Biotechnology Applications

Question 1 (12)

(a) Pathway (8) — award 1 mark per correct ordered step, max 8:

1. Determine the amino acid sequences of insulin A-chain (21 aa) and B-chain (30 aa); back-translate to DNA using codon-optimised sequences for E. coli. (1)
2. Chemically synthesise the two artificial genes (A-chain gene and B-chain gene). (1)
3. Insert each gene separately into a plasmid vector, fused downstream of the lacZ (β-galactosidase) gene under a promoter (inducible, e.g. lac). (1)
4. Transform the recombinant plasmids into separate E. coli cultures. (1)
5. Select transformants (antibiotic marker) and grow/induce (e.g. IPTG) to express fusion proteins. (1)
6. Harvest cells, lyse, and isolate the β-gal–chain fusion proteins; cleave off the chain with cyanogen bromide (CNBr) at methionine linker. (1)
7. Purify the individual A and B chains. (1)
8. Mix chains under oxidising conditions to form the correct disulfide bonds → functional insulin. (1)

(b) (2): Producing chains separately avoids relying on bacteria to fold and process proinsulin correctly (1); the two chains are combined in vitro and disulfide bonds (two inter-chain + one intra-A) are allowed to form, yielding the active mature hormone (1).

(c) (2): Bacteria lack the machinery for post-translational modifications such as glycosylation and lack the ER/Golgi processing/chaperone system (1). Insulin is not glycosylated and is a small peptide, so lack of glycosylation does not impair function — only correct disulfide bonding is needed, done chemically (1).

Question 2 (10)

(a) Bt mechanism (6):

• Bt (Bacillus thuringiensis) produces Cry protein as an inactive protoxin/crystal (1).
• When ingested by susceptible insect larva, the alkaline midgut pH solubilises the crystal (1).
• Gut proteases cleave the protoxin into the active toxin (1).
• Active toxin binds specific receptors on midgut epithelial cell membranes (present only in certain insects) (1).
• Toxin inserts and forms pores, disrupting ion balance / osmotic lysis of gut cells (1).
• Gut lining is destroyed → larva stops feeding and dies. Humans/non-target insects lack alkaline gut + specific receptors, so no activation/binding → harmless (1).

(b) Herbicide resistance (4):

• Glyphosate inhibits the enzyme EPSP synthase (EPSPS) in the shikimate pathway for aromatic amino acid synthesis (1).
• Normally this blocks synthesis of aromatic amino acids → plant dies (1).
• Resistant crops carry a transgene for a glyphosate-insensitive EPSPS (e.g. bacterial CP4 EPSPS) (1).
• The modified enzyme still functions despite glyphosate, so the crop survives while weeds die (1).

Question 3 (12)

(a) iPSC reprogramming (5):

• Start with a differentiated adult somatic cell (e.g. skin fibroblast) (1).
• Introduce a defined set of transcription factors — the Yamanaka factors: Oct4, Sox2, Klf4, c-Myc (OSKM) (2, all four = 2, partial = 1).
• These factors reactivate the endogenous pluripotency network / remodel epigenetic state, resetting the cell (1).
• Result: a cell restored to a pluripotent, embryonic-stem-cell-like state. Pluripotency = ability to differentiate into cell types of all three germ layers (ecto-, meso-, endoderm) (1).

(b) Cloning contrast (4):

• Shared technique: Somatic Cell Nuclear Transfer (SCNT) — nucleus of a somatic cell placed into an enucleated egg, stimulated to divide (1).
• Both generate a blastocyst genetically identical to the donor (1).
• Reproductive cloning: blastocyst implanted into a surrogate → whole cloned organism (e.g. Dolly) (1).
• Therapeutic cloning: inner cell mass harvested for patient-matched embryonic stem cells for therapy — no organism produced (1).

(c) Advantage (3): iPSCs are patient-derived, so cells/tissues are genetically matched → no immune rejection (1); they avoid the ethical problem of destroying embryos (1); they can be made from readily accessible adult cells in large amounts (1).

