Competency-Based Question Bank: Biotechnology: Principles and Processes

Section A: Competency-Based Multiple Choice Questions (Application & Analysis)

1. Analyze the Process: A student is performing a PCR reaction. After 30 cycles, he observes no amplification of the target gene. Upon checking the protocol, he finds he used human DNA polymerase instead of Taq polymerase. Why did the reaction fail? a) Human polymerase cannot add nucleotides. b) Human polymerase is not thermostable and denatures at 94°C during the denaturation step. c) Human polymerase requires a different pH. d) Primers do not bind to human polymerase. Answer: b) Human polymerase is not thermostable and denatures at 94°C during the denaturation step. Explanation: The first step of PCR is heating to ~94°C to separate strands. Normal polymerases (like human) denature and lose activity at this temperature. Taq polymerase is from a thermophile (Thermus aquaticus) and is heat-stable.

2. Evaluate the Cloning Strategy: You want to clone a human gene into E. coli. The gene contains introns. Which of the following should you use as the starting material to ensure the bacteria produces the correct protein? a) Genomic DNA b) mRNA c) cDNA (Complementary DNA made from mRNA) d) tRNA Answer: c) cDNA (Complementary DNA made from mRNA) Explanation: Bacteria (prokaryotes) lack the machinery (spliceosomes) to remove introns. Human genomic DNA has introns. cDNA is made from mature mRNA (where introns are already removed), so bacteria can translate it correctly.

3. Predict the Result: In Blue-White screening, a researcher inserts a gene of interest successfully into the lacZ gene of a plasmid vector. When these bacteria are plated on X-gal medium, the colonies will appear: a) Blue, because the enzyme is active. b) White, because the enzyme is inactivated (Insertional Inactivation). c) Blue, because the gene enhances enzyme activity. d) Colorless/Transparent because the bacteria die. Answer: b) White, because the enzyme is inactivated (Insertional Inactivation). Explanation: The lacZ gene produces beta-galactosidase which turns X-gal Blue. If a foreign gene is inserted into lacZ, the gene is broken (inactivated). No enzyme is made, so the colonies remain White.

4. Assertion (A): Restriction endonucleases are called "molecular scissors". Reason (R): They cut DNA at random locations to generate fragments. a) Both A and R are true and R is the correct explanation of A. b) Both A and R are true but R is not the correct explanation of A. c) A is true but R is false. d) A is false but R is true. Answer: c) A is true but R is false. Explanation: They are called scissors because they cut DNA, but they cut at specific recognition sequences (palindromes), not random locations.

5. Bioprocess Control: In a stirred-tank bioreactor, the "sparger" system malfunctions. Which parameter will be most immediately and critically affected? a) Temperature b) pH c) Dissolved Oxygen availability d) Foam level Answer: c) Dissolved Oxygen availability Explanation: The sparger bubbles sterile air/oxygen into the reactor. Without it, aerobic cells will suffocate due to lack of dissolved oxygen.

6. Identify the Enzyme: Which enzyme is essential for joining the "sticky ends" of a vector and a foreign gene to form a recombinant DNA molecule? a) DNA Polymerase b) Exonuclease c) DNA Ligase d) Alkaline Phosphatase Answer: c) DNA Ligase Explanation: Ligase forms the phosphodiester bonds between the sugar and phosphate groups of the two DNA fragments, sealing the nick.

7. Transformation Logic: Why are bacterial cells treated with Calcium Chloride ($CaCl_2$) before transformation? a) To kill the bacteria. b) To make the cell wall permeable to DNA (Competence). c) To provide nutrients for the DNA. d) To activate the restriction enzymes. Answer: b) To make the cell wall permeable to DNA (Competence). Explanation: Divalent cations like Calcium increase the efficiency with which DNA enters the bacterium through pores in its cell wall.

8. Analyze the Gel: In agarose gel electrophoresis, DNA fragments separate based on size. Where would you find the smallest DNA fragments? a) Near the cathode (negative electrode) where DNA was loaded. b) Near the anode (positive electrode), furthest from the well. c) In the middle of the gel. d) DNA does not move in an electric field. Answer: b) Near the anode (positive electrode), furthest from the well. Explanation: DNA is negatively charged and moves towards the positive anode. Smaller fragments move faster through the gel matrix and travel the furthest.

