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Monomer link to form polymers: condensation/hydrolysis

Overview
02 October 2014

14:40

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Cells
• 70% water
• 30% chemical
○ 15% protein
○ 4% small molecules
○ 6% RNA
○ 2% phospholipids
○ 1% DNA
○ 2% polysaccharides

Carbohydrates
Plant structure - cellulose
Plant storage - starch
Animal storage - glycogen
Animals eat for energy - starch

Starch
○ 2 polymers of glucose
 Amylose
 Amylopectin
○ Coiled up
Cellulose
○ Long, thin fibrils
○ Pack together
○ More than half of all organic carbon on
Earth

Nucleic acids
• In all cells
• Composed of nucleotides
• Store and transmit
heredity/genetic information
• DNA and RNA
• Genetic code
○ Instruction
 Direct cell
behaviour
 Encodes proteins
○ RNA directs protein
synthesis
• Function:
○ Storage
○ Transmission
○ Use
• Structure:
○ 5-C sugar
○ Phosphate group
○ 4/5 bases
 Adenine
 Thymine
 Guanine
 Cytosine
 Uracil

Proteins
• Polymers of amino acids
• Functions:
○ Structural support
○ Catalysis
○ Transport
○ Defence
○ Movement
○ Regulation
○ Storage
○ Hormonal
• Peptide bonds
○ Joins amino acids
○ Carboxyl and amino groups
• 20 amino acids
○ Distinguished by side chains
• Structure:
○ Primary - amino acids
○ Secondary- alpha helix, beta pleated
sheet
○ Tertiary - 3D structure
○ Quaternary - Multiple 3D structures,
prosthetic groups
• Affected by:
Denaturation
○ pH
○ Temperature

Biological M ^M M Page 1

Function is related to structure

Lipids
• Fats, oils, waxes, steroids
• Functions:
○ Energy storage
○ Protection and insulation
○ Structure in membranes
• Animal fat - solid at room temperature
• Oils and waxes
○ Oils liquid, waxes solid, at room temperature
○ Mostly in plants
• Fats and oils
○ Triglycerides
 Fatty acids and glycerol
○ Fats solid at room temperature
○ Oils liquid at room temperature
• Steroids
○ Role in reproductive hormones
○ Cholesterol
 Insoluble in water/blood
 Dietary
 Synthesised and broken down in liver
 HDL, blood to liver
 LDL, liver to blood --> plaques
 Part of cell membrane
• Non-polar
○ Do not dissolve
 Except for phospholipids, due to polar phosphate

Nucleic Acids
13 October 2014

11:07

History
• 19th century
○ Charles Darwin
○ Gregor Mendel
○ Johan Miescher
 Interested in white blood cells and phosphorous content
 Collected
 Washed in detergent
 Separated
 Removed proteins
 Precipitated "nuclein"
□ DNA and RNA

DNA - deoxyribose nucleic acid
RNA - ribonucleic acid
Structure
Both made of nucleotides, made of nucleosides
Base + ribose/deoxyribose = nucleoside
Nucleoside + phosphate = nucleotide

Bases

Nucleotides are bonded by strong covalent bonds
Phosphodiester

Naming nucleotides
Purine
• -ine to -osine
• Adenosine
• Guanosine
Pyramidine
• Cytodine
• Thymadine
• Uridine
Phosphate
• -5' monophosphate

Cytosine + deoxyribose + phosphate
Deoxy-cytodine-5'-monophosphate

If DNA then +deoxy

Formation
• Nucleic acid = polynucleotide
• Dehydration reaction
○ Water eliminated
Discovery
Fred Griffith - 1928
• Experiments with pneumonia
○ Rough strain - non virulent
○ Smooth strain - virulent
○ Heat killed smooth - non-deadly
○ Rough + heat-killed = deadly
"Something heat-stable from virulent bacteria can transform non-virulent bacteria to cause disease"

