1. Microbial Biotechnology
1.1. 1
1.1.1. Infectious agents
1.1.1.1. causes microbial diseases: minor irritants cause death
1.1.1.2. how do these cause diseases?
1.1.1.2.1. these agents destroy living cells/tissues of infected organism
1.1.1.2.2. they also release chemicals that interferes with normal activities of cells
1.1.1.3. Bioterrorism
1.1.1.3.1. refers to biological agents used as weapons to further personal or political agents
1.1.2. Therapeutic agents
1.1.2.1. are
1.1.2.1.1. Health Products
1.1.2.1.2. Vaccines
1.1.2.1.3. Drugs
1.1.2.1.4. Gene therapy using virus
1.1.2.2. Example
1.1.2.2.1. Penicillium Chrysogenum
1.1.3. Shape of Prokaryotes
1.1.3.1. Rod; bacillus [singular] ; bacillo [plural]
1.1.3.2. Corkscrew; spirillum [singular] ; spirilla [plural]
1.1.3.3. Spherical; coccus [singular] ; cocci [plural]
1.1.3.4. curved rods; Vibrio(s)
1.1.4. Binomial Naming System
1.1.4.1. Identification: Group/Family Genus Species Strain
1.1.4.2. E.g. Staphylococcus aureus
1.1.4.2.1. Genus Name: Staphylo
1.1.4.2.2. Shape: coccus
1.1.4.2.3. Species Name: Aureus
1.1.5. Pure Cultures
1.1.5.1. If bacteria allowed to grow it forms a colony
1.1.5.2. Pure Colony is colony that contains only one strain of microorganisms
1.1.5.3. All colony originates from one bacteria cell
1.1.5.3.1. Therefore genetically identical
1.1.5.4. All study requires (homogeneous) pure strains as a start.
1.1.6. Techniques to isolate bacteria
1.1.6.1. 16 streak technique
1.1.6.1.1. Isolating bacteria to study characteristics of each species
1.1.6.1.2. Repeat 16 streak to confirm if the colony ispure
1.1.6.2. Quantifying Bacteria
1.1.6.2.1. Serial Diffusion
1.1.6.2.2. CFU is the unit to indicate the number of bacteria that is capable of multiplying into a colony (cfu/ml)
1.1.7. Aseptic Techniques
1.1.7.1. Cover spread plate with cover
1.1.7.2. Flaming mouth of vessels
1.1.7.3. Dipping the L-shaped spreader in ethanal
1.1.7.4. Using spirit Lamp to provide sterile environment (working near flame)
1.2. 2
1.2.1. Bacteria Reproduction
1.2.1.1. Binary Fission
1.2.1.1.1. Typical bacterial cell with DNA
1.2.1.1.2. Cell enlarges and DNA replicates
1.2.1.1.3. New cell wall forms and the daughter DNA strands separate
1.2.1.1.4. Cells separate to form two genetically identical daughter cells
1.2.2. Bacterial Growth Curve
1.2.2.1. Lag Phase
1.2.2.1.1. DNA Replication
1.2.2.1.2. adapting to new environment
1.2.2.1.3. build up growth tools like enzymes
1.2.2.1.4. cells increase in size but do not divide
1.2.2.2. Log/Exponential Phase
1.2.2.2.1. exponential cell growth
1.2.2.2.2. highest reproduction and metabolic rate
1.2.2.3. Stationery Phase
1.2.2.3.1. slow down of growth due to depletion of nutrients
1.2.2.3.2. death rate = growth rate
1.2.2.4. Death Phase