9. Selectable Marker: A plasmid vector has two antibiotic resistance genes: Ampicillin ($amp^R$) and Tetracycline ($tet^R$). A foreign gene is inserted into the $tet^R$ site. A recombinant colony will be: a) Resistant to both Ampicillin and Tetracycline. b) Sensitive to both. c) Resistant to Ampicillin but sensitive to Tetracycline. d) Resistant to Tetracycline but sensitive to Ampicillin. Answer: c) Resistant to Ampicillin but sensitive to Tetracycline. Explanation: The insertion destroys the $tet^R$ gene (Insertional Inactivation), making it sensitive to Tetracycline. The $amp^R$ gene is untouched, so it remains resistant to Ampicillin.

10. Downstream Processing: Which of the following is not a part of downstream processing? a) Separation of the product. b) Purification of the product. c) Genetic engineering of the host cell. d) Clinical trials (quality control). Answer: c) Genetic engineering of the host cell. Explanation: Genetic engineering is "Upstream Processing" (creation of the strain). Downstream processing happens after the product has been made in the bioreactor (Separation, Purification, Formulation).

11. PCR Components: If you forget to add "Primers" to your PCR mixture, what will happen? a) Non-specific amplification will occur. b) No new DNA strands will be synthesized. c) The DNA will not denature. d) Taq polymerase will degrade the template. Answer: b) No new DNA strands will be synthesized. Explanation: DNA polymerase cannot start synthesis from scratch; it can only add nucleotides to an existing 3'-OH group provided by a primer.

12. Vector Choice: Which vector would be most suitable for transferring a gene into a plant cell? a) pBR322 b) Retrovirus c) Ti plasmid (Agrobacterium) d) Lambda Phage Answer: c) Ti plasmid (Agrobacterium) Explanation: The Ti plasmid from Agrobacterium tumefaciens naturally infects plants and transfers DNA. It is modified to be a safe vector for plants. Retroviruses are for animals.

13. Palindromic Sequence: Which of the following sequences is a palindrome (read same 5'->3' on both strands)? a) 5'-G A A T T C-3' b) 5'-C C T A G G-3' c) 5'-A T G C T G-3' d) 5'-A G C T-3' Answer: a) 5'-G A A T T C-3' Explanation: The complementary strand is 3'-C T T A A G-5'. Reading the complementary strand 5'->3' gives G A A T T C, which is identical to the top strand. (This is the EcoRI site).

14. Process Sequence: Correct order of rDNA steps: a) Isolation -> Ligation -> Cutting -> Transformation. b) Cutting -> Isolation -> Transformation -> Ligation. c) Isolation -> Cutting -> Ligation -> Transformation. d) Transformation -> Cutting -> Ligation -> Isolation. Answer: c) Isolation -> Cutting -> Ligation -> Transformation. Explanation: First get the DNA (Isolation), then cut it (Restriction), join it to vector (Ligation), and put it into host (Transformation).

15. Critical Thinking: Why is ethidium bromide used in gel electrophoresis? a) To cut the DNA. b) To visualize DNA under UV light (it intercalates). c) To make the DNA heavier. d) To give a negative charge to DNA. Answer: b) To visualize DNA under UV light. Explanation: Ethidium bromide intercalates between DNA bases and fluoresces bright orange under UV light, making the invisible DNA bands visible.

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Section B: Case-Study & Source-Based Questions

Case Study 1: The Insulin Factory

A biotech company wants to produce human insulin on a large scale. They isolate the human insulin gene. Scenario A: They insert the gene directly into a bacterial genome without a vector. Scenario B: They insert the gene into a high-copy-number plasmid vector and transform E. coli.

16. Evaluate: Which scenario is likely to produce insulin? a) Scenario A only. b) Scenario B only. c) Both. d) Neither. Answer: b) Scenario B only. Explanation: For foreign DNA to be maintained and replicated in a host, it must be linked to an "Origin of Replication" (ori). A plasmid vector provides this. Direct insertion without an ori (or integration mechanism) leads to loss of the DNA during cell division.

