, pigmentation, translucence, elevation.
Lab Backgrounds
Yogurt Lab: starter cultures Lactobacillus bulgaricus and Streptococcus thermophilus ferment lactose; yogurt standards: at least 8.25% solids not fat; fat content categories; stabilizers; demand for probiotic cultures.
Dental Caries & Microbial Flora: plaque bacterial communities; S. mutans as major caries contributor; plaque pH and fermentation of sugars; fluoride role; salivary restoration dynamics.
Gram Staining Lab: differentiates Gram-positive vs Gram-negative based on cell wall properties and dye retention; procedure steps and interpretation.
Aseptic Technique & Streaking Lab: steps to obtain well-isolated colonies; use of sterile loops, flame sterilization, plate handling, etc.
CHAPTER 4: BIOCHEMISTRY & PREPARING SOLUTIONS
4.1 Atoms
Atoms are composed of protons, neutrons (nucleus) and electrons (electron cloud). Protons determine identity (Z, atomic number).
Nucleus held together by the strong force; electrons occupy orbitals; electron configuration predicts chemical behavior.
4.2 The Periodic Table of Elements
Elements organized by increasing atomic number; cells contain symbol, atomic number, mass, and name; groups/families share similar valence electron counts; trends: atomic radius, ionization energy, electronegativity, electron affinity.
Protons = atomic number; Neutrons = atomic mass − atomic number; Electrons = atomic number.
4.3 What is a Mole & Why are Moles Used?
Mole is a unit defined by Avogadro’s number: particles per mole.
One mole of carbon = 12.0 g; mass of solute = molarity × volume × molar mass:
Relationship: moles ↔ mass; molarity (M) = moles per liter (mol/L).
Example: Chromium oxide CrO₂:
4.4 Mixing Solutions
Concentration concepts: grams per liter (g/L), molarity (M), parts per million (ppm), percent composition.
Percent solution:
Measure solute mass, measure total solution mass,
Serial dilutions: used for achieving very small final concentrations; concept of fixed dilution factor.
CHAPTER 5: BASIC DNA STRUCTURE
5.1 Basic Information
DNA stands for Deoxyribonucleic Acid; largest cellular molecule; genetic code contained in nucleotide sequence; inheritance through reproduction.
5.2 History of Research
Griffith (1928): transformation with dead virulent bacteria transforming live non-virulent bacteria; Avery–McCarty–MacLeod (1944): DNA as the transforming material; Chargaff (late 1940s): A=T and C=G rule; Rosalind Franklin (1951): X-ray crystallography revealed DNA structure; Hershey–Chase (1952): DNA as genetic material; Watson & Crick (1953): DNA double-helix model with antiparallel strands; Nobel prizes awarded in 1962 (crick/wilkins) and 1968–1980s to various contributors.
5.3 DNA Structure
Nucleotides consist of sugar (deoxyribose), phosphate, and a nitrogenous base (A, T, G, C).
Backbone: sugar–phosphate; bases pair via hydrogen bonds (A–T with 2, G–C with 3).
Strands are antiparallel (5' to 3' vs 3' to 5'); 3' end lacks phosphate, 5' end has phosphate; sequence written 5'→3' along each strand.
Base pairing maintains equal amounts of A/T and G/C across the two strands.
5.4 DNA Extraction
Steps overview: lyse cells to release DNA; degrade proteins; precipitate DNA with salt; wash with cold alcohol; re-suspend; confirm via electrophoresis.
Applications include FISH, T-RFLP, sequencing; DNA extraction can target cheek cells, bacteria, viruses, etc.
CHAPTER 6: DNA PROFILING & GEL ELECTROPHORESIS
6.1 A Brief Historical Introduction
Electrophoresis evolved from paper to gel-based methods; gels provide sieving through a porous matrix; DNA movement is length-dependent.
6.2 Agarose Gel Electrophoresis of Nucleic Acids
Nucleic acids migrate toward the positive electrode; shorter fragments move faster; effective size range for dsDNA in agarose ~100 bp to 25,000 bp; very long fragments exhibit limiting mobility.