1.2.2.4.1. exponential death rate
1.2.2.4.2. death rate > growth rate
1.2.3. Methods of bacterial enumeration
1.2.3.1. Total Cell Count
1.2.3.1.1. Haemocytometer
1.2.3.2. disadvantage of total cell count method
1.2.3.3. Viable Cell Count
1.2.3.3.1. overview
1.2.3.3.2. Spread Plate Method
1.2.3.3.3. concept of Serial Dilution
1.2.4. Culturing Microbes
1.2.4.1. Macroelements
1.2.4.1.1. required in relatively large quanity
1.2.4.1.2. to build cell structures and help with metabolism
1.2.4.2. Micronutrients [trace elements]
1.2.4.2.1. required in trace amount to help enzymes function
1.2.4.3. places where nutrients go
1.2.4.3.1. carbon in all biomolecules
1.2.4.3.2. hydrogen and carbon in all biomolecules
1.2.4.3.3. nitrogen in proteins and nucleic acid
1.2.4.3.4. phosphorus, sulphur in proteins nucleic acid and organic molecules
1.2.4.4. Culture Medium
1.2.4.4.1. Contain all the nutrients required by the organism for growth
1.2.4.4.2. Can be classified into the following types
1.2.4.5. Bacterial Colony Appearance
1.3. 3
1.3.1. Morphology
1.3.1.1. Cell
1.3.1.2. Colony
1.3.2. Classification due to Gram Staining (cell wall)
1.3.2.1. Cell Wall
1.3.2.2. Gram Staining
1.3.2.2.1. Staining bacteria differently base on the properties of their cell wall
1.3.2.2.2. Gram-Positive
1.3.2.2.3. Gram-Negative
1.3.2.2.4. Procedure
1.3.2.3. KOH Test
1.3.2.3.1. Rapid Taste (same purpose as Gram Staining)
1.3.2.3.2. Gram Negative bacteria --> thinner cell wall --> easily lysed in 3% potassium hydroxide --> DNA strand exude --> visible by lifting (sticky)
1.3.2.4. Gram Positive
1.3.2.4.1. 90% peptidoglycan, high amount of teichoic acids
1.3.2.5. Gram Negative
1.3.2.5.1. 10% peptidoglycan have outer membrane called Lipopolysaccharides (LPS)
1.3.2.6. gram staining > KOH Test
1.3.2.6.1. why? Gram Staining allows us to see bacterial cell morphology
1.3.3. Classification based on sensitivity due to antibiotics
1.3.3.1. Antibiotics
1.3.3.1.1. Functions
1.3.3.1.2. Characteristics
1.3.3.2. certain antibiotics might have harmful side effects/toxicity when taken by humans due to the antibiotics affecting biological system of patients
1.3.4. Mode of action of antibiotics
1.3.4.1. inhibitors of cell wall synthesis
1.3.4.1.1. Sugar Molecules form the backbone of peptidoglycan layers
1.3.4.1.2. transpeptidase (enzyme) links up the layers via tetrapeptides (forming bacterial cell wall, peptidoglycan)
1.3.4.1.3. Penicillin
1.3.4.2. inhibitors of protein synthesis
1.3.4.2.1. many antibiotics bind specifically to bacterial ribosomes
1.3.4.2.2. Tetracylines
1.3.4.3. inhibitors of nucleic acid synthesis
1.3.4.4. inhibitors of cell membrane
1.3.5. DNA (nucleus) --(transcription through RNA Polymerase)--> MRNA (messenger) --(translation in ribosome)--> Protein (ribosomes in cytoplasm)
