Exam 2025 Practice

Applied Molecular Cellular Biology

📘 Applied Exam Practice — Complete Theoretical Summary (Exam-Focused)

This document is essentially an exam walkthrough + meta-guidance explaining:

what kinds of questions appear,
what theoretical understanding is expected,
and why certain answers earn points.

The exam tests conceptual understanding, data interpretation, and methodological reasoning, not memorization of wording.

🧠 1. Paper Overview Questions (Big-Picture Understanding)

✍️ “Describe what this paper is about (in your own words)”

What examiners want:

A clear summary, not copied text
Correct biological context
Awareness of the broader goal

Core theory:

The paper focuses on recombinant insulin production in yeast
Yeast is used as an expression system
The ultimate goal is cheap, scalable insulin production

🔑 Key insight: Saying only “they made insulin in yeast” is incomplete. You must connect expression system → application (biomanufacturing, medicine, food biotech).

🧫 2. Why Use Yeast Instead of E. coli?

⚖️ Expression System Comparison

Feature	Yeast 🧬	E. coli 🦠
Cell type	Eukaryote	Prokaryote
Protein folding	✅ Better	❌ Often misfolded
Disulfide bonds	✅ Yes	❌ Limited
Secretion	✅ Possible	❌ Difficult
PTMs	Partial	None

Theory tested:

Protein folding matters for functional insulin
Yeast has eukaryotic chaperones
Yeast can secrete proteins, simplifying purification

🧠 Exam logic: Yeast bridges the gap between bacterial simplicity and mammalian complexity.

🧬 3. Why Produce Insulin from Multiple Species? 🐄🐔🐖

Core theoretical motivation:

Insulin is a growth factor
Different species’ cells respond best to species-matched growth factors
Applications include:
- Cultured meat
- Serum-free media

🧪 Ethical & practical angle

Replaces fetal calf serum (FCS):
- Expensive
- Ethically problematic
- Batch-variable

🔑 Exam takeaway: This is not just molecular biology — it’s biotechnology + ethics + scalability.

🧬 4. Plasmid Map Interpretation (Classic Exam Question)

You must identify functional elements, not just name them.

Typical components:

Promoter (e.g. galactose promoter)
Gene of interest (insulin)
Signal peptide (for secretion)
Selection markers (yeast & bacterial)
Origin of replication (yeast vs E. coli)
Terminator

⚠️ Galactose promoter nuance

Expression occurs without galactose
Reason: leaky expression / engineered regulation
You are not expected to explain regulation failure unless relevant

📌 Exam scoring:

Listing components = OK
Explaining function = better
Understanding why present = best

🧩 5. Why Replace the C-Peptide with HL18?

Insulin biology refresher:

Native insulin contains a C-peptide
C-peptide is cleaved during maturation

HL18 advantages:

Improves expression
Enhances secretion
Simplifies processing

🧠 Key principle: Protein engineering is used to optimize production, not just mimic biology.

🧪 6. DTT + β-Mercaptoethanol in SDS-PAGE

🔬 What does this show?

Insulin chains are linked by disulfide bonds
Reducing agents break these bonds
Result:
- Non-reduced → one band
- Reduced → multiple chains

🧠 Exam logic: You must connect chemical treatment → molecular structure → gel pattern.

🧲 7. IMAC (Immobilized Metal Affinity Chromatography)

Principle:

His-tagged proteins bind metal ions (Ni²⁺, Co²⁺)
Elution via imidazole competition

📌 Why this matters on the exam:

Even if not taught in class, you must:
- Recognize the method
- Understand why it works

🧠 Meta-lesson: You are expected to prepare unfamiliar methods in advance.

📊 8. Figure Interpretation & Poor Figure Legends

SDS-PAGE figure criticism:

Missing:
- Clear lane labels
- Ladder identity
- Experimental conditions

📌 What examiners reward:

Noticing missing controls
Suggesting improvements
Demonstrating scientific literacy

📈 9. MTT Assay: Why Only a Small Effect?

