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DSE Biology Diagnostic: Microorganisms and Biotechnology

Unit Test 1: Bacterial and Viral Structure​

Question

(a) Describe the structure of a typical bacterial cell, naming and stating the function of five structural features. [5 marks]

(b) Explain why viruses are described as obligate intracellular parasites and why they are not considered living organisms. [3 marks]

(c) A student claims that viruses can be killed by antibiotics. Evaluate this claim. [2 marks]

Worked Solution

(a) Five structural features of a typical bacterial cell:

  1. Cell wall: Made of peptidoglycan (murein). Provides mechanical strength and prevents the cell from bursting by osmotic lysis. It is not made of cellulose (unlike plant cell walls).

  2. Cell surface membrane: A phospholipid bilayer (partially permeable) that controls the movement of substances in and out of the cell. It also contains respiratory enzymes for aerobic respiration.

  3. Circular DNA (nucleoid): A single, circular DNA molecule found free in the cytoplasm (not enclosed in a nucleus). It carries the genetic information needed for the cell's basic functions.

  4. Plasmid: Small, additional circular rings of DNA separate from the main nucleoid. Plasmids often carry genes for antibiotic resistance and can be transferred between bacteria (conjugation). Plasmids are widely used as vectors in genetic engineering.

  5. Flagellum (flagella): A long, whip-like projection used for locomotion. It rotates to propel the bacterium through liquid environments.

(Alternative: slime capsule for protection against phagocytosis; pili/fimbriae for attachment; mesosomes for respiration; 70S ribosomes for protein synthesis.)

(b) Viruses are obligate intracellular parasites because they cannot carry out any metabolic processes on their own. They have no ribosomes, no enzymes for energy production, and no ability to synthesise proteins. They can only reproduce inside a living host cell, hijacking the host cell's metabolic machinery (ribosomes, ATP, nucleotides) to replicate their genetic material and synthesise viral proteins.

Viruses are not considered living because they:

  • Do not carry out respiration or any metabolic reactions.
  • Cannot reproduce independently (they require a host cell).
  • Do not grow or develop.
  • Show no response to stimuli.
  • They are essentially just nucleic acid (DNA or RNA) in a protein coat (capsid) -- an infectious particle rather than a living cell.

(c) The claim is incorrect. Antibiotics target structures and processes specific to bacteria (e.g. cell wall synthesis, 70S ribosomes, bacterial DNA replication). Viruses do not have cell walls, ribosomes, or their own metabolic enzymes. Therefore, antibiotics have no effect on viruses. Viral infections are treated with antiviral drugs (which target viral-specific processes such as reverse transcriptase or viral protease) or managed through the immune system (vaccines for prevention).

Unit Test 2: Fermentation and Bioreactor Design​

Question

Yeast (Saccharomyces cerevisiae) is used in the industrial production of ethanol by fermentation.

(a) Write the word equation for the fermentation of glucose by yeast. [1 mark]

(b) Describe the conditions that must be maintained in a bioreactor (fermenter) for optimal ethanol production by yeast, and explain the importance of each condition. [5 marks]

(c) Explain why the fermentation process must be carried out under aseptic (sterile) conditions and describe two methods used to achieve this. [3 marks]

Worked Solution

(a) Glucose→Ethanol+Carbon dioxide\text{Glucose} \to \text{Ethanol} + \text{Carbon dioxide}

(b) Conditions for optimal ethanol production in a bioreactor:

  1. Optimal temperature (approximately 30∘C30^{\circ}C): Yeast enzymes work best at this temperature. Higher temperatures denature enzymes; lower temperatures slow the rate of reaction.

  2. Optimal pH (slightly acidic, approximately pH 5--6): The pH must be maintained within the optimum range for yeast enzyme activity. Buffers may be added to the culture medium.

  3. Anaerobic conditions: Fermentation is an anaerobic process. Oxygen must be excluded from the bioreactor because if oxygen is present, yeast will carry out aerobic respiration instead, producing CO2CO_{2} and water rather than ethanol. The bioreactor may be sealed or flushed with nitrogen gas.

  4. Nutrient supply: A constant supply of glucose (the substrate) must be maintained. The nutrient medium must also contain minerals, nitrogen sources (e.g. ammonium salts), and vitamins for yeast growth.

  5. Efficient stirring/mixing: The bioreactor must have a stirrer to ensure the culture is well mixed. This distributes nutrients evenly, maintains uniform temperature and pH throughout, and keeps yeast in suspension for maximum contact with the substrate.

(Additional: product removal -- ethanol must be removed as it accumulates because high ethanol concentrations become toxic to yeast.)

(c) Aseptic conditions are essential because:

If contaminating microorganisms (bacteria, fungi) enter the bioreactor, they can compete with the yeast for nutrients, produce unwanted by-products, or contaminate the product. This reduces the yield and quality of the ethanol and may require the entire batch to be discarded.

Two methods for maintaining aseptic conditions:

  1. Sterilisation of the bioreactor and medium: The bioreactor and the nutrient medium are sterilised using steam at high temperature and pressure (autoclaving at 121∘C121^{\circ}C for 15--20 minutes) before use, killing all microorganisms.

  2. Sterile air filtration: Any air entering the bioreactor (e.g. for cooling or mixing) is passed through HEPA filters or other sterile filters that remove microorganisms, preventing contamination from the air supply.

(Alternative: all entry/exit ports have sterile valves; the culture medium is sterilised before inoculation.)

