Microorganisms and Biotechnology

Classification of Microorganisms

Microorganisms are organisms that are too small to be seen clearly with the naked eye. They are found in all three domains of life and include bacteria, fungi, and viruses (though viruses are not considered true living organisms).

Bacteria (Prokaryotes)

Structure:

Feature	Description
Cell wall	Made of peptidoglycan (murein); provides shape and protection
Cell membrane	Phospholipid bilayer; controls movement of substances
Cytoplasm	Contains ribosomes (70S), plasmids (small circular DNA), and metabolic enzymes
Nucleoid region	Contains a single, circular, naked DNA molecule (not enclosed by a nuclear envelope)
Flagella	Some bacteria have whip-like flagella for locomotion
Pili (fimbriae)	Hair-like appendages for attachment to surfaces or conjugation
Capsule	Some bacteria have a slimy outer layer for protection against desiccation and phagocytosis
Plasmids	Small circular DNA molecules carrying additional genes (e.g., antibiotic resistance)

Shapes of bacteria:

Shape	Description	Example
Cocci	Spherical	Staphylococcus
Bacilli	Rod-shaped	Escherichia coli
Spirilla	Spiral	Treponema pallidum
Vibrio	Comma-shaped	Vibrio cholerae

Reproduction:

• Binary fission (asexual): The DNA replicates, and the cell divides into two identical daughter cells. This is the primary method of reproduction. Under optimal conditions (adequate nutrients, suitable temperature), bacteria can divide every 20 minutes.

$1 \to 2 \to 4 \to 8 \to 16 \to \ldots \to 2^n$

Where $n$ is the number of divisions.

Growth curve of bacteria:

Phase	Description
Lag phase	Bacteria adapt to new environment; no significant growth
Log (exponential) phase	Rapid cell division; population doubles at constant rate
Stationary phase	Growth rate equals death rate; nutrients become limiting
Decline (death) phase	Death rate exceeds growth rate; waste accumulates, nutrients depleted

Calculating bacterial population:

If a bacterium divides every 20 minutes, after $n$ divisions:

$\mathrm{Population} = 2^n$

Number of divisions in time $t$ minutes:

$n = \frac{t}{20}$

Fungi (Eukaryotes)

Structure:

• Fungi can be unicellular (yeasts) or multicellular (moulds, mushrooms)
• Multicellular fungi consist of mycelium -- a network of thread-like structures called hyphae
• Hyphae may be septate (divided by cross-walls) or aseptate (coenocytic -- no cross-walls)
• Cell walls are made of chitin (not cellulose)
• Fungi store carbohydrate as glycogen
• Fungi are heterotrophic -- they obtain nutrients by absorption (saprophytic or parasitic)

Reproduction:

Asexual reproduction:

• Yeast: Buds form on the parent cell; the bud grows and eventually separates
• Moulds: Spores are produced asexually by mitosis at the tips of specialised hyphae (sporangia). Spores are lightweight and dispersed by wind.

Sexual reproduction:

• Involves fusion of haploid nuclei from two parent hyphae
• Can involve the formation of specialised sexual structures
• Produces genetically varied spores by meiosis

Viruses

Viruses are not considered living organisms because they do not carry out any life processes on their own. They are obligate intracellular parasites -- they can only reproduce inside a host cell.

Structure:

Component	Description
Genetic material	Either DNA or RNA (never both); single-stranded or double-stranded
Protein coat (capsid)	Protective outer layer made of protein subunits (capsomeres)
Envelope	Some viruses have an additional lipid envelope derived from the host cell membrane
Attachment proteins	Proteins on the surface that bind to specific receptors on host cells

No cellular structure: Viruses have no cytoplasm, no ribosomes, no cell membrane, no organelles.

Sizes: Viruses are much smaller than bacteria, typically 20-400 nm (bacteria are 1-5 micrometres). Electron microscopy is required to visualise viruses.

Reproduction (lytic cycle):

1. Attachment: Virus attaches to specific receptor proteins on the host cell surface
2. Injection: Viral genetic material is injected into the host cell (or the entire virus enters)
3. Replication: The virus uses the host cell's ribosomes, enzymes, and raw materials to replicate its genetic material and synthesise viral proteins
4. Assembly: New viral particles are assembled inside the host cell
5. Release: Host cell lyses (bursts), releasing new viruses that can infect other cells

Lysogenic cycle (some viruses, called temperate phages):

1. Viral DNA is inserted into the host cell's genome (becomes a prophage)
2. The viral DNA is replicated along with the host DNA during cell division
3. Under certain conditions (stress), the prophage enters the lytic cycle

Comparison of Microorganisms

Feature	Bacteria	Fungi	Viruses
Cell type	Prokaryotic	Eukaryotic	Acellular
Nucleus	No true nucleus	True nucleus	None
DNA	Circular, naked	Linear, in nucleus	DNA or RNA
Ribosomes	70S	80S	None
Cell wall	Peptidoglycan	Chitin	No cell wall
Living?	Yes	Yes	No
Reproduction	Binary fission	Spores, budding	Inside host cell only
Size	1-5 micrometres	5-100 micrometres (hyphae)	20-400 nm
Metabolism	Independent	Independent	Dependent on host cell
Antibiotics	Effective	Some effective	Not effective

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Useful Microorganisms

Food Production

Yoghurt:

• Microorganism: Lactobacillus bulgaricus and Streptococcus thermophilus
• Process:
  1. Milk is pasteurised (heated to ~72 degrees C for 15 seconds) to kill harmful bacteria
  2. Milk is cooled to ~40-45 degrees C (optimal for the bacteria)
  3. Starter culture (Lactobacillus) is added
  4. Bacteria ferment lactose (milk sugar) to lactic acid: $\mathrm{C}_{12}\mathrm{H}_{22}\mathrm{O}_{11} \mathrm{ (lactose)} + \mathrm{H}_2\mathrm{O} \to 4\mathrm{C}_3\mathrm{H}_6\mathrm{O}_3 \mathrm{ (lactic acid)}$
  5. Lactic acid lowers the pH, causing milk proteins (casein) to coagulate, giving yoghurt its thick texture
  6. Yoghurt is refrigerated to slow further fermentation

Cheese:

• Microorganism: Lactobacillus species (for lactic acid) + rennet (enzyme from calf stomach or microbial source) + various moulds (e.g., Penicillium roqueforti for blue cheese)
• Process:
  1. Milk is pasteurised
  2. Starter bacteria convert lactose to lactic acid
  3. Rennet is added to coagulate casein (curds form)
  4. Curds are cut, drained, and pressed to remove whey
  5. Curds are ripened (aged) -- bacteria and moulds break down proteins and fats, developing flavour

Bread:

• Microorganism: Saccharomyces cerevisiae (baker's yeast)
• Process:
  1. Yeast is mixed with flour, water, and sugar
  2. Yeast carries out anaerobic respiration (fermentation): $\mathrm{C}_6\mathrm{H}_{12}\mathrm{O}_6 \xrightarrow{\mathrm{yeast}} 2\mathrm{C}_2\mathrm{H}_5\mathrm{OH} + 2\mathrm{CO}_2$
  3. CO$_2$ gas produced causes the dough to rise
  4. The dough is baked -- alcohol evaporates, CO$_2$ expands further, setting the bread structure

Alcohol (Beer and Wine):

• Microorganism: Saccharomyces cerevisiae (brewer's yeast)
• Process:
  1. Sugars (from malted barley for beer, or grape juice for wine) are fermented by yeast
  2. Yeast carries out anaerobic respiration: $\mathrm{C}_6\mathrm{H}_{12}\mathrm{O}_6 \xrightarrow{\mathrm{yeast}} 2\mathrm{C}_2\mathrm{H}_5\mathrm{OH} + 2\mathrm{CO}_2$
  3. The ethanol produced is the alcohol in the beverage
  4. Fermentation is carried out in anaerobic conditions (absence of oxygen)

Biogas:

• Microorganism: Methanogenic bacteria (Methanobacterium)
• Process:
  1. Organic waste (manure, plant material) is placed in an anaerobic digester
  2. Bacteria break down the organic matter in the absence of oxygen
  3. Methane (CH$_4$) is produced as the main component of biogas
  4. Biogas can be used as fuel for cooking, heating, or electricity generation
  5. The remaining slurry can be used as fertiliser

Antibiotics

Definition: Antibiotics are chemical substances produced by microorganisms (usually fungi or bacteria) that kill or inhibit the growth of other microorganisms.

Worked Example: Antibiotic Action

A student investigates the effect of two antibiotics (penicillin and streptomycin) on two species of bacteria (Staphylococcus aureus and Escherichia coli). The results are shown as zones of inhibition (clear area around the antibiotic disc):

Antibiotic	Zone of inhibition for S. aureus (mm)	Zone of inhibition for E. coli (mm)
Penicillin	25	0
Streptomycin	18	20

(a) Explain why penicillin is effective against S. aureus but not E. coli.