Possible explanations:

Medium already contains growth factors
Insulin receptors may be saturated
MTT measures metabolism, not proliferation directly

Better experimental design:

Serum-free conditions
Washout step
More sensitive assays

🧠 Key principle: Negative or weak results ≠ failure — they require interpretation.

🔁 10. Alternative Proliferation Assays

Valid answers include:

Cell counting
BrdU / EdU incorporation
Wound-healing assays
Colony formation

📌 Exam rule: Any method is valid if the biological reasoning is sound.

🧬 11. Antibody Specificity (aa 30–40)

Western blot logic:

Size ≠ identity
Antibody binding confirms protein identity

Cross-species recognition:

Region 30–40 is conserved
Antibody cannot distinguish human vs cow vs pig insulin

🧠 Key takeaway: Epitope conservation determines antibody specificity.

🧠 12. Does Insulin Origin Need to Match Cell Type?

Answer: No strong evidence

Data shows similar effects across species
No clear species-specific advantage

📌 Exam insight: You must challenge assumptions using data, not intuition.

🧬 13. Yeast Genome Integration & Marker Recycling

Step 1: Homologous recombination

Linear DNA integrates efficiently in yeast

Step 2: Marker removal

Avoids metabolic burden
Selection via 5-FOA / URA3 counter-selection

🧠 Exam challenge: This tests genetic engineering logic, not memorization.

🧬 14. DNA Sequencing & BLAST Interpretation

Core rules:

DNA is read 5′ → 3′
Databases may store reverse complements

Common mistakes:

Ignoring strand orientation
Comparing sequences backwards

📌 Scoring tip: Evaluate each option — partial credit is possible.

🌰 15. Detecting Amygdalin (Bitter Almond Taste)

Valid detection methods:

ELISA (if antibodies exist)
Mass spectrometry
NMR
Chemical extraction

😂 Bonus logic:

Taste ≠ quantitative but shows chemical awareness

🐛 16. DNA Damage Assays in C. elegans

Possible approaches:

Reporter strains
DNA damage markers
Apoptosis assays
Long-range PCR

🧠 Key idea: Genotoxicity = phenotype + molecular readout.

🦠 17. Pathogenic Bacteria as Biopesticides

Data interpretation:

EM images show epithelial destruction
Indicates toxin activity

⚠️ Practical conclusion:

Not acceptable for human food
Ethical & regulatory implications matter

🔍 18. Expansion Microscopy

Principle:

Sample embedded in polymer gel
Gel expands isotropically (~4–5×)
Improves resolution beyond diffraction limit

🍞 Analogy:

Raisins in expanding dough

🚰 19. Aquaporins: Structure & Function

Structure:

6 transmembrane helices
Tetrameric assembly

Functions:

Water transport
Cell migration
Adhesion
Cancer relevance

🧠 Why multiple AQPs?

Different tissues
Different regulation
Different permeability

⚗️ 20. Synthetic Aquaporins & Inhibitors

Observations:

Synthetic AQPs increase migration
Hg²⁺ inhibits both natural & synthetic AQPs
DA-18 selectively inhibits synthetic AQPs

Mechanistic reasoning:

Charge complementarity
Channel blockage

🧬 21. Therapeutic Strategies for Aquaporin Disorders

Acceptable answers:

Small-molecule modulators
RNA-based approaches
CRISPR-Cas9
Trafficking correction

📌 Exam rule: Creativity is allowed if biologically plausible.

🧠 22. Organoids & Conserved Gene Function

Strategy:

Edit human stem cells (CRISPR)
Generate brain organoids
Assess dopaminergic neurons
Rescue with orthologs

🧠 Key concept: Model systems test functional conservation, not just sequence similarity.

📝 Final Exam Strategy (Explicitly Stated in File)

✅ Answer all questions ✅ Start simple, then add detail ❌ Don’t get stuck on one hard question ❌ Don’t skip supplementary material

Quiz

Score: 0/30 (0%)

Q0. What is the primary scientific goal of producing insulin in yeast described in the document?

To study yeast metabolism

To replace all bacterial expression systems

To enable cheaper and scalable insulin production

To improve yeast genetic stability

Q1. Why is yeast often preferred over E. coli for recombinant insulin production?