(died in 1941)
Oswald Avery
Control

Chloroform - no
protein

Enzyme - no carbs

DNase - no DNA

DNA ppte - only
DNA

Rough and heatkilled smooth

Rough and heatkilled smooth

Rough and heatkilled smooth

Rough and heatkilled smooth

Rough and heatkilled smooth

Mouse died

Mouse died

Mouse died

Mouse survives

Sequences
• 5' to 3'
• 5' phosphate group attached to 1 pentose ring
• 3' unreacted -OH at C3, reacts with phosphate

Mouse died

Transforming principle = DNA

Erwin Chargaff
• Interested in sperm
• Isolated DNA
○ Salmon and sea urchin
1
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3
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Extracts DNA
Hydrolyses with acid
Separates nucleotide - paper chromatography
Concentrations of each measured using spectrophotometry

• Results
○ Molar ratio
 Adenine:Thymine = 1
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 Purines:Pyramidine = 1
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64-1
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58-1
...
Purines always pair with pyrimidines

Biological M ^M M Page 2

Structure
• Incorrect theories
○ Leven (1910) - Tetranucleotide
 Proven wrong by Chargaff
○ Pauling and Corey (1953) - Triple Helix
• Rosalind Franklin's X-ray crystallographs of DNA led Crick and Watson to develop their theory of
the double helix
• Double helix
• AT, CG
• Antiparallel
• Constant sugar phosphate backbone
• dCMP (deoxy-cytodine-5'monophosphate) - dAMP - dCMP - dGMP (deoxy-guanosine-5'
monophosphate) - dTMP
○ 5' - CACGT - 3'
○ 3' - GTGCA - 5'
• Reversible bonding
○ H bonds
• Semi-conservative duplication
○ Each new strand is 1/2 original
• 1 turn of the helix is 34 Angstroms
• Structure confirmed
○ Scanning tunnelling microscope

The central dogma of molecular biology
DNA makes RNA makes Protein
• Replication - DNA to DNA
○ DNA polymerase
• Transcription - DNA to RNA
○ RNA polymerase
• Translation - RNA to protein
○ Ribosome

Amino acids are produced by codons
moving through ribosomes
Degenerate code

Biologist's Toolkit
20 October 2014

11:04

What drives scientific progress
• New problems
• New ideas
• New tools
Making progress, tackling problems, and proving ideas

Preparative work
Preparing biological molecules for analysis
Qualitative work
Identifying the presence of biological molecules
Quantitative work
Quantifying the number of biological molecules

Qualitative
It is easier if you separate compounds
• Lysis
○ Breaking up cells, organs, tissues
 Detergents (SDS)
 Bead beating, heat, grinding, ultrasound
• Solvent Extraction
○ Partitioning
○ Immiscible
 Hydrophilic
 Hydrophobic
○ Soluble
○ Chromatography
 Different affinity for mobile and solid phase
○ Purifying proteins
 Green fluorescent protein (GFP)
 Impurities washed from column
 Only GFP in column
 Different solvent used to wash it out (elution)
• Centrifugation
○ Differential centrifugation
 Dense particles sediment more easily
□ Supernatant - lighter stuff at the top
□ Pellet - denser particles
○ Density gradient centrifugation
 Liquids of varying density
 Solid matter collects at the boundary
between liquids
 Cells, bits of cells, and molecules can be
separated by density
• Electrophoresis
○ Separating molecules by size and/or shape
○ Often used for separating DNA or RNA fragments
DNA and RNA are uniformly negative due to the
phosphate backbone
The will be drawn towards a positive electrode
○ DNA/RNA gel electrophoresis
 Agarose gel used
 Smaller molecules will move through faster
than bigger molecules
 Equally negative molecules will be separated
by size
 DNA can be stained using fluorescent dyes
 Control markers are used for comparison

Quantitative
• When is qualitative not enough?
○ Change in concentration
○ Yield
○ Dose-dependent
○ Compliance