2. Labs!!
2.1. Aseptic Techniques and 16 streak
2.1.1. Aseptic Techniques are used to prevent contamination in cultures
2.1.2. principles of aseptic techniques
2.1.2.1. growth medium and container must be sterilised as soon as medium is prepared
2.1.2.2. container must always be covered to prevent the entrance of of microorganisms
2.1.2.3. instruments and fluids that touch inside of container, sterile medium or culture, must be sterilised first
2.1.2.4. must not contaminate your work area with cultures
2.1.3. Types of aseptic Techniques
2.1.3.1. Flame
2.1.3.1.1. when vessels are open, you need to work beside a spirit lamp
2.1.3.1.2. this sets up convection current which circulates air and contaminants in the air away from entering the culture
2.1.3.2. Flaming mouth of bottle
2.1.3.2.1. vessels must be open for least possible amount of time
2.1.3.2.2. after opening sterile bottle, mouth of bottle must be flamed immediately
2.1.3.2.3. flaming allows convection current to be away from opening preventing bacteria away from nearing bottle
2.1.4. 16 Streak Plate Technique
2.1.4.1. to separate different microorganisms to study small colonies
2.1.4.2. used to increase the distance of Colony Forming Units to isolate different types of microorganisms
2.1.4.3. all colonies from one single bacteria and all bacteria in colony would have identical characteristics
2.1.4.4. procedure
2.1.4.4.1. flame inoculating loop until red hot (sterilised)
2.1.4.4.2. dip inoculating loop into source/medium
2.1.4.4.3. do 4 streaks in one segment with lid covering and immediately close
2.1.4.4.4. flame loop again
2.1.4.4.5. for the next 12 streaks no need to dip into source
2.2. Pipettes
2.2.1. general
2.2.1.1. precision instrument to take precise and small amounts of liquids
2.2.1.2. tips can be autoclaved
2.2.1.3. comes in different ranges
2.2.2. usage of micropipettes
2.2.2.1. check the volume you want to take in
2.2.2.2. push the plunger to the first stop
2.2.2.3. insert tip into liquid
2.2.2.4. release plunger gently
2.2.2.5. push plunger to 1st stop to expel liquid (if liquid remains a drop on the pipette, tap/touch the tip on the side of vessel)
2.2.2.6. push plunger till second stop to completely expel liquid
2.2.3. usage of serological pipettes
2.2.3.1. used to take larger amount of liquids
2.2.3.2. the tips are sterile and you attach by shoving the tip into the pipette with care
2.2.3.3. take in liquids by rolling the wheel
2.2.3.4. release liquid through the wheel of lever
2.2.4. conversion of units
2.2.4.1. µl = microlitre
2.2.4.2. 1ml = 1000µl
2.2.4.3. 1µl = 1/1000ml = 0.001ml
3. Biomolecules
3.1. Definition: Biomolecules are organic compounds meaning they all contain hydrogen and carbon.
3.2. Why all biomolecules have carbon in them?
3.2.1. - Carbon 4th most abundant element in universe
3.2.2. - Carbon is special since it can make a maximum of 4 bonds
3.2.3. - Ability to produce long carbon chains like octane
3.2.4. - Ability to produce rings forms such as cyclohexane
3.3. Functions of Biomolecules
3.3.1. Store energy (carbs & lipids)
3.3.2. Formation of cell membrane (phospholipids)
3.3.3. Provide structural support (cellulose)
3.3.4. Help control chemical reaction in organisms
3.3.5. Store Hereditary information (nucleic acid)
3.4. Polymers
3.4.1. Polymers are macromolecules made of of repeating units of monomers
3.4.2. Monomer --> Polymer --> Full Organism
3.4.3. Synthesis of Polymers