(b) Explain why streptomycin is effective against both species.

(c) Why are antibiotics not effective against viruses?

Solution

(a) Penicillin inhibits cell wall synthesis by targeting peptidoglycan. S. aureus is a Gram-positive bacterium with a thick peptidoglycan cell wall, making it susceptible. E. coli is a Gram-negative bacterium with a thinner peptidoglycan layer protected by an outer membrane, which limits penicillin's access to its target.

(b) Streptomycin inhibits protein synthesis by binding to the 30S ribosomal subunit. Both S. aureus and E. coli have 70S ribosomes (with 30S subunits) that are the target of streptomycin. Since all bacteria have similar ribosomes, streptomycin can affect both species.

(c) Viruses have no cell wall (so cell wall-targeting antibiotics like penicillin are irrelevant) and no ribosomes (so protein synthesis-targeting antibiotics like streptomycin are irrelevant). Viruses use the host cell's ribosomes and machinery to replicate. Antibiotics target bacterial-specific structures and processes that viruses do not possess.

Discovery: Alexander Fleming discovered penicillin in 1928 from the mould Penicillium notatum.

Antibiotic	Source	Mechanism
Penicillin	Penicillium (fungus)	Inhibits cell wall synthesis in bacteria
Streptomycin	Streptomyces (bacterium)	Inhibits protein synthesis (30S ribosome)
Tetracycline	Streptomyces (bacterium)	Inhibits protein synthesis (30S ribosome)
Erythromycin	Streptomyces (bacterium)	Inhibits protein synthesis (50S ribosome)

Important notes:

• Antibiotics are effective against bacteria but NOT against viruses (viruses have no cell wall and no ribosomes; they use host cell machinery)
• Misuse of antibiotics (not completing the full course, using them for viral infections) contributes to antibiotic resistance

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Harmful Microorganisms

Pathogens

A pathogen is any microorganism that causes disease. Pathogenic microorganisms include certain bacteria, fungi, and viruses.

Transmission of Pathogens

Route of Transmission	Description	Example
Direct contact	Physical contact with an infected person	Skin infections, warts
Droplet infection	Coughing, sneezing releases droplets containing pathogens	Flu, TB, common cold
Water-borne	Ingestion of contaminated water	Cholera, typhoid
Food-borne	Ingestion of contaminated food	Salmonella, E. coli food poisoning
Vector-borne	Transmitted by another organism (e.g., mosquito)	Malaria (mosquito), Dengue
Blood-borne	Through contaminated blood or needles	HIV, Hepatitis B
Sexually transmitted	Through sexual contact	HIV, Syphilis, Gonorrhoea

Diseases Caused by Pathogens

Disease	Pathogen Type	Pathogen	Transmission	Prevention
Tuberculosis	Bacterium	Mycobacterium tuberculosis	Droplet infection	BCG vaccine; good ventilation
Cholera	Bacterium	Vibrio cholerae	Water-borne	Clean water; hygiene; oral rehydration
Salmonellosis	Bacterium	Salmonella spp.	Food-borne	Proper cooking; hygiene
Gonorrhoea	Bacterium	Neisseria gonorrhoeae	Sexually transmitted	Condoms; avoid sharing needles
Influenza	Virus	Influenza virus	Droplet infection	Vaccination; hygiene
COVID-19	Virus	SARS-CoV-2	Droplet, airborne	Vaccination; masks; hygiene
HIV/AIDS	Virus	Human Immunodeficiency Virus	Blood-borne, sexual	Safe sex; avoid shared needles
Ringworm	Fungus	Tinea species	Direct contact	Good hygiene; avoid sharing towels
Athlete's foot	Fungus	Tinea pedis	Direct contact	Keep feet dry; avoid shared footwear
Malaria	Protozoan	Plasmodium (protozoan)	Mosquito vector	Mosquito nets; insect repellent; antimalarial drugs

How Pathogens Cause Disease

Bacteria cause disease by:

• Producing toxins that damage body tissues (e.g., Vibrio cholerae produces an enterotoxin that causes water to be secreted into the intestines, leading to severe diarrhoea)
• Damaging body cells directly
• Multiplying inside the body and overwhelming the immune system

Viruses cause disease by:

• Invading host cells and using the cell's machinery to replicate
• Bursting host cells (lytic cycle) to release new viral particles, destroying tissues
• Triggering an immune response that itself causes damage (e.g., inflammation)

Fungi cause disease by:

• Producing spores that can be inhaled or contact the skin
• Growing on or in body tissues, causing damage and irritation
• Producing toxins

Cholera as a Case Study

Pathogen: Vibrio cholerae (bacterium)

Transmission: Ingestion of contaminated water or food (faecal-oral route)

Mechanism of disease:

1. Vibrio cholerae is ingested with contaminated water
2. The bacteria survive the acidic stomach (some are killed, but not all)
3. In the small intestine, the bacteria attach to the intestinal wall and multiply
4. They produce a cholera toxin (enterotoxin)
5. The toxin causes chloride ion channels in the intestinal epithelial cells to remain open
6. Chloride ions (Cl$^-$) are secreted into the intestinal lumen
7. This lowers the water potential in the lumen, causing water to follow by osmosis from the blood and tissues into the intestine
8. Result: severe, watery diarrhoea; rapid dehydration; loss of ions (electrolytes)

Treatment:

• Oral Rehydration Solution (ORS): Contains water, glucose, and electrolytes (Na$^+$, K$^+$, Cl$^-$). The glucose is co-transported with Na$^+$ across the intestinal epithelium, and water follows by osmosis, rehydrating the patient.
• Intravenous drip in severe cases
• Antibiotics can reduce the duration of the illness

Prevention:

• Clean, treated water supply
• Proper sanitation and sewage disposal
• Good personal hygiene
• Vaccination (available but not universally used)

Body's Defence Mechanisms

First line of defence (non-specific):

• Skin: Physical barrier; secretes sebum (antimicrobial); sweat contains lysozyme
• Mucous membranes: Trap pathogens; mucus in the respiratory tract traps dust and microbes
• Cilia: Hair-like structures in the trachea that sweep mucus upwards and outwards
• Stomach acid: HCl (pH ~2) kills most ingested pathogens
• Tears: Contain lysozyme (enzyme that breaks down bacterial cell walls)

Second line of defence (non-specific):

• Phagocytosis: Phagocytes (neutrophils, macrophages) engulf and digest pathogens
  1. Phagocyte recognises the pathogen (non-self)
  2. Phagocyte engulfs the pathogen by surrounding it with its membrane (forming a phagosome)
  3. Lysosomes fuse with the phagosome, releasing digestive enzymes (lysozymes, proteases)
  4. The pathogen is digested and destroyed
• Inflammation: Tissue damage triggers release of histamine, causing vasodilation and increased capillary permeability. More phagocytes and plasma proteins arrive at the site.

Third line of defence (specific -- immune response):

• Cell-mediated immunity: T lymphocytes destroy virus-infected cells and cancer cells
• Humoral immunity: B lymphocytes produce antibodies specific to the antigen

Antibiotic Resistance

How antibiotic resistance develops:

1. In any bacterial population, there is genetic variation (some bacteria carry resistance genes, e.g., on plasmids)
2. When antibiotics are used, susceptible bacteria are killed
3. Resistant bacteria survive and reproduce
4. The resistance gene is passed on to offspring (vertical transmission) and to other bacteria via conjugation (horizontal transmission)
5. Over time, the resistant strain becomes dominant

Mechanisms of resistance:

• Production of enzymes that break down the antibiotic (e.g., beta-lactamase breaks down penicillin)
• Altering the target site of the antibiotic
• Pumping the antibiotic out of the cell (efflux pumps)
• Reducing membrane permeability to the antibiotic

Superbugs: Bacteria resistant to multiple antibiotics (e.g., MRSA -- Methicillin-Resistant Staphylococcus aureus)

Reducing antibiotic resistance:

• Only use antibiotics when prescribed by a doctor
• Complete the full course of antibiotics (do not stop early even if symptoms improve)
• Do not use antibiotics for viral infections
• Develop new antibiotics
• Reduce antibiotic use in agriculture

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Biotechnology

Genetic Engineering

Genetic engineering (recombinant DNA technology) is the direct manipulation of an organism's genome to produce desired characteristics.

Basic process:

1. Isolation of the desired gene: The gene of interest is identified and extracted from the donor organism's DNA using restriction enzymes (restriction endonucleases).

2. Cutting DNA with restriction enzymes: Restriction enzymes cut DNA at specific recognition sequences (palindromic sequences). For example, the enzyme EcoRI cuts at GAATTC, producing "sticky ends" (single-stranded overhangs).

3. Preparing the vector: A vector (carrier) is used to transfer the gene into the host organism. Common vectors include plasmids (from bacteria) and viruses. The same restriction enzyme is used to cut the vector, producing complementary sticky ends.