17. Reasoning: Why does Scenario A fail? a) Bacteria degrade human DNA instantly. b) The DNA cannot replicate because it lacks an "Origin of Replication" (ori) which the vector provides. c) The gene is too large. d) Bacteria don't have insulin receptors. Answer: b) The DNA cannot replicate because it lacks an "Origin of Replication"... Explanation: Without an ori, the DNA is not copied when the bacterium divides, so daughter cells won't have the gene.

18. Optimization: To maximize production in Scenario B, the company uses a Stirred-tank Bioreactor. What is the specific role of the "Stirrer"? a) To cut the DNA. b) To maintain even temperature and oxygen availability throughout the tank. c) To kill unwanted bacteria. d) To precipitate the insulin. Answer: b) To maintain even temperature and oxygen availability throughout the tank. Explanation: The stirrer mixes the contents, ensuring all cells get equal access to nutrients and oxygen (homogeneity) and preventing heat hotspots.

Case Study 2: The Antibiotic Selection

A student creates a recombinant plasmid by inserting gene X into the BamHI site of the pBR322 vector. The BamHI site is located within the Tetracycline resistance gene ($tet^R$). The vector also has an Ampicillin resistance gene ($amp^R$).

19. Predict: What will be the phenotype of the transformants containing the recombinant plasmid? a) $amp^R$ $tet^R$ b) $amp^S$ $tet^S$ c) $amp^R$ $tet^S$ d) $amp^S$ $tet^R$ Answer: c) $amp^R$ $tet^S$ Explanation: They have the plasmid, so they are resistant to Ampicillin ($amp^R$). However, the insertion of Gene X broke the Tetracycline gene, making them sensitive to Tetracycline ($tet^S$).

20. Method: How will the student select these recombinant bacteria from non-recombinants? a) They will grow on Tetracycline plates. b) They will grow on Ampicillin plates but die on Tetracycline plates (Replica Plating). c) They will be blue in color. d) They will be fluorescent. Answer: b) They will grow on Ampicillin plates but die on Tetracycline plates. Explanation: This is the method of "Replica Plating". Non-recombinants are $amp^R tet^R$ (grow on both). Recombinants are $amp^R tet^S$ (die on tet). The ones that die on the tet plate are the ones you want (recovered from the master amp plate).

21. Troubleshooting: If the student finds colonies growing on both Ampicillin and Tetracycline plates, what does this indicate? a) The ligation was successful. b) The ligation failed, and the plasmid re-circularized without the gene (Non-recombinant). c) The bacteria mutated. d) Gene X confers tetracycline resistance. Answer: b) The ligation failed, and the plasmid re-circularized... Explanation: If they grow on tetracycline, the $tet^R$ gene is still intact. This means Gene X did not insert; the vector just closed back up. These are non-recombinants.

Case Study 3: The Crime Scene

Investigators find a tiny drop of blood at a crime scene. The amount of DNA is too small for standard fingerprinting.

22. Strategy: Which technique must be used first to make the sample usable? a) Gel Electrophoresis. b) PCR (Polymerase Chain Reaction). c) Southern Blotting. d) Centrifugation. Answer: b) PCR (Polymerase Chain Reaction). Explanation: PCR is used to amplify (make millions of copies of) a specific DNA segment from a minute sample.

23. Process: During this technique, the temperature is raised to 94°C. What happens to the DNA at this step? a) It replicates. b) It denatures (strands separate). c) Primers anneal. d) It gets cut by restriction enzymes. Answer: b) It denatures (strands separate). Explanation: High temperature breaks the hydrogen bonds between the two DNA strands, separating them into single strands.

24. Enzyme: Why is the enzyme from Thermus aquaticus used in this specific process? a) It is faster than human enzymes. b) It is thermostable and survives the 94°C heating step. c) It proofs-read errors. d) It works at 0°C. Answer: b) It is thermostable and survives the 94°C heating step. Explanation: Taq polymerase is heat-stable. Ordinary polymerases would be destroyed by the denaturation heat, requiring fresh enzyme every cycle. Taq survives the whole process.

