Biotechnology Applications

Question 1 (12)

(a) (3)

• Bacteria (prokaryotes) lack the machinery to correctly fold proinsulin and cleave the C-peptide / form correct disulfide bonds (1).
• Expressing chains separately gives simpler, well-defined products (1).
• The A and B chains are then joined chemically in vitro via disulfide bonds to form mature functional insulin (1).

(b) (3) Purified yield per batch = $4.2\ \text{g/L} \times 500\ \text{L} = 2100\ \text{g}$ (1). This is 68% of expressed: expressed $= 2100 / 0.68 = 3088.2\ \text{g}$ (1). $= 3.09\ \text{kg}$ (1).

(c) (3) Purified mass $= 2100\ \text{g} = 2\,100\,000\ \text{mg}$ (1). Doses $= 2\,100\,000 / 0.6$ (1) $= 3\,500\,000$ doses (1).

(d) (3) — any TWO, 1.5 each (round to 3):

• Constant high expression imposes a metabolic burden, slowing cell growth → lower total biomass/yield (1.5).
• Continuous protein production may cause toxicity / inclusion body aggregation or plasmid instability/loss without selection timing (1.5).
• Cannot decouple growth phase from production phase — cells cannot first reach high density before producing (1.5).

Question 2 (12)

(a) (3)

• Cry protoxin is solubilised and activated only in the insect gut's alkaline pH (1).
• Activated toxin binds specific receptors on insect midgut epithelial cells, forming pores → cell lysis / death (1).
• Humans have acidic stomachs and lack these gut receptors, so the protein is digested harmlessly (1).

(b) (4)

• Refuge = block of non-Bt maize where susceptible pests survive and breed (1).
• Rare resistant insects surviving on Bt crop mate with abundant susceptible insects from refuge (1).
• Resistance alleles are usually recessive, so heterozygous offspring remain susceptible / killed by Bt (1).
• This keeps resistance-allele frequency low, delaying spread through population (1).

(c) (3) Independent events: $P(\text{both}) = (2\times10^{-3})\times(2\times10^{-3}) = 4\times10^{-6}$ (2). Stacking requires an insect to acquire two rare independent resistances simultaneously — vastly less likely, so resistance evolves far slower (1).

(d) (2)

• Herbicide resistance: crop carries an enzyme/altered target that tolerates a chemical spray (e.g. glyphosate-insensitive EPSPS), protecting the crop from the herbicide (1).
• Bt resistance: crop produces its own insecticidal toxin that kills feeding pests directly (1).

Question 3 (12)

(a) (6) — 2 marks each part:

• (i) mRNA vaccine delivers mRNA encoding S (in lipid nanoparticles); subunit delivers the purified S protein itself (2).
• (ii) mRNA: antigen made inside the patient's own cells; subunit: antigen made in vitro in bioreactors (e.g. yeast/insect cells) then injected (2).
• (iii) mRNA advantage: very fast to design/manufacture from sequence alone, no cell culture of protein needed. Subunit advantage: no genetic material injected / more stable storage / established safety, easier acceptance (2).

(b) (4)

• Immunise animal with S; isolate B/plasma cells producing the antibody (1) — contribute antibody specificity (1).
• Fuse with myeloma (cancer) cells (1) — contribute immortality / indefinite division (1).
• (Fused hybridoma is selected, cloned, and screened for the neutralising antibody.)

(c) (2)

• Monoclonal antibody gives passive immunity: ready-made antibodies act immediately to neutralise virus → treatment (1).
• Vaccine gives active immunity: stimulates patient's own immune response + memory cells for future protection → prophylactic (1).

Question 4 (12)

(a) (4)

• Skin cells reprogrammed to iPSCs by introducing pluripotency transcription factors (e.g. Oct4, Sox2, Klf4, c-Myc) (1).
• iPSCs are pluripotent → can be differentiated into β-cells (1).
• Because cells come from the patient's own genome, they are genetically matched (1).
• So MHC/self-antigens match → immune system does not recognise them as foreign → no/low rejection (1).

(b) (4)

• Therapeutic cloning: end-product is cells/tissues (e.g. via SCNT to make patient-matched stem cells) for treatment (1) + no whole organism produced (1).
• Reproductive cloning: end-product is a whole cloned organism/individual (1).
• Ethical concern (reproductive): e.g. cloning humans, welfare/high abnormality rate, identity/consent issues (1).

(c) (4)

• Organoid = a small 3D self-organising tissue-like structure grown in vitro from stem cells that mimics an organ's structure/function (2).
• Two advantages (1 each): more physiologically realistic 3D architecture/cell interactions than flat 2D; patient-specific so predicts that individual's drug response; better models tissue-level effects/drug diffusion. (any 2)

Question 5 (12)

(a) (3) — any THREE, 1 each:

• Temperature — optimal for enzyme/organism activity, avoid denaturation.
• pH — kept optimal for microbial metabolism.
• Dissolved O₂ / aeration (or agitation) — supplies oxygen and mixing for aerobic metabolism (for ethanol, control anaerobic conditions / mixing).
• Nutrient/substrate feed — maintains growth. (Also foam control, sterility.)

(b) (4) Moles glucose $= 1\,000\,000\ \text{g} / 180 = 5555.6\ \text{mol}$ (1). Ethanol moles $= 2 \times 5555.6 = 11\,111.1\ \text{mol}$ (1). Mass $= 11\,111.1 \times 46 = 511\,111\ \text{g}$ (1) $\approx 511\ \text{kg}$ (1).

(c) (2) Actual $= 0.82 \times 511.1 = 419\ \text{kg}$ (2).

(d) (3)

• Engineer bacteria carrying hydrocarbon-degrading enzyme genes (e.g. alkane monooxygenase) to break down oil (1).
• Add a genetic circuit/reporter: a contaminant-responsive promoter linked to a reporter gene (e.g. GFP / luciferase) that switches on in proportion to pollutant level (1).
• This engineered biosensor produces a measurable signal reporting contamination while degradation proceeds (1).

[
  {"claim":"Q1b expressed insulin ~3088 g", "code":"purified=4.2*500; expressed=purified/0.68; result = abs(expressed-3088.235)<1.0"},
  {"claim":"Q1c doses = 3.5 million", "code":"doses=(4.2*500*1000)/0.6; result = doses==3500000"},
  {"claim":"Q2c both-resistance prob = 4e-6", "code":"p=(Rational(2,1000))**2; result = p==Rational(4,1000000)"},
  {"claim":"Q5b theoretical ethanol ~511.1 kg", "code":"mol_glu=1000000/180; mol_eth=2*mol_glu; mass=mol_eth*46/1000; result = abs(mass-511.11)<0.5"},
  {"claim":"Q5c actual ethanol ~419 kg", "code":"actual=0.82*(2*(1000000/180)*46/1000); result = abs(actual-419.11)<1.0"}
]