4. Ligation: The cut gene and cut vector are mixed. The complementary sticky ends pair up by hydrogen bonding. DNA ligase seals the sugar-phosphate backbone, forming a recombinant DNA molecule.

5. Transformation: The recombinant DNA is introduced into the host cell (e.g., bacterial cell). In bacteria, this can be done by:

   • Heat shock (briefly heating cells in the presence of calcium chloride)
   • Electroporation (electric pulse creates temporary pores in the cell membrane)

6. Selection and culture: Transformed cells are identified using marker genes (e.g., antibiotic resistance). Only cells that have taken up the plasmid survive on antibiotic-containing media. Successfully transformed cells are cultured to produce the desired product.

7. Expression: The host cell's machinery (ribosomes, enzymes) transcribes and translates the inserted gene, producing the desired protein.

Example: Production of Human Insulin by Genetic Engineering

Before genetic engineering, insulin for diabetic patients was extracted from pig and cow pancreases. This had issues: limited supply, allergic reactions in some patients, ethical concerns.

Process:

1. The human insulin gene is identified (on chromosome 11)
2. mRNA for insulin is extracted from human pancreatic cells
3. Reverse transcriptase enzyme is used to produce complementary DNA (cDNA) from the mRNA
4. The cDNA is cut with restriction enzymes to produce sticky ends
5. A bacterial plasmid is cut with the same restriction enzyme
6. The insulin cDNA is inserted into the plasmid using DNA ligase
7. The recombinant plasmid is introduced into E. coli bacteria by transformation
8. Transformed bacteria are selected and cultured in large fermenters
9. The bacteria produce human insulin as they grow and divide
10. Insulin is extracted, purified, and packaged for medical use

Advantages:

• Identical to human insulin (no immune rejection)
• Large-scale, continuous production
• Cheaper than extraction from animal sources
• No ethical issues with animal use

Genetically Modified (GM) Crops

What are GM crops?

Crops that have had their genome modified by genetic engineering to introduce desirable traits.

Examples:

GM Crop	Modification	Benefit
Bt cotton	Contains Bacillus thuringiensis gene	Produces toxin that kills insect pests; reduces pesticide use
Golden rice	Contains beta-carotene gene	Produces vitamin A precursor; combats vitamin A deficiency
Herbicide-resistant soybeans	Resistant to glyphosate herbicide	Farmers can spray herbicide to kill weeds without harming crop
Virus-resistant papaya	Resistant to papaya ringspot virus	Crop is protected from viral disease
Delayed-ripening tomatoes	Modified ripening gene	Longer shelf life; reduces food waste

Benefits of GM crops:

• Increased crop yield
• Reduced pesticide use (Bt crops)
• Enhanced nutritional content (Golden rice)
• Tolerance to environmental stresses (drought, salinity)
• Disease resistance

Concerns about GM crops:

• Potential long-term health effects (not yet fully understood)
• Gene flow to wild relatives (creating "superweeds")
• Impact on biodiversity (monocultures; harm to non-target organisms)
• Ethical concerns about manipulating nature
• Corporate control of food supply (patents on seeds)
• Development of pest resistance (pests evolving resistance to Bt toxin)

Cloning

Definition: Cloning is the production of genetically identical copies of an organism or cell.

Types of cloning:

1. Natural cloning:

• Asexual reproduction in bacteria (binary fission), plants (runners, tubers, bulbs), fungi (spores)
• Identical twins in humans (a single zygote splits into two)

2. Artificial cloning in plants (tissue culture):

1. A small piece of tissue (explant) is taken from the parent plant
2. The explant is placed on a nutrient medium containing growth hormones (auxins and cytokinins)
3. The explant develops into a mass of undifferentiated cells called a callus
4. The callus is transferred to a different medium that stimulates differentiation into shoots and roots
5. Plantlets are transferred to soil and grown into mature plants
6. All resulting plants are genetically identical to the parent (clones)

Advantages: Rapid propagation of desirable plants; disease-free plants; conservation of rare species; year-round production.

3. Artificial cloning in animals:

Embryo splitting:

• A fertilised egg (zygote) is allowed to divide into a few cells in vitro
• The cells are separated and each is implanted into a surrogate mother
• Each develops into a genetically identical offspring

Somatic Cell Nuclear Transfer (SCNT) -- Dolly the sheep (1996):

1. A body cell (somatic cell) is taken from the donor animal
2. The nucleus is extracted from this cell
3. An unfertilised egg cell (ovum) is taken from another animal and its nucleus is removed (enucleated)
4. The donor nucleus is inserted into the enucleated egg cell
5. The egg cell is stimulated (by electric shock) to divide
6. The developing embryo is implanted into a surrogate mother
7. The offspring is genetically identical to the donor of the nucleus (not the egg donor)

Advantages of animal cloning:

• Reproduction of animals with desirable traits (high milk yield, disease resistance)
• Conservation of endangered species
• Production of genetically identical animals for research

Disadvantages/ethical issues:

• Low success rate (Dolly was 1 success out of 277 attempts)
• Cloned animals may have health problems (premature aging, organ defects)
• Reduced genetic diversity
• Ethical concerns about playing God; welfare of cloned animals

Polymerase Chain Reaction (PCR)

PCR is a laboratory technique used to amplify (make many copies of) a specific segment of DNA.

Requirements:

• DNA template (the DNA to be copied)
• Taq polymerase: A heat-stable DNA polymerase from Thermus aquaticus (a bacterium from hot springs); synthesises new DNA strands
• Primers: Short, single-stranded DNA sequences that are complementary to the regions flanking the target DNA; they define where amplification begins
• Free nucleotides (dATP, dTTP, dCTP, dGTP): Building blocks for new DNA strands
• Thermal cycler: A machine that rapidly changes the temperature

PCR Cycle (repeated 25-35 times):

1. Denaturation (94-96 degrees C): The double-stranded DNA is heated, causing the hydrogen bonds between the two strands to break. The DNA separates into two single strands.

2. Annealing (50-65 degrees C): The temperature is lowered. Primers bind (anneal) to their complementary sequences on the single-stranded DNA by hydrogen bonding.

3. Extension (72 degrees C): Taq polymerase extends the primers by adding nucleotides in the 5' to 3' direction, synthesising new complementary DNA strands.

After each cycle, the amount of DNA doubles:

$\mathrm{After } n \mathrm{ cycles: } \mathrm{Number of DNA molecules} = 2^n$

Starting from 1 DNA molecule:

Cycles ($n$)	DNA Molecules ($2^n$)
1	2
5	32
10	1,024
20	1,048,576
30	1,073,741,824

Applications of PCR:

• Forensic science (amplifying DNA from tiny crime scene samples)
• Medical diagnosis (detecting pathogens, genetic disorders)
• Paternity testing
• Evolutionary biology (amplifying ancient DNA)
• Genetic fingerprinting

Genetic Fingerprinting (DNA Profiling)

Genetic fingerprinting is a technique used to identify individuals based on their unique DNA pattern.

Principle: Non-coding regions of DNA contain repetitive sequences called Variable Number Tandem Repeats (VNTRs) or Short Tandem Repeats (STRs). The number of repeats at each locus varies between individuals.

Process:

1. DNA is extracted from a sample (blood, saliva, hair root)
2. PCR is used to amplify specific STR loci
3. The DNA fragments are separated by gel electrophoresis (smaller fragments travel further through the gel)
4. The resulting pattern of bands is compared between samples

Applications:

• Forensic identification (matching crime scene DNA to suspects)
• Paternity/maternity testing
• Identifying victims of disasters
• Establishing evolutionary relationships between species

Ethical Issues in Biotechnology

Issue	Arguments For	Arguments Against
Genetic engineering	Cures genetic diseases; improves crop yield	Unforeseen consequences; playing God; designer babies
GM crops	Reduces pesticide use; solves malnutrition	Superweeds; corporate control; unknown health risks
Cloning	Preserves endangered species; medical research	Low success rate; animal welfare; loss of diversity
Stem cell research	Potential cures for degenerative diseases	Destruction of embryos; alternative methods exist
Genetic screening	Early detection of diseases	Discrimination; psychological impact; privacy
Gene therapy	Treats genetic disorders at source	Somatic vs germline; off-target effects; cost
Genetically modified animals	Better models for research; pharmaceuticals	Animal welfare; environmental release concerns

Worked Example: Genetic Engineering Process

A scientist wants to produce human insulin using genetically engineered E. coli bacteria.

(a) Explain why reverse transcriptase is used to obtain the insulin gene rather than cutting it directly from human DNA.

(b) Explain the role of the plasmid in this process.

(c) How are transformed bacteria identified from non-transformed ones?