3.4.3.1. Dehydration/Condensation Reaction: removal of water molecule to connect monomers into polymers (enzymes and energy required)
3.4.3.2. Hydrolysis Reaction: addition of water molecule to disassemble polymers into monomers (enzymes required)
3.5. Carbohydrates
3.5.1. Functions
3.5.1.1. Store energy
3.5.1.2. Important building blocks of other biomolucules
3.5.1.3. structural components of a cell
3.5.2. Classes of Carbohydrates
3.5.2.1. Monosaccharides Single sugar molecule with 3-7 carbon molecules
3.5.2.2. Disaccharides Two monosaccharides joined during dehydration reaction
3.5.2.3. Polysaccharides Complex polymers of monosaccharides
3.6. Lipids
3.6.1. long-chained hydrocarbon molecules with the carboxylic acid (-COOH) functional group
3.6.2. not polymers but can be formed through dehydration and broken down through hydrolysis
3.6.3. greasy and oily compounds e.g. triglycerides, phospholipids, cholesterols, steroids
3.6.4. Triglyceride
3.6.4.1. Structure: a glycerol bonded with 3 fatty acids
3.6.4.2. Functions
3.6.4.2.1. energy storage
3.6.4.2.2. insulation
3.6.4.2.3. cushioning of organs and tissues
3.6.5. Phospholipids
3.6.5.1. Structure: a glycerol linked to a phosphate group and 2 fatty acid chains
3.6.5.2. Function
3.6.5.2.1. main component of cell membrane
3.6.6. Cholestrol
3.6.6.1. Structure: 4 interconnected carbon rings (special)
3.6.6.2. Functions
3.6.6.2.1. stabilize cell membrane
3.6.6.2.2. used by the body to synthesise hormones as signalling molecules
3.6.6.2.3. made into bile to help in digestion
3.6.7. Saturated VS Unsaturated Fat
3.6.7.1. Saturated: no double bond, solid at room temp, originate from animals
3.6.7.2. Unsaturated: one or more double bonds, liquid at room temp, originate from plants
3.7. Proteins
3.7.1. Consists of C, H, O and N elements
3.7.2. Functions
3.7.2.1. Mechanical Support/Movement
3.7.2.2. Enzymes
3.7.2.3. Transport
3.7.2.4. Defense
3.7.2.5. Body Regulatory
3.7.2.5.1. hormones
3.7.2.6. Support
3.7.2.6.1. keratin, collagen
3.7.3. Amino Acids
3.7.3.1. building blocks of protein
3.7.3.2. joined together via peptide bonds to form a polypeptide chain
3.7.3.3. Synthesis and Degradation of Protein
3.7.3.4. All proteins are folded into a specific 3d shape. Loss of Structure = Loss of function
4. Cells
4.1. Basic Properties of Cells
4.1.1. can replicate themselves
4.1.2. metabolize catabolize: break down compounds to produce simple compounds and elements anabolize: synthesize molecules required by cells
4.1.3. respond to stimuli
4.1.4. die
4.2. Eukaryotic Cells
4.2.1. Overview
4.2.1.1. membrane -bound nucleus
4.2.1.2. membrane-bound organelles
4.2.1.3. compartmentalize
4.2.1.4. cytoskeleton
4.2.2. Structures of Eukaryotic Cell
4.2.2.1. Cell Membrane
4.2.2.1.1. Aka Plasma Membrane
4.2.2.1.2. Structure
4.2.2.1.3. Fun Fact!!!
4.2.2.1.4. Is known to have a Fluid Mosaic Model
4.2.2.1.5. Functions
4.2.2.1.6. Movement of substances in cell membrane
4.2.2.2. Nucleus
4.2.2.2.1. Structure
4.2.2.2.2. Functions
4.2.2.3. Ribosomes
4.2.2.3.1. Structure
4.2.2.3.2. Functions
4.2.2.3.3. Location
4.2.2.4. Rough and Smooth Endoplasmic Reticulum
4.2.2.4.1. RER
4.2.2.4.2. SER
4.2.2.5. Golgi Apparatus
4.2.2.5.1. Structure
4.2.2.5.2. Functions
4.2.2.6. Mitochondria
4.2.2.6.1. Structure
4.2.2.6.2. Functions
4.2.3. PLANT CELL EXCLUSIVE STRUCTURES
4.2.3.1. Plant Vacuole
4.2.3.1.1. Structure
4.2.3.1.2. Fucntions
4.2.3.2. Chloroplasts
4.2.3.2.1. Structure
4.2.3.2.2. Functions
4.3. Prokaryotic Cells
4.3.1. Prokaryotes
4.3.1.1. refers to bacteria
4.3.1.2. small