Concept of a linear DNA ladder and “nested sets” in sequencing contexts.
6.3 DNA Fingerprinting (Beginner's Guide)
Six steps: extract DNA, cut with restriction enzymes, separate fragments on gel, transfer to paper (Southern blot), probe with radioactive or labeled DNA, visualize via film.
Probes exploit tandem repeats (STRs or VNTRs) to generate a distinctive pattern.
Forensics uses STRs (short tandem repeats) and VNTRs (variable number tandem repeats) for identity testing.
6.4 RFLPs (Restriction Fragment Length Polymorphisms)
Exploits variations in DNA sequences; digestion with restriction enzymes yields different fragment patterns; historically foundational to DNA profiling; now largely supplanted by PCR-based methods due to sensitivity requirements.
6.5 VNTRs
Regions with tandem repeats; length variations across individuals; used in identification and forensic analysis.
6.6 DNA Fingerprinting Technology / DNA Profiling
Practical uses: crime scene analysis, paternity testing, missing persons, historical investigations; privacy concerns.
Candy Electrophoresis Lab Background; Paternity Testing Lab Background; Restriction Digestion Analysis of Lambda DNA Lab Background; (Crime Scene) DNA Fingerprinting Using Restriction Enzymes Lab Background – Applied labs accompanying the topics above.
CHAPTER 7: DNA REPLICATION
7.1 Introduction to Replication
DNA replication produces two identical copies from one molecule; semi-conservative replication; origins of replication; replication forks expand bidirectionally.
7.2 Short Review of DNA Structure
Double helix; nucleotides with sugar, phosphate, and base; base pairing; antiparallel strands; 3' and 5' ends; directionality of synthesis.
7.3 DNA Polymerase
Polymerases extend existing 3' end; require a primer; DNA Polymerase III builds new strands; energy from hydrolysis of phosphate bonds in dNTPs.
7.4 Replication Process
Three phases: initiation (origins opened by initiator proteins; AT-rich regions easier to unwind), elongation (leading strand synthesized continuously, lagging strand via Okazaki fragments; RNA primers removed and replaced with DNA; ligation fixes nicks), termination (for prokaryotes, forks meet; for eukaryotes telomeres). Telomeres shorten in somatic cells; telomerase maintains telomeres in germ cells.
7.5 DNA Replication Proteins
Key players: Helicase, DNA Polymerase III, clamp, SSBs, Topoisomerase, DNA Gyrase, DNA Ligase, RNA Primase, Telomerase.
CHAPTER 8: PCR
8.1 Introduction to PCR
PCR amplifies a targeted DNA region using DNA polymerase and primers; generates billions of copies from small starting material.
8.2 History of PCR
Early work (Kleppe et al., 1971) described in vitro DNA amplification; Mullis (1983) conceived PCR; Nobel Prize (1993) for Mullis; PCR transformed genetic testing, mapping, sequencing, etc.
8.3 PCR Step-by-Step
Cycle steps: denaturation at ~94°C (separates strands), annealing ~54–60°C (primers bind), extension ~72°C (DNA polymerase extends).
Each cycle doubles the amount of target DNA; after ~30–40 cycles, substantial amplification occurs; analysis via gel electrophoresis.
A typical cycle yields exponential growth: after n cycles, copies ≈ 2^n (assuming 100% efficiency).
8.4 PCR Cycles Review
Mapping of cycle steps: Denaturing (C), Annealing (A), Extension (B) as shown in practice.
8.5 PCR-Type DNA Testing
Two main methods: PCR-based amplification for analysis; non-amplified (RFLP) methods when sufficient DNA is available; PCR is more sensitive but more prone to contamination.
8.6 Taq Polymerase
Thermostable DNA polymerase from Thermus aquaticus enables high-temperature cycling; eliminates need to replenish enzyme after denaturation steps.
8.7 What is PCR Used For?
Applications include DNA fingerprinting, pathogen detection, genetic disease diagnosis, sequencing support, and forensic testing.