Solution

(a) Human genomic DNA contains introns (non-coding regions) that are transcribed into pre-mRNA but are spliced out during mRNA processing. Bacteria (prokaryotes) lack the splicing machinery to remove introns. Reverse transcriptase is used to make complementary DNA (cDNA) from mature mRNA, which already has introns removed. The cDNA contains only exons (coding sequences) and can be directly expressed in bacteria.

(b) The plasmid acts as a vector -- a carrier that transfers the insulin gene into the bacterial cell. The same restriction enzyme cuts both the insulin cDNA and the plasmid, producing complementary sticky ends. DNA ligase joins them, creating a recombinant plasmid. The plasmid also contains a promoter sequence (so bacterial RNA polymerase can transcribe the gene) and a marker gene (e.g., antibiotic resistance) for selection.

(c) The plasmid contains a marker gene for antibiotic resistance (e.g., ampicillin resistance). After transformation, bacteria are grown on agar containing the antibiotic. Only bacteria that have successfully taken up the plasmid (with the antibiotic resistance gene) survive. Non-transformed bacteria (without the plasmid) die on the antibiotic-containing medium.

Somatic gene therapy: Modifying body cells to treat disease. Changes are NOT passed to offspring.

Germline gene therapy: Modifying gametes or early embryos. Changes ARE passed to offspring. Currently not permitted in most countries due to ethical concerns.

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Common Pitfalls

1. Calling viruses living organisms: Viruses are NOT alive. They do not respire, feed, excrete, grow, or reproduce independently. They are obligate intracellular parasites.

2. Confusing bacteria and viruses in treatment: Antibiotics work against bacteria, NOT viruses. Prescribing antibiotics for viral infections (e.g., common cold, flu) is ineffective and contributes to antibiotic resistance.

3. Stating that all bacteria are harmful: The vast majority of bacteria are harmless or beneficial. Fewer than 1% of bacterial species are pathogenic.

4. Confusing fungal and bacterial cell walls: Fungal cell walls are made of chitin. Bacterial cell walls are made of peptidoglycan. Plant cell walls are made of cellulose. Do not mix these up.

5. Forgetting that viruses contain EITHER DNA OR RNA: A virus contains one or the other, never both. Do not write that a virus contains "DNA and RNA."

6. Confusing binary fission and mitosis: Binary fission is asexual reproduction in prokaryotes. It is NOT the same as mitosis (which occurs in eukaryotes). Binary fission does not involve spindle formation or chromosomes in the same way.

7. Stating that the insulin gene was cut directly from DNA: In practice, the insulin gene is obtained from mRNA using reverse transcriptase to make cDNA. This is because eukaryotic genes contain introns that bacteria cannot splice out. cDNA has no introns.

8. Confusing restriction enzymes and DNA ligase: Restriction enzymes CUT DNA at specific sequences. DNA ligase JOINS DNA fragments together (seals the sugar-phosphate backbone). They have opposite functions.

9. Saying PCR can amplify a whole genome: PCR amplifies a specific segment of DNA (defined by the primers). It does not amplify the entire genome. Whole genome amplification uses different methods.

10. Writing that Taq polymerase is from humans: Taq polymerase is obtained from Thermus aquaticus, a thermophilic bacterium. It is used because it is heat-stable and does not denature at 94-96 degrees C, unlike human DNA polymerase.

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Problem Set

Problem 1: A culture of bacteria divides by binary fission every 20 minutes. The initial population is 500 bacteria.

(a) Calculate the population after 2 hours.

(b) Calculate the population after 3 hours.

(c) How long does it take for the population to reach 32,000?

If you get this wrong, revise: Classification of Microorganisms -- Bacteria (Reproduction; Growth curve)

Solution

(a) Number of divisions in 2 hours (120 minutes): $n = 120 / 20 = 6$

Population = $500 \times 2^6 = 500 \times 64 = 32\,000$

(b) Number of divisions in 3 hours (180 minutes): $n = 180 / 20 = 9$

Population = $500 \times 2^9 = 500 \times 512 = 256\,000$

(c) We need $500 \times 2^n = 32\,000$

$2^n = 32\,000 / 500 = 64 = 2^6$

$n = 6$

Time = $6 \times 20 = 120$ minutes = 2 hours.

Problem 2: Explain how antibiotic resistance develops in a bacterial population. Use the concepts of genetic variation, natural selection, and inheritance in your answer.

If you get this wrong, revise: Harmful Microorganisms -- Antibiotic Resistance

Solution

1. Genetic variation: Within any bacterial population, random mutations occur during DNA replication. Some bacteria may acquire a mutation that gives them resistance (e.g., a gene for beta-lactamase). Resistance genes can also be transferred via plasmids during conjugation.

2. Selection pressure: When an antibiotic is administered, it creates a selection pressure. Susceptible bacteria are killed or their growth is inhibited.

3. Natural selection: Bacteria carrying the resistance gene survive the antibiotic treatment. These resistant bacteria have a selective advantage and can continue to grow and reproduce.

4. Inheritance: Resistant bacteria reproduce by binary fission, passing the resistance gene to all offspring (vertical transmission). The gene can also be transferred to other bacteria via conjugation (horizontal transmission), spreading resistance through the population.

Problem 3: A scientist starts with 10 DNA molecules and runs 30 cycles of PCR.

(a) How many DNA molecules will be present after 30 cycles?

(b) If the DNA segment is 300 base pairs long, what is the total number of base pairs of amplified DNA produced?

If you get this wrong, revise: Biotechnology -- Polymerase Chain Reaction (PCR)

Solution

(a) After 30 cycles: $N = 10 \times 2^{30} = 10 \times 1\,073\,741\,824 = 10\,737\,418\,240$ DNA molecules

(b) Total base pairs = $10\,737\,418\,240 \times 300 = 3.221 \times 10^{12}$ base pairs

Problem 4: Describe the steps involved in producing human insulin using genetically engineered bacteria. Explain why cDNA is used rather than genomic DNA.

If you get this wrong, revise: Biotechnology -- Genetic Engineering; Example: Production of Human Insulin

Solution

1. mRNA is extracted from human pancreatic beta cells. Reverse transcriptase synthesises cDNA from the mRNA (removing introns).
2. The same restriction enzyme cuts both the insulin cDNA and a bacterial plasmid, producing complementary sticky ends.
3. DNA ligase seals the cDNA into the plasmid, creating a recombinant plasmid.
4. The recombinant plasmid is introduced into E. coli by transformation (heat shock or electroporation).
5. Transformed bacteria are selected using a marker gene (antibiotic resistance) on the plasmid.
6. Successfully transformed bacteria are cultured in large fermenters, producing human insulin.

cDNA is used because genomic DNA contains introns that bacteria cannot splice out. cDNA contains only the coding sequence (exons) and can be directly expressed in bacteria.

Problem 5: Explain how Vibrio cholerae causes severe diarrhoea and why oral rehydration solution (ORS) is an effective treatment.

If you get this wrong, revise: Harmful Microorganisms -- Cholera as a Case Study; Body's Defence Mechanisms

Solution

Vibrio cholerae attaches to the intestinal epithelium and produces a cholera toxin. This toxin causes chloride ion channels to remain open, leading to continuous secretion of Cl$^-$ into the intestinal lumen. This lowers the water potential in the lumen, causing water to follow by osmosis from the blood and tissues into the intestine. Result: severe watery diarrhoea and rapid dehydration.

ORS is effective because it contains glucose and Na$^+$. The sodium-glucose co-transporter (SGLT1) on intestinal epithelial cells actively co-transports Na$^+$ and glucose from the lumen into the cell. Water follows by osmosis into the cells and then into the blood. This rehydrates the patient even though the cholera toxin is still causing Cl$^-$ secretion.

Problem 6: For each of the following statements, identify whether it applies to bacteria, fungi, or viruses: (a) Has a cell wall (b) Contains ribosomes (c) Can reproduce independently outside a host (d) Stores genetic material as RNA (e) Has a true nucleus (f) Can be killed by antibiotics.

If you get this wrong, revise: Classification of Microorganisms -- Comparison of Microorganisms

Solution

(a) Bacteria (peptidoglycan) and fungi (chitin). Viruses have no cell wall. (b) Bacteria (70S) and fungi (80S). Viruses have no ribosomes. (c) Bacteria and fungi. Viruses require a host cell. (d) Viruses (some have RNA). Bacteria and fungi always have DNA. (e) Fungi only. Bacteria have a nucleoid region; viruses have none. (f) Bacteria only. Fungi are treated with antifungals; viruses are unaffected by antibiotics.

Problem 7: A country is considering allowing cultivation of Bt cotton. Discuss two benefits and two risks.

If you get this wrong, revise: Biotechnology -- Genetically Modified (GM) Crops

Solution

Benefits:

1. Reduced pesticide use: Bt cotton produces its own Bt toxin, reducing chemical sprays. This lowers costs and reduces environmental harm to non-target organisms.
2. Increased yield: By reducing pest damage, crop yields are higher and more reliable, increasing farmer income.