Example
Alkaline lysis extraction of plasmid DNA from bacteria
• Adding sodium hydroxide and a detergent (sodium dodecyl sulphate SDS)
○ pH to 11-12
○ Bacteria break open
○ DNA becomes single stranded
○ Proteins (including DNase) denature
• Acetic acid
○ pH to 4
• Centrifuge
○ Supernatant - plasmids
 Small and circular strands stay soluble and rebind quickly due to high tension
○ Pellet - chromosomal DNA and proteins
 Chromosomal DNA rebinds wrong - tangled mess
• Chromatography used to purify the solute
○ Positively charged silica in the top
○ Centrifugation
 Supernatant - mostly plasmid stuck to beads
 Pellet - dissolved chromosomal DNA gets through the column
○ Elution liquid and centrifugation
 Pure plasmids can be separated

Accuracy and Precision
• Accuracy
○ How close is it to the true value
• Precision
○ Consistently in the same place

UV-Visible Spectroscopy
• Molecule absorbs radiation
• Excited state
○ Photons interact with electrons
○ Move to higher energy levels
○ Emit excess energy as heat or light
Spectrofluorimetry
• Natural fluorescence
• Artificial stain
• DNA
○ Series of peaks given
○ Genome can be sequenced

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Identification
• Mass
• Composition
• 3D structure

Nuclear Magnetic Resonance Imaging
• Alignment of nuclei to magnetic field
• Absorb energy
• Energy required depends on
○ Field strength
○ Nearby atoms
• Release of energy to return to original state

Mass Spectrometry
• Clue to identity
• Detection and identification in mixture
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Vapourised
Ionised
Accelerated
Deflected
Detected

• Heavier particles go in a straighter line
• The energy needed to deflect the particles allow the
mass to be calculated
• Calibration
○ Testing against standards of known quantity

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Beer-Lambert Law
Relates absorbance to concentration

A=
Absorbance = molar absorptivity (Lmol-1cm-1) x concentration molL-1) x
distance travelled by light

Biological M ^M M Page 3

Spectroscopy
• Interaction of chemical compounds with
electromagnetic radiation
○ Change in nuclear arrangement
○ Molecular radiation
○ Vibrations
○ Excitation of electrons
• Identification by peak absorption
○ Amino - 198nm
○ Carbonyl - 186nm and 280nm

 Limit of Detection (outside of 'noise')
 Limit of Quantification
 Limit of Linearity

Measurements, Units, and Solutions
27 October 2014

10:59

As scientists we measure natural phenomena
We need to use the same scales of measurement
We always use the Systeme Internationale

A = Ampere
K = Kelvin
Mol = moles
s = second
cd = candela
Kg = kilogram
m = metre

Solutions
• Container - often volumetric flask
• Solvent - polar or non-polar
• Solute

Percentage Solution
Mass (g)/Volume (ml) x 100 = % solution (g100ml-1)
Weight per volume
Can be volume/volume
Liquid solute
Can be weight/weight

Moles
Number of moles = weight (g)/molecular weight
18
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1-0
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youtube
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Coli must also be true of an elephant" - Monod
Monod • Microbes prefer glucose
• A pause in bacterial growth occurs when all glucose has been consumed, before the bacteria can switch to
consuming another sugar - diauxic shift
• The glucose consumption genes are always turned on, other sugars must be switched on
• Sugar presence regulates which genes are expressed
• Adaptive regulation of genes

E
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lac operon

Repressor protein sits on operator, preventing expression
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Glucose blocks lactose transport, default "off" position giving priority
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XGal is broken down to a blue pigment by
lactases
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• Blue-white screening can be used to test for lactose intolerance and lac gene plasmid uptake
○ Only cells with plasmids grown on antibiotic treated agar
○ The plasmids carry the lac gene
○ The lac gene is broken if a DNA fragment is ligated into its middle - lactose intolerant - white culture
○ No fragment --> lac works --> XGal turns blue
○ White colonies contain the desired fragment
So we can swap genes between organisms, and even make them produce proteins
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 New system:
□ Asperigillus migar is a fungus used in
foodstuffs and is considered safe
□ Researchers measured which genes are
turned on by arsenic exposure
□ They discovered one gene, which makes a
protein pump to remove arsenic, is
expressed 200x if arsenic is present
□ Green Fluorescent Protein was fused to
the pump
□ Arsenic causes the fungus to glow green
○ Paper based Ebola test
 Blood sample on strip
 Colour change
□ Presence
□ Strain
 Synthetic gene networks which turn on or off in
the presence of Ebola
○ Diesel from E
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coli
• Prospects
○ Potential solutions to heath and envornment
challenges
○ Limited by imagination and genetic diversity
○ Risks? Environmental/terrorism/health impacts
○ Ethics - should we be doing this?