Question 4 (10)

(a) Hybridoma steps (6) — 1 mark each:

1. Immunise a mouse with the target antigen. (1)
2. Antigen stimulates B-lymphocytes; harvest antibody-producing B-cells from the spleen. (1)
3. Fuse B-cells with immortal myeloma cells (using PEG) → hybridomas. (1)
4. Grow in HAT selection medium to select fused hybridomas only. (1)
5. Screen and single-cell clone to isolate the hybridoma making the desired specific antibody (monoclonal). (1)
6. Culture the selected clone (or grow in bioreactor) and purify the secreted monoclonal antibody. (1)

(b) HAT selection (4):

• HAT = Hypoxanthine, Aminopterin, Thymidine. Aminopterin blocks the de novo nucleotide synthesis pathway (1).
• Cells must then use the salvage pathway (enzyme HGPRT) to make nucleotides (1).
• Myeloma cells are HGPRT-deficient → cannot use salvage → die in HAT; unfused B-cells have HGPRT but are mortal → die out naturally in culture (1).
• Hybridomas gain HGPRT from the B-cell parent (survive HAT) and immortality from the myeloma parent → grow indefinitely (1).

Question 5 (8)

(a) mRNA vaccine (5):

• Lipid nanoparticle (LNP) encapsulates and protects mRNA, aids cellular uptake / endosomal escape into cytoplasm (1).
• Host ribosomes translate the mRNA into the antigen protein (e.g. spike) (1).
• Antigen is presented on MHC / displayed, triggering both antibody (B-cell) and T-cell responses → immunity/memory (1).
• Modified nucleosides (e.g. pseudouridine) reduce innate immune degradation and increase translation (1).
• mRNA is transient and stays in cytoplasm — it does not enter the nucleus and cannot integrate into the genome (no reverse transcriptase / no DNA intermediate) (1).

(b) Comparison (3):

• mRNA vaccine: delivers genetic instructions; antigen is made inside the patient's own cells (1).
• Recombinant subunit vaccine: delivers the ready-made antigen protein itself (1).
• The protein is produced beforehand in a cell/culture system (bacteria, yeast, insect cells) and purified, then injected (1).

Question 6 (8)

(a) Four bioreactor parameters (4) — 1 mark each (any 4):

• Temperature — optimal enzyme/growth kinetics; excess denatures proteins. (1)
• pH — maintained by buffering/acid-base addition for enzyme activity & cell health. (1)
• Dissolved O₂ / aeration & stirring — supplies oxygen for aerobic culture; mixing homogenises. (1)
• Nutrient/substrate feed — controlled feed avoids substrate inhibition and maintains growth. (Also accept foam control, agitation rate, cell density.) (1)

(b) Bioremediation (4):

• Definition: use of living organisms (microbes/plants) to remove, degrade, or detoxify environmental pollutants (2).
• Example: oil-degrading bacteria (e.g. Pseudomonas, "superbug") break down hydrocarbons in oil spills — enzymes oxidise alkanes/aromatics to CO₂ and water; OR Deinococcus/engineered microbes reducing/immobilising heavy metals or degrading pesticides (2).

[
  {"claim": "Insulin A-chain has 21 residues and B-chain has 30 residues, total 51 residues in mature insulin",
   "code": "A=21; B=30; result = (A+B == 51)"},
  {"claim": "Mature insulin has 3 disulfide bonds (2 interchain + 1 intrachain A)",
   "code": "interchain=2; intrachain=1; result = (interchain+intrachain == 3)"},
  {"claim": "Four Yamanaka reprogramming factors (OSKM)",
   "code": "factors={'Oct4','Sox2','Klf4','cMyc'}; result = (len(factors) == 4)"},
  {"claim": "HAT medium named for its three components Hypoxanthine, Aminopterin, Thymidine",
   "code": "hat=['Hypoxanthine','Aminopterin','Thymidine']; result = (len(hat) == 3)"}
]