Risks:

1. Development of resistance: Bollworm populations may evolve resistance to Bt toxin, rendering the crop ineffective (similar to antibiotic resistance).
2. Gene flow: The Bt gene could transfer to wild relatives through cross-pollination, potentially creating "superweeds" that harm non-target insects.

Problem 8: Compare natural cloning, plant tissue culture cloning, and animal cloning by nuclear transfer. Give one advantage and one disadvantage of each.

If you get this wrong, revise: Biotechnology -- Cloning

Solution

Natural cloning (asexual reproduction): Advantage: Rapid population increase (no mate needed). Disadvantage: No genetic variation, so the population is vulnerable to the same disease.

Plant tissue culture: Advantage: Large numbers of identical, disease-free plants can be produced quickly. Disadvantage: All plants are genetically identical (same disease susceptibility); requires sterile lab conditions.

Animal cloning (SCNT): Advantage: Can reproduce animals with desirable traits; could help conserve endangered species. Disadvantage: Very low success rate; cloned animals often have health problems; ethical concerns about animal welfare.

Problem 9: Explain the lytic cycle of a bacteriophage. Why are viruses not considered living organisms?

If you get this wrong, revise: Classification of Microorganisms -- Viruses (Reproduction; Comparison table)

Solution

Lytic cycle:

1. Attachment: The bacteriophage attaches to specific receptor proteins on the bacterial cell surface.
2. Injection: Viral DNA is injected into the host cell.
3. Replication: The virus uses the host cell's ribosomes, enzymes, and raw materials to replicate its DNA and synthesise viral proteins.
4. Assembly: New viral particles are assembled inside the host cell.
5. Release: The host cell lyses (bursts), releasing new phages that infect other cells.

Viruses are not living because they do not carry out any life processes independently: they do not respire, feed, grow, excrete, or reproduce on their own. They are obligate intracellular parasites that can only replicate inside a host cell, using the host's machinery.

Problem 10: Describe how yoghurt is produced, naming the microorganism involved and the chemical change that occurs.

If you get this wrong, revise: Useful Microorganisms -- Food Production (Yoghurt)

Solution

Yoghurt is produced using Lactobacillus bulgaricus and Streptococcus thermophilus. The process:

1. Milk is pasteurised (heated to ~72 degrees C for 15 seconds) to kill harmful bacteria.
2. Milk is cooled to ~40-45 degrees C (optimal for the bacteria).
3. Starter culture is added.
4. The bacteria ferment lactose (milk sugar) to lactic acid. The chemical equation: lactose + H$_2$O $\to$ 4 lactic acid.
5. Lactic acid lowers the pH, causing milk proteins (casein) to coagulate, giving yoghurt its thick texture.
6. Yoghurt is refrigerated to slow further fermentation.

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Industrial Microbiology

Industrial Production Using Microorganisms

Microorganisms are used in large-scale industrial processes to produce a wide range of products. The key advantages of using microorganisms include: rapid growth, easy cultivation, ability to use cheap raw materials, and the production of valuable metabolites.

Fermentation Technology

Batch fermentation:

• All nutrients (substrate) are added at the beginning
• Microorganisms grow through lag phase, exponential phase, stationary phase, and death phase
• Products are harvested at the end of the process
• The fermenter is cleaned and sterilised before the next batch
• Advantages: simple; suitable for small-scale production; easy to control contamination
• Disadvantages: downtime between batches; lower productivity; conditions change over time (nutrient depletion, waste accumulation)

Continuous fermentation:

• Nutrients are continuously added, and products are continuously removed
• Microorganisms are maintained in the exponential (log) phase of growth, maximising productivity
• Advantages: higher productivity; no downtime; consistent product quality
• Disadvantages: risk of contamination over long periods; difficult to maintain optimal conditions; mutations can reduce product quality over time

Primary vs Secondary Metabolites:

Type	Description	Production Phase	Examples
Primary metabolites	Substances produced during active growth (exponential phase); essential for growth and survival	Exponential/log phase	Amino acids, ethanol, organic acids (citric acid), enzymes
Secondary metabolites	Substances produced after the main growth phase (stationary phase); not essential for growth	Stationary phase	Antibiotics (penicillin), pigments, toxins

Antibiotic Production: Penicillin

Penicillin is produced by the fungus Penicillium chrysogenum in a fed-batch fermentation process.

Process:

1. Penicillium chrysogenum is grown in a large fermenter (stainless steel vessel, 50,000-200,000 dm$^3$) containing a sterile nutrient medium (sugar, nitrogen source, minerals)
2. The fermenter is continuously stirred and aerated to ensure even distribution of nutrients and oxygen
3. Temperature is maintained at approximately 25-27 degrees C (optimal for fungal growth and penicillin production)
4. pH is maintained at approximately 6.5-7.0 by adding acid or alkali
5. During the exponential growth phase, the fungus grows rapidly but produces little penicillin (primary metabolism)
6. After the growth phase, when nutrients begin to deplete, the fungus switches to secondary metabolism and begins producing penicillin
7. Glucose is fed in slowly (fed-batch): high glucose concentrations repress penicillin production, so glucose is added gradually to maintain a low but steady concentration
8. The fermentation runs for approximately 6-8 days
9. Penicillin is extracted from the filtrate using solvent extraction or ion-exchange chromatography

Key points for DSE:

• Penicillin is a secondary metabolite -- it is produced during the stationary phase, not during active growth
• High glucose concentration represses penicillin synthesis (catabolite repression), so glucose is added slowly (fed-batch)
• The process requires aseptic (sterile) conditions to prevent contamination by other microorganisms

Enzyme Production Using Microorganisms

Microorganisms are the main source of industrial enzymes because they can be cultured in large quantities and their enzyme production can be optimised through genetic modification and selection.

Enzyme	Source Microorganism	Industrial Application
Proteases	Bacillus subtilis, Bacillus licheniformis	Biological detergents (break down protein stains); food processing (tenderising meat, cheese making)
Amylases	Bacillus amyloliquefaciens, Aspergillus niger	Starch processing (convert starch to sugars); brewing; baking (improve dough quality)
Lipases	Candida rugosa, Thermomyces lanuginosus	Biological detergents (break down fat stains); food processing (flavour development in cheese)
Pectinases	Aspergillus niger	Fruit juice clarification (break down pectin, which causes cloudiness)
Lactase	Kluyveromyces lactis	Production of lactose-free milk for lactose-intolerant individuals
Cellulases	Trichoderma reesei	Biofuel production (break down cellulose in plant biomass to glucose for fermentation); textile industry (stone-washing denim)
Taq polymerase	Thermus aquaticus	PCR (polymerase chain reaction) -- heat-stable DNA polymerase that can withstand the high temperatures of PCR cycles

Bioremediation

Bioremediation is the use of microorganisms to clean up contaminated environments (soil, water).

Application	Microorganism	Mechanism
Oil spill cleanup	Pseudomonas putida, Alcanivorax	Break down hydrocarbons in crude oil into less harmful substances; can be enhanced by adding nutrients (biostimulation)
Heavy metal removal	Pseudomonas, Bacillus	Accumulate heavy metals (lead, cadmium, mercury) from contaminated soil and water
Sewage treatment	Mixed microbial communities	Break down organic matter in wastewater through aerobic and anaerobic processes
Pesticide degradation	Pseudomonas, Bacillus	Produce enzymes that degrade organophosphate and carbamate pesticides
Plastic degradation	Ideonella sakaiensis	Produces PETase enzyme that breaks down PET plastic into monomers

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Antibiotic Resistance: Detailed Mechanisms

How Bacteria Become Resistant

Mechanism	Molecular Basis	Antibiotic Affected
Enzymatic inactivation	Bacteria produce enzymes that chemically modify or destroy the antibiotic molecule	Beta-lactamase breaks down penicillin
Target modification	Mutations alter the molecular target of the antibiotic (e.g., altered ribosomes, altered penicillin-binding proteins)	MRSA (altered PBP2a); rifampicin resistance (altered RNA polymerase)
Efflux pumps	Membrane proteins actively pump the antibiotic out of the cell, reducing its intracellular concentration	Tetracycline resistance; multi-drug resistance in Gram-negative bacteria
Reduced permeability	Mutations reduce the number or size of porins in the outer membrane, limiting antibiotic entry	Gram-negative resistance to beta-lactams and quinolones
Biofilm formation	Bacteria embedded in a protective extracellular matrix; reduced penetration of antibiotics and reduced metabolic activity	Chronic infections; catheter-associated infections

Horizontal Gene Transfer of Resistance

Antibiotic resistance genes can spread between bacteria of different species through horizontal gene transfer, much faster than vertical transmission (parent to offspring via binary fission).