8.8 Limitations of PCR
Plateau effect due to inhibitors and depletion of reagents; end-point quantification challenges; contamination risk.
8.9 Using PCR to Sequence DNA – The Sanger Method
Manual sequencing via Sanger method uses ddNTP terminators; four reactions with each ddNTP; polyacrylamide gel separation; visualization via autoradiography or fluorescence.
8.10 Fully Automated Sequencing – Computer Mediated Sequencing
Modern automated sequencing uses fluorescent labels and laser detection; high-throughput sequencing in a single lane possible; require careful data interpretation for signal quality.
PV92 Gene Lab Background; DNA Sequencing Lab Background – practical labs accompanying the PCR & sequencing chapters.
CHAPTER 9: RECOMBINANT DNA TECHNOLOGY
9.1 Ribonucleic Acid (RNA)
RNA overview: roles in transcription, translation, and regulation; mRNA, tRNA, rRNA; RNA can also have catalytic roles (ribozymes); RNA-based regulation relates to various diseases.
9.2 RNA Structure
RNA bases: A, G, C, U; single-stranded; ribose sugar; differences from DNA (no T); structural implications.
9.3 Protein Synthesis
Central dogma: DNA → RNA → protein; transcription adds RNA copy; translation uses ribosomes to synthesize polypeptides; codons (triplets) specify amino acids; tRNA anticodons pair with mRNA codons; ribosome sites A, P, E; methionine as start codon; N- and C- termini orientation; post-translational modifications.
9.4 Manipulating DNA
DNA manipulation and gene transfer enable production of recombinant proteins (e.g., insulin, growth hormone). Bacterial transformation serves as a foundation for biotech production.
9.5 Central Dogma of DNA
Re-statement of DNA → RNA → Protein; gene as DNA sequence; mRNA as template; translation yields phenotype.
CHAPTER 10: GENETIC ENGINEERING
10.1 What is Genetic Engineering?
Recombinant DNA technology: altering genes to create GMOs; major phases: synthesis of recombinant DNA and gene cloning.
10.2 Techniques of Genetic Engineering
Gene isolation, vector selection, cloning of desired gene, gene transfer, selection of transformants, replication of recombinant DNA in host, and propagation of clones; in practice, two routes: DNA cloning in a vector or using PCR for amplification.
10.3 Restriction Enzymes
Restriction enzymes cut DNA at specific sites; recognition sequences are palindromic; most cuts generate sticky ends; Type II restriction enzymes cut within recognition sequences.
Enzymes named after organism of origin (e.g., EcoRI). Methylases protect host DNA from restriction.
Gel electrophoresis separates fragments by size to analyze digestion patterns.
10.4 DNA Ligase
DNA ligase seals nicks to complete DNA backbone after restriction fragment joining; works with compatible ends.
10.5 Vectors
Vectors carry foreign DNA into host cells; must have origin of replication, selective marker, cloning sites, suitable size, and transcription control elements; circular plasmids are common
10.6 Plasmids
Common vectors (e.g., pBR322); features include ori, multiple restriction sites, antibiotic resistance markers; used to clone and propagate recombinant DNA; transformation efficiency and selection strategies.
10.7 Gene Transfer
Methods: heat shock, electroporation, viral delivery, plant transformation via Agrobacterium Ti plasmid, gene gun, microinjection, liposomes; markers used to identify transformed cells (e.g., antibiotic resistance).
Transformation efficiency statistics and selection strategies; on/off inducible promoters for regulated gene expression (e.g., IPTG induction of lac operon in plasmids; T7 RNA polymerase system).
10.8 Genetic Technology in Perspective
Historical overview from Descartes to Boyer & Cohen; contributions of Berg, Cohen, and others; definition from European Federation of Biotechnology: integrating natural science and organisms for products and services.
10.9 Principles of Genetic Engineering
Two core techniques: genetic engineering and sterile bioprocessing; safe design to avoid unwanted gene transfer; risk management and containment.