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Section C: Creating, Designing & Critical Thinking

25. Designing a Vector: You need to design a new cloning vector for yeast.

• Task: List 3 essential components your vector must possess to function.
• Explanation: Briefly explain the function of each. Answer:

1. Origin of Replication (ori): A sequence where replication starts, allowing the plasmid to replicate independently within the yeast cell.
2. Selectable Marker: A gene (e.g., antibiotic resistance) to identify and select the cells that have successfully taken up the vector (transformants).
3. Cloning Sites (Recognition Sites): Specific sequences for restriction enzymes where the foreign gene can be inserted.

26. Troubleshooting a Bioreactor: Problem: The cell growth in your bioreactor has suddenly stopped, and the pH has dropped significantly.

• Diagnosis: What could be the cause?
• Action Plan: Propose two steps to correct this or prevent it in the next batch. Answer:
• Diagnosis: The pH drop usually indicates the accumulation of acidic metabolic waste (like lactic acid or CO2) or bacterial contamination (which often produce acids). Oxygen limitation can also cause anaerobic fermentation producing acids.
• Action Plan:
  1. Check Aeration: Increase the sparging/stirring rate to ensure sufficient oxygen supply (prevent anaerobic fermentation).
  2. pH Control: Ensure the automatic pH control system (buffer addition) is working.
  3. Sterility Check: Test for contamination. If contaminated, the batch must be discarded and the vessel sterilized.

27. Analogy Creation: Create a real-world analogy for Recombinant DNA Technology.

• Components to map: DNA, Restriction Enzyme, Ligase, Vector, Host. Answer:
• Analogy: Publishing a new recipe in a cookbook.
• DNA: The text of a new recipe (e.g., "Insulin Cake").
• Restriction Enzyme: Scissors used to cut the recipe out of a magazine and to cut a blank space in the cookbook.
• Ligase: Glue/Tape used to paste the new recipe into the cookbook.
• Vector: The Cookbook itself (a carrier).
• Host: The Chef/Kitchen that takes the cookbook and actually bakes the cake (Expression).

28. Ethical Evaluation: "Biopiracy" involves using bio-resources of a country without authorization.

• Scenario: A company patents a turmeric-based healing cream. Indians have used turmeric for centuries.
• Task: Argue why this patent might be unjust based on "Traditional Knowledge". Answer:
• Argument: Patents are granted for novel inventions. Since the healing properties of turmeric are well-documented in ancient Indian texts (Ayurveda) and used traditionally, this knowledge is "Prior Art" (public knowledge). Therefore, it is not a "new" invention, and patenting it steals from the collective heritage of the Indian people without giving them credit or benefit (Biopiracy).

29. Visualizing the Process: Draw a schematic of Agarose Gel Electrophoresis.

• Label: Wells, Direction of migration, Anode (+), Cathode (-), Small fragments, Large fragments.
• Challenge: Indicate where a circular plasmid would run compared to a linear DNA of the same size. Answer: (Visual Description)
• Setup: Wells are at the top (Negative/Cathode).
• Direction: DNA moves downwards towards the Bottom (Positive/Anode).
• Separation: Large fragments stay near the top (slow). Small fragments are near the bottom (fast).
• Challenge: A Supercoiled Circular Plasmid is compact and moves faster (further down) than a linear DNA of the same weight, which experiences more drag. (Note: Nicked/relaxed circles run slower).

30. Formulating a Hypothesis: Observation: Restriction enzymes in bacteria cut foreign viral DNA but do not cut the bacteria's own DNA, even though the recognition sequences might be present.

• Hypothesis: How does the bacterium protect its own DNA? Answer:
• Hypothesis (Modification): The bacterium possesses a modification enzyme (Methylase) that adds a methyl group ($CH_3$) to the nucleotides (Adenine or Cytosine) within the recognition sequence of its own DNA.
• Mechanism: This methylation changes the shape of the site just enough that the restriction enzyme cannot bind or cut it, effectively "masking" the site from its own scissors.

31. Critical Comparison: Compare PCR inside a test tube vs. DNA Replication inside a living cell.

• Task: List 2 similarities and 2 major differences. Answer:
• Similarities:
  1. Both synthesize DNA in the 5' to 3' direction.
  2. Both require a Template strand and building blocks (dNTPs).
• Differences:
  1. Unwinding: PCR uses Heat (Denaturation) to separate strands; Cells use the enzyme Helicase.
  2. Primers: PCR uses DNA primers (synthesized chemically); Cells use RNA primers (made by Primase).