Method	Description
Conjugation	Direct transfer of plasmids (circular DNA carrying resistance genes) between two bacteria connected by a pilus
Transformation	Uptake of free DNA from the environment (released when bacteria die); the DNA may contain resistance genes
Transduction	Bacteriophages (viruses) carry bacterial DNA from one cell to another during infection; the DNA may include resistance genes

Plasmids are particularly important in the spread of antibiotic resistance because:

• They can carry multiple resistance genes at once (multi-drug resistance)
• They can be transferred between different species of bacteria via conjugation
• They replicate independently of the bacterial chromosome, so they are maintained even without selection pressure (though there is a metabolic cost)

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Additional Problem Set

Problem 11: Describe the process of penicillin production by Penicillium chrysogenum in a fermenter. Explain why glucose is added slowly (fed-batch) rather than all at once.

If you get this wrong, revise: Industrial Microbiology -- Antibiotic Production

Solution

Penicillium chrysogenum is cultured in a large stirred-tank fermenter with controlled conditions (temperature 25-27 degrees C, pH 6.5-7.0, continuous aeration and stirring). During the exponential growth phase, the fungus grows rapidly but produces little penicillin. Once growth slows and nutrients deplete (stationary phase), the fungus switches to secondary metabolism and begins producing penicillin.

Glucose is added slowly (fed-batch) because high glucose concentrations repress penicillin biosynthesis through catabolite repression. When glucose is abundant, the fungus preferentially uses it for growth and does not produce secondary metabolites. By adding glucose gradually, the glucose concentration is maintained at a low level that supports fungal metabolism without repressing penicillin synthesis. This maximises penicillin yield. The fed-batch approach also allows the fermentation to be run for longer (6-8 days) compared to a simple batch process, further increasing total production.

Problem 12: Explain how bacteria can become resistant to the antibiotic penicillin through (a) the production of beta-lactamase and (b) alteration of penicillin-binding proteins. Describe how resistance genes can spread between different species of bacteria.

If you get this wrong, revise: Antibiotic Resistance -- Detailed Mechanisms

Solution

(a) Beta-lactamase production: A mutation in the bacterial genome (or acquisition of a plasmid carrying the bla gene) enables the bacterium to produce the enzyme beta-lactamase. This enzyme hydrolyses the beta-lactam ring in penicillin, rendering the antibiotic inactive. The modified penicillin molecule can no longer bind to its target and is excreted from the cell. The bacterium survives despite the presence of the antibiotic.

(b) Altered penicillin-binding proteins (PBPs): Penicillin normally works by binding to PBPs on the bacterial cell membrane, inhibiting the transpeptidase enzyme that cross-links peptidoglycan during cell wall synthesis. A mutation in the gene encoding PBPs can alter the protein's shape, reducing penicillin's ability to bind. The transpeptidase continues to function, allowing the bacterium to synthesise its cell wall normally even in the presence of penicillin. This is the mechanism of MRSA resistance.

Horizontal gene transfer: Resistance genes can spread between different bacterial species through:

• Conjugation: A plasmid carrying resistance genes is transferred through a pilus from a donor bacterium to a recipient
• Transformation: Free DNA containing resistance genes (released from dead bacteria) is taken up by competent bacteria
• Transduction: A bacteriophage transfers bacterial DNA (including resistance genes) from one bacterium to another during infection

These mechanisms allow resistance to spread much more rapidly than vertical transmission alone (binary fission), creating multi-drug-resistant strains that pose serious public health threats.

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Genetic Engineering: Detailed Techniques

Restriction Enzymes

Restriction endonucleases are enzymes that cut DNA at specific recognition sequences (usually 4-8 base pairs long). They are essential tools in genetic engineering because they allow precise cutting of DNA at known locations.

Feature	Description
Recognition sequence	A specific palindromic DNA sequence (reads the same 5' to 3' on both strands)
Cut type	Most produce "sticky ends" (single-stranded overhangs); some produce "blunt ends" (cut straight across both strands)
EcoRI example	Recognition sequence: GAATTC; cuts between G and A on both strands, producing sticky ends with 5' overhangs of AATT
Specificity	Each enzyme recognises only one specific sequence; over 3,000 different restriction enzymes have been identified

Sticky ends vs blunt ends:

• Sticky ends: The single-stranded overhangs are complementary between fragments cut with the same enzyme. This allows DNA fragments from different sources to be joined together because the complementary bases pair by hydrogen bonding. Sticky ends are preferred in genetic engineering because they allow directional insertion (the DNA can only be inserted in one orientation).
• Blunt ends: Both strands are cut at the same position. Blunt ends can be joined to any other blunt end (regardless of the enzyme used), but this is less efficient and allows insertion in either orientation.

DNA Ligase

DNA ligase joins DNA fragments by catalysing the formation of phosphodiester bonds between adjacent nucleotides. It is used to:

• Join the target gene into the plasmid vector after both have been cut with the same restriction enzyme
• Seal nicks in the sugar-phosphate backbone of DNA

Polymerase Chain Reaction (PCR)

PCR is a technique for amplifying a specific segment of DNA exponentially, producing millions of copies from a tiny starting sample.

Requirements:

• Template DNA: The DNA containing the target sequence to be amplified
• Primers: Two short single-stranded DNA sequences (15-30 bases each) that are complementary to the regions flanking the target sequence on opposite strands. Forward primer binds to the start of the target; reverse primer binds to the complementary strand at the end of the target
• Taq polymerase: A heat-stable DNA polymerase from Thermus aquaticus (a bacterium living in hot springs). It can withstand the high temperatures (95 degrees C) used in PCR without denaturing
• Free nucleotides: dATP, dTTP, dCTP, dGTP (the building blocks for DNA synthesis)
• Buffer: Contains Mg$^{2+}$ ions (cofactor for Taq polymerase) and optimal pH

PCR cycle (repeated 25-35 times):

1. Denaturation (95 degrees C, 30 seconds): The double-stranded DNA template is heated to separate it into two single strands
2. Annealing (55-65 degrees C, 30 seconds): The temperature is lowered to allow primers to bind (anneal) to their complementary sequences on the single-stranded template by hydrogen bonding
3. Extension (72 degrees C, 30-60 seconds): Taq polymerase synthesises new DNA strands from the primers, using the template as a guide. The rate is approximately 1000 bases per minute

After n cycles: $2^n$ copies of the target sequence are produced (assuming 100% efficiency).

Applications of PCR:

Application	Description
Forensic science	Amplifying DNA from tiny blood samples or hair follicles for DNA fingerprinting and criminal identification
Medical diagnosis	Detecting pathogen DNA in patient samples (e.g., HIV, tuberculosis, COVID-19)
Genetic testing	Detecting genetic mutations associated with inherited diseases (e.g., cystic fibrosis, Huntington's disease)
Evolutionary biology	Amplifying ancient DNA from fossils; comparing DNA sequences between species
Paternity testing	Comparing DNA profiles between child, mother, and alleged father
Gene cloning	Amplifying a specific gene for insertion into a vector

Gel Electrophoresis

Gel electrophoresis separates DNA fragments by size. DNA is negatively charged (due to phosphate groups) and moves towards the positive electrode.

Process:

1. DNA samples are cut with restriction enzymes, producing fragments of different lengths
2. DNA fragments are loaded into wells in an agarose gel
3. An electric current is applied across the gel
4. DNA fragments migrate through the gel towards the positive electrode
5. Smaller fragments move faster and further; larger fragments move slower and less far
6. The DNA is visualised by staining with a fluorescent dye (e.g., ethidium bromide) and viewing under UV light

Applications:

• DNA fingerprinting: Comparing the banding patterns of DNA fragments between individuals. Every individual (except identical twins) has a unique DNA profile due to variations in non-coding DNA (short tandem repeats, STRs)
• PCR product verification: Confirming that PCR amplified the correct size fragment
• Analysing genetic disorders: Identifying the presence or absence of specific alleles

Worked Example: Genetic Engineering -- Producing Human Insulin in Bacteria

Describe the steps involved in producing human insulin using genetic engineering, explaining why each step is necessary.

Solution

1. Isolation of the insulin gene: mRNA for human insulin is extracted from pancreatic beta cells. Reverse transcriptase is used to make complementary DNA (cDNA) from the mRNA. cDNA is used instead of genomic DNA because eukaryotic genes contain introns, and bacteria cannot remove introns (they lack spliceosomes). If genomic DNA were used, the protein produced would include intron-encoded amino acids and would be non-functional.

2. Cutting the DNA: The insulin cDNA and a plasmid vector are both cut with the same restriction enzyme (e.g., EcoRI). This produces complementary sticky ends on both the cDNA and the plasmid.

3. Ligation: DNA ligase joins the insulin cDNA into the plasmid by catalysing phosphodiester bonds between the sticky ends, creating a recombinant plasmid.

4. Transformation: The recombinant plasmid is introduced into E. coli bacteria by making the bacterial cells permeable (using heat shock or electroporation). Not all bacteria take up the plasmid.