Lipids
06 November 2014

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15:03

Not a defined functional group
Soluble in non-polar solvents
Very limited solubility in water
Few polar or charged groups

• Amphipathic - mix of polar and non-polar groups
• Hydrolysable and non-hydrolysable

Compartmentalisation of eukaryotic cells
• Lysosomes
○ Bilayer
○ Inner and outer aqueous zones
• Plasma membranes
• Golgi apparatus
• Nucleus
• SER
• RER
• Nucleolus
• Chromatin
• Mitochondria

Where?
• In all living cells
• Adipocytes
○ Lipid storage
• Germinating plant seeds
○ Energy reserve
• Biological membranes
• Blood
• Better than sugars for energy
○ Carbons more reduced
○ Hydrophobic
 Do not dissolve
 Can be stored

Waxes
• Plants
• Animals
○ Vertebrates
 Ducks
○ Invertebrates
 Insects
□ Bees

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Fatty Acids
• All hydrolysable lipids contain fatty acids
• Long chains

Sperm Wales
• Spermaceti
○ Blubbery mass - 18000kg
○ 1/3 of weight

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Human Body
• Vitamins D, A, K
• Bile salts
• Eicosanoids - local hormones
• Androgens and oestrogens
• Glycerophospholipids, sphingolipids, and cholesterol - membranes
• Brain
○ High concentration of lipids in CNS
 Second only to adipose tissue
○ Cell signally
○ Tissue physiology
○ Lipid imbalances
 Neurological disorders
 CNS injury
○ Lipid damage
 Oxidative stress
 Polyunsaturated fatty acids
□ Lipid peroxidation

• Saturated and unsaturated fatty acids
Steroids

○ Cis double bonds prevent the fatty acids from lining up, reducing packing
density

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○ ω (is the last carbon in the chain
○ ω - [no
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• Formation of assemblies
○ Synergistic action
• Diversity
○ Rigidity vs
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 Side chains stick out

○ L-isomer more common
• 20 amino acids
○ 20 side chains
○ Size
○ Shape
○ Charge
○ H-bonding
○ Hydrophobia
○ Chemical reactivity
• Essential aas in diet

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○ pH changes the proportion, and thus the overall structure
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 Ferritin is almost entirely alpha helices
○ Primary structures can line up, running in alternate directions,
joined by hydrogen bonds
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g
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g
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00)
○ Lactose 0
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• Sugars are also involved in other molecules
○ Glucosamine - involved in chitin

Biological M ^M M Page 15

Water and Hydrogen Bonding
01 December 2014

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11:04

Essential for life
Cells 75-95% water
Covalent
Solid, liquid and gas

Ionic vs
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4 bonds per water molecule
• DNA
○ Between strands
○ Pyramidine to purine
• Proteins
○ Folding
 Brings groups together
 Interaction between C=O and H-N
□ Amino acid side chains

Biological M ^M M Page 17

Redox
01 December 2014

11:21

Oxidation reaction
• Addition of oxygen
○ 2H2+O2-->2H2O
○ H oxidised
 Electrons lost to oxygen
• Loss of electrons
○ Cu --> Cu2+ + 2eReduction reactions
• Gain of electrons
• Loss of oxygen

Oxidation
Is
Loss
Reduction
Is
Gain
OIL RIG

Redox
• Reduction and oxidation simultaneously
• Cellular respiration
C6H12O6 + 6O2 --> 6CO2 + 6H2O
NAH+ + H <--> NADH

Redox at electrode surfaces
• Redox reaction in solution consumes and releases electrons in equal quantities at each electrode

Redox Based Biosensor
• E
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diabetes blood-sugar sensor
1
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Glucose converted to glucondactone by an enzyme
3
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The electron is transported to an electrode
5

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