4.3.1.3. simple
4.3.1.4. absence of nucleus
4.3.1.5. absence of membrane-bound nucleus
4.3.2. Structure of Prokaryotic Cell
4.3.2.1. Cell Wall
4.3.2.1.1. Structure
4.3.2.1.2. Functions
4.3.2.2. Cell Membrane
4.3.2.3. Cytoplasm
4.3.2.3.1. Structure
4.3.2.3.2. Contains
4.3.2.4. Pilus
4.3.2.4.1. Structure
4.3.2.4.2. Function
4.3.2.5. Fimbrae
4.3.2.5.1. Structure
4.3.2.5.2. Function
4.3.2.6. Glycocalyx
4.3.2.6.1. Structure
4.3.2.6.2. Function
4.3.2.7. Flagella
4.3.2.7.1. Structure
4.3.2.7.2. Function
4.3.2.8. Endospores
4.3.2.8.1. Structure
4.3.2.8.2. Function
5. Gene Technology
5.1. 1
5.1.1. Structure and Function of DNA
5.1.1.1. Building block of DNA
5.1.1.1.1. Nucleotides
5.1.1.1.2. DNA is a type fo nucleic acid
5.1.1.1.3. Monomer
5.1.1.1.4. Macromolecule
5.1.1.1.5. Nucelotides connect together to form a long chain
5.1.1.1.6. DNA IS A DOUBLE-STRANDED HELIX
5.1.1.1.7. two strands of polynucleotide are antiparallel to each other
5.1.1.2. Functions of DNA
5.1.1.2.1. Store genetic information on how to make proteins
5.1.1.2.2. DNA has the ability to
5.1.2. DNA, Genes, Chromosomes & Genome
5.1.2.1. Each DNA molecule has many genes
5.1.2.1.1. Each DNA molecule coiled into highly compact structure called chromosome.
5.1.3. DNA Replication
5.1.4. Mutation
5.1.4.1. Proteins detrrmine characteristics of an organism
5.1.4.1.1. proteins made up of amino acid
5.1.4.1.2. proteins have many functions
5.1.4.1.3. DNA sequence determine sequence of amino acids, which in turn determine type of protein and its function
5.1.4.1.4. Mutation → change of amino acid sequence, (may result in) lost 3D structure → lost function
5.1.4.1.5. Denaturation → lost 3D structure → lost function
5.1.4.2. Point-mutation
5.1.4.2.1. base-pair in the gene is substituted, added or deleted
5.1.4.2.2. the consequence depends on how the change in codon affects the protein
5.1.4.2.3. Effects of point mutation
5.2. 2
5.3. 3
5.3.1. how do we see plasmid? (does our DNA have our desired gene of interest?)
5.3.1.1. Agarose Gel Electrophoresis
5.3.2. DNA is negatively charged since phosphate group in DNA is negatively charged.
5.3.3. materials required for agarose gel electrophoresis
5.3.3.1. An electrophoresis chamber and power supply
5.3.3.2. Gel casting tray and comb
5.3.3.3. Agarose gel
5.3.3.4. Electrophoresis buffer e.g. Tris-borate-EDTA (TBE)
5.3.3.5. Loading dye
5.3.3.6. DNA staining agent e.g. SYBR Green
5.3.3.7. UV Transilluminator
5.3.4. Steps:
5.3.4.1. Makes the gel
5.3.4.1.1. materials required
5.3.4.1.2. Steps
5.3.4.2. Set up electrophoresis box
5.3.4.2.1. materials required
5.3.4.2.2. Steps
5.3.4.3. Electricity time!!!
5.3.4.3.1. 1. Place back cord from electrohporesis into matching outlet of power supply
5.3.4.3.2. Repelled by negative charge the DNA strand moves towards the positive charge
5.3.4.4. Stain the gel
5.3.4.4.1. 1. Use stain called ethidium bromide binds to DNA and shows up flourescent light
5.3.4.4.2. 2. Drop mold into staining solution
5.3.4.4.3. 3. Drop the mold into UV Light Box
5.3.5. - higher percentage of agarose - smaller pore size - allows better separation and resolution of smaller DNA fragments
6. helicase unwinds helix and seperates two strands - material required for DNA replication
6.1. Template DNA
6.1.1. provides template for synthesis of new DNA strands
6.2. Helicase
6.2.1. unwinds helix and separates both strands
6.3. DNA Polymerase
6.3.1. synthesise new DNA strands
6.4. dNTPs
6.4.1. building blocks for new DNA strands