5. Selection: Bacteria that took up the plasmid are selected using an antibiotic resistance marker gene on the plasmid. When grown on agar containing the antibiotic, only transformed bacteria (with the plasmid) survive.

6. Culture and expression: Transformed bacteria are grown in large fermenters. They transcribe and translate the insulin gene, producing human insulin (as proinsulin, which is later processed to active insulin).

7. Purification: Insulin is extracted from the bacterial culture and purified for medical use.

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The Carbon Cycle and Microorganisms

Role of Microorganisms in Carbon Cycling

Process	Microorganisms Involved	Description
Decomposition	Bacteria and fungi (saprotrophs)	Break down dead organic matter (plants, animals, waste); release CO$_2$ through respiration; recycle carbon and nutrients back into the ecosystem
Carbon fixation	Cyanobacteria; chemosynthetic bacteria	Convert inorganic carbon (CO$_2$ or bicarbonate) into organic compounds
Methanogenesis	Methanogenic archaea (strict anaerobes)	Produce methane (CH$_4$) from organic matter in anaerobic environments (wetlands, rice paddies, landfill sites, guts of ruminants); methane is a potent greenhouse gas (25x more warming potential than CO$_2$ over 100 years)
Methane oxidation	Methanotrophic bacteria	Oxidise methane (CH$_4$) to CO$_2$ and water in aerobic environments (soil, water); help to mitigate methane emissions

Decomposition in Detail

Decomposition is the breakdown of dead organic matter by decomposers (bacteria and fungi). The rate of decomposition depends on:

Factor	Faster Decomposition	Slower Decomposition
Temperature	Warm (higher enzyme activity)	Cold (lower enzyme activity)
Moisture	Damp (provides medium for enzyme activity and decomposer growth)	Dry (decomposers need water; desert conditions slow decomposition)
Oxygen	Aerobic (aerobic decomposers are more efficient)	Anaerobic (slower; produces methane instead of CO$_2$)
C:N ratio of the material	Low (nitrogen-rich material, e.g., fresh grass)	High (carbon-rich material, e.g., wood, autumn leaves); the material is tough and low in nitrogen (a limiting nutrient for decomposer growth)
Surface area	Large (e.g., cut or shredded material)	Small (e.g., whole logs)

Compost production: A practical application of decomposition. Organic waste (food scraps, garden waste) is piled in a compost heap; bacteria and fungi decompose the organic matter, producing a nutrient-rich compost that can be used as fertiliser. Regular turning of the heap introduces oxygen (promoting aerobic decomposition) and speeds up the process.

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Antibiotics: Mechanisms and Resistance in Detail

Mechanisms of Action

Mechanism	Description	Example
Inhibit cell wall synthesis	Interfere with the synthesis of peptidoglycan; weaken the cell wall; the bacterium bursts due to osmotic pressure (only affect growing bacteria)	Penicillin (binds to and inhibits transpeptidase enzymes that cross-link peptidoglycan); vancomycin; cephalosporins
Inhibit protein synthesis	Bind to bacterial 70S ribosomes (which differ from eukaryotic 80S ribosomes) and interfere with translation	Tetracycline (binds to 30S subunit, prevents tRNA binding); erythromycin (binds to 50S subunit, prevents translocation); streptomycin
Inhibit DNA replication	Interfere with DNA gyrase (topoisomerase II) which is needed for DNA supercoiling during replication	Ciprofloxacin; nalidixic acid
Disrupt cell membrane	Insert into the bacterial cell membrane, forming pores that cause leakage of cellular contents	Polymyxin B (binds to lipopolysaccharide in Gram-negative outer membrane)
Inhibit metabolic pathways	Act as antimetabolites (structural analogues of essential metabolites); competitively inhibit enzymes in metabolic pathways	Sulfonamides (competitive inhibitors of dihydropteroate synthase, blocking folate synthesis); trimethoprim

Antibiotic Spectrum

Type	Description	Example
Narrow-spectrum	Effective against only a limited range of bacteria (specific Gram-positive or Gram-negative)	Penicillin V (Gram-positive); vancomycin (Gram-positive); isoniazid (mycobacteria)
Broad-spectrum	Effective against a wide range of bacteria (both Gram-positive and Gram-negative)	Tetracycline; ampicillin; ciprofloxacin; chloramphenicol

Why Antibiotics Do Not Affect Viruses

Reason	Explanation
No cell wall	Many antibiotics target bacterial cell wall synthesis; viruses have no cell wall (they have a protein capsid, not peptidoglycan)
No ribosomes	Many antibiotics target bacterial 70S ribosomes; viruses do not have ribosomes (they use the host cell's ribosomes to translate their mRNA)
No independent metabolism	Many antibiotics target bacterial metabolic pathways; viruses have no metabolism of their own (they rely entirely on the host cell's metabolic machinery)
Viruses are inside host cells	Viruses replicate inside host cells; an antibiotic that interferes with viral replication would also need to affect the host cell, causing unacceptable side effects

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Biotechnology in Agriculture

Genetically Modified (GM) Crops

Crop	Modification	Benefit	Controversy
Bt maize / Bt cotton	Gene from Bacillus thuringiensis inserted; plant produces Bt toxin protein that kills insect pests (lepidopteran larvae)	Reduced need for chemical insecticides; higher yields	Potential harm to non-target insects (e.g., butterflies); development of resistance in pests
Golden Rice	Genes for beta-carotene synthesis inserted; rice grains produce beta-carotene (precursor of vitamin A)	Addresses vitamin A deficiency in developing countries (a major cause of childhood blindness)	Intellectual property concerns; acceptance by local communities; long-term environmental effects
Roundup Ready soybeans	Gene for glyphosate resistance inserted (EPSPS enzyme); crop can be sprayed with glyphosate herbicide without being killed	Simplifies weed control; higher yields	Development of herbicide-resistant weeds ("superweeds"); dependence on single herbicide company
Virus-resistant papaya	Gene from papaya ringspot virus inserted; plant is resistant to the virus	Saved the Hawaiian papaya industry from ringspot virus	Public concern about eating viral genes
Herbicide-resistant oilseed rape	Modified to be resistant to specific herbicides	Easier weed management in crop fields	Gene flow to wild relatives; creation of "superweeds"

Methods of Introducing Genes into Plants

Method	Description
Agrobacterium-mediated transformation	Agrobacterium tumefaciens is a soil bacterium that naturally transfers a segment of its Ti (tumour-inducing) plasmid (T-DNA) into plant cells, causing crown gall disease. Scientists replace the tumour-causing genes with the desired gene and use the bacterium as a natural gene delivery vector
Biolistics (gene gun)	Microscopic gold or tungsten particles coated with DNA are fired at high speed into plant cells using a gene gun; some particles penetrate the nucleus and the DNA is incorporated into the plant genome
Protoplast transformation	The plant cell wall is removed (using cellulase enzyme) to create protoplasts; DNA is introduced using electroporation or PEG (polyethylene glycol); the cell wall is then regenerated

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Common Pitfalls

• Antibiotics are produced by MICROORGANISMS, not by pharmaceutical companies from scratch. Most antibiotics are derived from fungi (e.g., penicillin from Penicillium) or bacteria (e.g., streptomycin from Streptomyces). Pharmaceutical companies isolate, purify, and mass-produce them
• Broad-spectrum antibiotics kill BOTH Gram-positive and Gram-negative bacteria, including beneficial bacteria in the gut. This can cause diarrhoea and secondary infections (e.g., Clostridium difficile infection). Narrow-spectrum antibiotics target specific bacteria and cause less disruption to the normal microbiome
• Antibiotic resistance is a NATURAL phenomenon accelerated by human activity. Resistance genes exist naturally in soil bacteria (they produce antibiotics to compete with other bacteria and need resistance genes to protect themselves). Overuse and misuse of antibiotics in medicine and agriculture SELECT for resistant bacteria, increasing the frequency of resistance genes in the population
• Bt crops produce the Bt toxin protein, NOT live bacteria. The plant cells synthesise the Bt protein from the inserted bacterial gene. When an insect eats the plant tissue, the toxin is activated in the insect's alkaline gut and kills the insect by forming pores in its gut lining**

tip

tip Ready to test your understanding of Microorganisms and Biotechnology? The diagnostic test contains the hardest questions within the DSE specification for this topic, each with a full worked solution.

Unit tests probe edge cases and common misconceptions. Integration tests combine Microorganisms and Biotechnology with other biology topics to test synthesis under exam conditions.

See Diagnostic Guide for instructions on self-marking and building a personal test matrix.

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Industrial Biotechnology Applications

Bioremediation

Bioremediation is the use of microorganisms (bacteria, fungi, algae) to break down or remove environmental pollutants.