Yeast grows faster than E. coli

Yeast allows proper protein folding and disulfide bond formation

Yeast plasmids are smaller

Yeast requires fewer nutrients

Q2. What is a major reason for producing insulin from multiple animal species?

To test yeast mutation rates

To study insulin evolution

To support species-specific cell growth in applications like cultured meat

To improve antibody specificity

Q3. Which ethical concern is addressed by producing recombinant insulin instead of using fetal calf serum?

Environmental pollution

Animal welfare and slaughter

Antibiotic resistance

Genetic instability

Q4. What is the function of a signal peptide in the yeast expression construct?

Enhances transcription rate

Allows plasmid replication

Directs the protein for secretion

Confers antibiotic resistance

Q5. Why can a galactose promoter show expression even without galactose?

Because galactose is present in trace amounts

Due to leaky or engineered promoter regulation

Because yeast cannot repress transcription

Because glucose activates the promoter

Q6. What is the purpose of replacing the C-peptide with the HL18 peptide?

To increase insulin molecular weight

To improve secretion and expression efficiency

To change insulin receptor binding

To reduce immunogenicity

Q7. Why does reducing SDS-PAGE show multiple insulin-related bands?

Proteins aggregate during electrophoresis

Disulfide bonds are broken, separating insulin chains

The gel concentration is too low

Insulin is degraded by SDS

Q8. What is the fundamental principle of IMAC purification?

Charge-based separation

Size-based filtration

Affinity of His-tags for metal ions

Hydrophobic interaction

Q9. Why is linear DNA often preferred over plasmids for yeast genome integration?

Linear DNA replicates faster

Yeast cannot take up plasmids

Linear DNA enhances homologous recombination efficiency

Plasmids are toxic to yeast

Q10. Why is marker recycling important in yeast genetic engineering?

To reduce mutation rates

To avoid excessive metabolic burden from marker expression

To increase plasmid copy number

To improve transcription accuracy

Q11. What key mistake commonly occurs when interpreting DNA BLAST results?

Ignoring GC content

Misreading the E-value scale

Failing to consider strand orientation (5′ to 3′)

Using too short sequences

Q12. Why does an MTT assay sometimes show only a small response to insulin treatment?

MTT is insensitive to insulin

Cells may already be saturated with growth factors

Insulin degrades MTT

MTT only measures apoptosis

Q13. Why is protein size alone insufficient to confirm protein identity?

Proteins often migrate anomalously

Different proteins can share similar molecular weights

Gels cannot resolve small differences

All of the above

Q14. Why can antibodies targeting insulin residues 30–40 not distinguish species origin?

The antibody is non-specific

That region is conserved across species

Insulin is identical in all species

The epitope is masked

Q15. True or False: The exam prioritizes memorization of exact wording over conceptual understanding.

True

False

Q16. True or False: Weak experimental effects are considered invalid results in the exam context.

True

False

Q17. True or False: IMAC purification relies on covalent bonding between the protein and the column.

True

False

Q18. True or False: Expansion microscopy improves resolution by physically enlarging the biological sample.

True

False

Q19. True or False: Aquaporins are composed of six transmembrane helices and form tetramers.

True

False

Q20. True or False: Mercury ions inhibit both natural and synthetic aquaporins.

True

False

Q21. True or False: DA-18 selectively inhibits synthetic aquaporins more than natural ones.

True

False

Q22. True or False: DNA damage in C. elegans can be assessed indirectly through apoptosis assays.

True

False

Q23. True or False: Taste testing alone is a sufficient quantitative method to detect amygdalin.

True

False

Q24. True or False: The use of pathogenic bacteria in food products was proposed as a realistic solution.

True

False

Q25. True or False: Synthetic aquaporins can enhance cell migration in wound-healing assays.

True

False

Q26. True or False: Marker genes can consume a large fraction of yeast protein production capacity.

True

False

Q27. True or False: BLAST sequence comparison must consider reverse-complement sequences.

True

False

Q28. True or False: The exam allows partial credit for logically correct but incomplete answers.

True

False

Q29. True or False: Organoids can be used to test functional conservation of genes identified in C. elegans.

True

False