Type	Description	Example
In situ bioremediation	Treatment occurs at the site of contamination without excavating the polluted material	Adding nutrients (bio-stimulation) or microorganisms (bio-augmentation) to contaminated soil or groundwater
Ex situ bioremediation	Contaminated material is excavated and treated elsewhere (e.g., in a bioreactor or landfill)	Contaminated soil is removed and treated in a controlled facility

Pollutant	Microorganism Used	Mechanism
Oil spills	Pseudomonas, Alcanivorax	Hydrocarbon-degrading bacteria break down oil components into less harmful substances
Heavy metals	Certain bacteria, fungi	Microorganisms can accumulate, transform, or immobilise heavy metals (e.g., reducing Cr(VI) to less toxic Cr(III))
Pesticides	Various soil bacteria	Enzymes produced by bacteria break down pesticide molecules
Sewage and wastewater	Mixed microbial communities	Aerobic and anaerobic bacteria break down organic matter in sewage treatment plants
Plastic degradation	Ideonella sakaiensis	Bacterium discovered in 2016 that can break down PET (polyethylene terephthalate) plastic using PETase enzymes

Biofuels

Biofuel	Source Material	Description	Advantages	Disadvantages
Bioethanol	Sugarcane, maize, wheat	Produced by yeast fermentation of sugars; can be blended with petrol or used pure (E85)	Renewable; reduces CO2 emissions	Competes with food production; lower energy density than petrol
Biodiesel	Vegetable oils, animal fats	Produced by transesterification of oils with methanol; can be used in diesel engines	Renewable; biodegradable	Competes with food production; higher NOx emissions
Biogas (methane)	Organic waste, manure, sewage	Produced by anaerobic digestion of organic matter by methanogenic archaea; used for heating and electricity generation	Uses waste materials; reduces landfill	Lower energy density; methane leaks are potent greenhouse gas
Biobutanol	Sugarcane, maize, cellulose	Produced by bacterial fermentation (e.g., Clostridium acetobutylicum); can be used in petrol engines without modification	Higher energy density than ethanol; can use existing infrastructure	More expensive to produce; less efficient fermentation

Biotechnology in Medicine

Application	Description	Example
Recombinant insulin	Human insulin gene inserted into E. coli or yeast; bacteria/yeast produce human insulin for diabetics	Humulin (recombinant human insulin)
Recombinant vaccines	Pathogen antigens produced by genetically modified organisms; safer than live attenuated vaccines	Hepatitis B vaccine (yeast-produced surface antigen); HPV vaccine
Monoclonal antibodies	Identical antibodies produced by a single clone of B cells (hybridoma technology); used in diagnosis, cancer therapy, autoimmune diseases	Trastuzumab (breast cancer); adalimumab (rheumatoid arthritis); rapid COVID-19 antigen tests
Gene therapy	Functional copies of a defective gene are delivered to a patient's cells using a viral vector	Treatment of severe combined immunodeficiency (SCID); retinal dystrophy
Stem cell therapy	Embryonic or adult stem cells are used to replace damaged or diseased cells and tissues	Bone marrow transplants for leukaemia; potential for treating Parkinson's, spinal cord injury
PCR in diagnosis	Polymerase chain reaction used to amplify and detect pathogen DNA in patient samples	COVID-19 testing; HIV viral load testing; genetic disease screening
ELISA	Enzyme-linked immunosorbent assay; uses antibodies and enzymes to detect specific antigens or antibodies in blood samples	HIV testing; pregnancy testing (hCG detection); COVID-19 antibody testing

Monoclonal Antibody Production (Hybridoma Method)

1. Immunisation: A mouse is injected with the target antigen, stimulating B cells to produce antibodies against it
2. B cell extraction: B cells are removed from the mouse's spleen
3. Cell fusion: The B cells are fused with myeloma cells (cancerous B cells that divide indefinitely) using polyethylene glycol (PEG) or electrofusion
4. Hybridoma cells: The fused cells are called hybridomas; they combine the antibody-producing ability of B cells with the immortality of myeloma cells
5. Selection: Cells are grown in HAT medium -- only hybridoma cells survive (unfused B cells die naturally; unfused myeloma cells are killed by the HAT medium because they lack the enzyme HGPRT)
6. Screening: Individual hybridoma cells are cultured separately and tested to identify those producing the desired antibody
7. Cloning: The selected hybridoma is cloned to produce a large population of identical cells, all producing the same monoclonal antibody
8. Large-scale production: The hybridoma cells are grown in large bioreactors; antibodies are harvested from the culture medium

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Microbiology Techniques

Aseptic Technique

Aseptic technique is essential when working with microorganisms in the laboratory to prevent contamination of cultures and protect the operator from potentially pathogenic organisms.

Technique	Description	Purpose
Flaming the loop	The inoculating loop is heated until it glows red hot in a Bunsen burner flame	Sterilises the loop before and after use
Working near a Bunsen flame	All procedures are carried out close to the upward current of hot air created by a Bunsen burner	Creates a sterile zone; airborne contaminants are drawn away from the work area
Sterilising equipment	Petri dishes, pipettes, and culture media are sterilised by autoclaving (high-pressure steam at 121 degrees C for 15 minutes)	Kills all microorganisms including endospores
Opening Petri dishes minimally	Petri dish lids are only partially lifted and never placed face-down on the bench	Prevents airborne contamination of the agar surface
Disinfecting surfaces	The bench surface is wiped with 70% ethanol before and after the procedure	Kills any microorganisms on the bench

Growing Microorganisms in Culture

Component	Description
Culture medium	Contains nutrients required for microbial growth (carbohydrates, nitrogen source, minerals, vitamins)
Agar	A polysaccharide derived from seaweed; used as a solidifying agent in culture media; melts at ~85 degrees C and solidifies at ~42 degrees C
Nutrient broth	Liquid culture medium; used for growing large numbers of bacteria in suspension
Nutrient agar	Solid culture medium in Petri dishes; used for obtaining isolated colonies of bacteria
Selective media	Contains substances that inhibit the growth of certain microorganisms while allowing others to grow
Differential media	Contains indicators that allow different types of microorganisms to be distinguished by their appearance
Incubation	Cultures are typically incubated at 25-30 degrees C (for environmental bacteria) or 37 degrees C (for human pathogens) for 24-48 hours
Inverted Petri dishes	Cultured plates are stored upside down to prevent condensation from dripping onto the agar surface

Measuring Bacterial Growth

Method	Description	Advantages	Disadvantages
Direct count (microscopy)	Counting bacteria in a known volume under a microscope using a haemocytometer (counting chamber)	Quick; does not require incubation	Cannot distinguish living from dead cells
Viable count (colony counting)	Diluting a sample, spreading it on agar plates, incubating, and counting the colonies that grow (each colony originates from a single living cell)	Only counts living (viable) cells; widely used	Requires incubation time (24-48 hours); colonies may not be visible if very small
Turbidity (optical density)	Measuring the cloudiness (turbidity) of a liquid culture using a spectrophotometer -- more cells = more turbid	Quick; non-destructive; can monitor growth continuously	Cannot distinguish living from dead cells; less accurate at very low or very high densities
Dry mass	Filtering a known volume of culture, drying the filter paper and cells, and weighing	Direct measurement of biomass	Destructive; time-consuming; cannot monitor same culture continuously

Bacterial Growth Curve

Phase	Description	Rate of Division	Rate of Death
Lag phase	Bacteria are adapting to the new environment; synthesising enzymes and other molecules needed for growth; no significant increase in cell number	Low/zero	Low
Exponential (log) phase	Bacteria are dividing at their maximum rate; population doubles at regular intervals (generation time)	Maximum	Low
Stationary phase	Growth rate equals death rate; nutrients are becoming depleted; toxic waste products are accumulating; population size stabilises	Decreasing	Increasing
Death (decline) phase	Death rate exceeds growth rate; nutrients exhausted; toxic waste products at high levels; population declines	Near zero	High

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Common Pitfalls

• Antibiotics are produced by microorganisms, NOT by the human body. Penicillin is produced by the fungus Penicocillium; streptomycin by Streptomyces
• Antibiotics target bacterial cells, NOT viruses. Viruses have no cell walls, ribosomes, or metabolic processes that antibiotics can target. Taking antibiotics for a viral infection is ineffective and contributes to antibiotic resistance
• Bacteriostatic antibiotics STOP bacterial growth (do not kill); bactericidal antibiotics KILL bacteria. Knowing the difference is important for treating immunocompromised patients (who need bactericidal antibiotics since their immune system cannot finish the job)**
• In PCR, the enzyme used is Taq polymerase (from Thermus aquaticus), NOT DNA polymerase from human cells. Taq polymerase is heat-stable and does not denature at the high temperatures (95 degrees C) used in PCR**
• Restriction enzymes cut DNA at specific recognition sequences, producing either blunt ends or sticky ends. Sticky ends (with single-stranded overhangs) are more useful in genetic engineering because they facilitate the joining of DNA fragments from different sources**
• In bioremediation, "in situ" means treating the pollution on-site, NOT in a laboratory or bioreactor. "Ex situ" means removing the contaminated material for treatment elsewhere**