Unit Test 3: Genetic Engineering in Biotechnology​

Question

Genetically modified (GM) crops have been developed to possess various advantageous traits.

(a) Describe the process of creating a GM crop that is resistant to an insect pest (e.g. Bt cotton, which produces a toxin from Bacillus thuringiensis that kills insect larvae). [5 marks]

(b) Explain two potential environmental concerns associated with growing GM crops. [4 marks]

(c) A GM bacterium has been engineered to produce human growth hormone (HGH). Explain why this is preferable to extracting HGH from human pituitary glands. [3 marks]

Worked Solution

(a) Process for creating Bt cotton:

  1. Isolation of the Bt toxin gene: The gene from Bacillus thuringiensis that codes for the Bt toxin (Cry protein) is identified and cut out using a restriction enzyme, producing sticky ends.

  2. Preparation of the vector: A plasmid (e.g. from Agrobacterium tumefaciens -- Ti plasmid) is cut open with the same restriction enzyme, producing complementary sticky ends.

  3. Ligation: The Bt toxin gene is inserted into the plasmid and joined using DNA ligase, forming a recombinant plasmid.

  4. Transformation: The recombinant plasmid is introduced into Agrobacterium tumefaciens bacteria, which naturally infects plant cells and transfers the T-DNA (containing the Bt gene) into the plant cell's genome.

  5. Plant tissue culture: The transformed plant cells are grown in tissue culture using plant growth regulators (auxins and cytokinins) to regenerate whole cotton plants that now contain and express the Bt toxin gene.

  6. Selection: Plants that have successfully incorporated the Bt gene are selected using genetic markers (e.g. antibiotic resistance genes on the plasmid).

  7. Testing: The GM cotton plants are tested to confirm that they produce the Bt toxin in their tissues and that the toxin is effective against the target insect pest.

(b) Two environmental concerns:

  1. Gene flow to wild relatives: The GM crop could cross-pollinate with wild, non-GM plant relatives nearby, transferring the resistance gene to wild populations. This could create "superweeds" that are resistant to insect pests but also have a competitive advantage over native plants, potentially disrupting natural ecosystems and reducing biodiversity.

  2. Impact on non-target organisms: Although the Bt toxin is specific to certain insect pests, there is concern that it may also harm non-target, beneficial insects (e.g. pollinators like bees, butterflies) that come into contact with the GM crop. This could have cascading effects on food webs and ecosystem stability.

(Alternative: development of pest resistance to the Bt toxin over time through natural selection; reduction in crop diversity if only GM varieties are grown.)

(c) Advantages of producing HGH using GM bacteria:

  1. Quantity: GM bacteria can be grown in large-scale fermenters to produce unlimited quantities of HGH, whereas extraction from human pituitary glands is limited by the availability of donor glands.

  2. Safety: Extracting HGH from human pituitary glands carries the risk of transmitting human diseases (e.g. Creutzfeldt-Jakob disease, CJD, which occurred historically from contaminated HGH extracts). HGH from GM bacteria is free from human pathogens.

  3. Purity and consistency: Recombinant HGH is chemically identical to natural human HGH and can be produced with high purity and batch-to-batch consistency. Extraction from pituitary glands may result in variable purity and contamination with other proteins.

Integration Test 1: Microorganisms in Food Production and Safety​

Question

Yeast is used in both bread-making and brewing.

(a) Explain the role of yeast in bread-making. Why is the dough left to rise in a warm place before baking? [4 marks]

(b) In brewing, fermentation is followed by pasteurisation. Explain what pasteurisation is and why it is necessary. [3 marks]

(c) A food poisoning outbreak is traced to contamination of chicken by Salmonella bacteria. Describe three ways in which contamination could have occurred during food handling and preparation, and explain how each could have been prevented. [6 marks]

Worked Solution

(a) Role of yeast in bread-making:

Yeast carries out anaerobic respiration (fermentation) of sugars present in the dough. The products are:

  • Carbon dioxide: Trapped in the dough by the elastic gluten network (formed from glutenin and gliadin proteins in wheat flour), causing the dough to rise (increase in volume). The CO2CO_{2} creates air pockets that give bread its light, spongy texture after baking.
  • Ethanol: Produced alongside CO2CO_{2} but evaporates during baking.

The dough is left to rise in a warm place (approximately 2525--35∘C35^{\circ}C) because this is close to the optimum temperature for yeast enzyme activity. At this temperature, yeast respires rapidly, producing CO2CO_{2} quickly and causing the dough to rise efficiently. If too cold, the reaction is too slow; if too hot, yeast enzymes denature.

(b) Pasteurisation is a process of heating a food or liquid (e.g. beer, milk) to a specific temperature (typically 6060--72∘C72^{\circ}C) for a specific time, then cooling it rapidly.

Why it is necessary: Pasteurisation kills most harmful microorganisms (pathogenic bacteria) and many spoilage organisms in the product, extending its shelf life and ensuring it is safe for consumption. In brewing, pasteurisation prevents residual yeast and bacteria from continuing to ferment sugars in the bottled product, which would alter the taste and could cause bottles to burst due to excess CO2CO_{2} pressure.

(c) Three ways contamination could occur and prevention:

  1. Cross-contamination from raw to cooked food: Raw chicken (which commonly carries Salmonella on its surface) could contaminate ready-to-eat foods, cutting boards, or utensils. Prevention: Use separate cutting boards and utensils for raw meat and ready-to-eat foods. Never place cooked food on a surface that has held raw chicken without washing it thoroughly first.

  2. Insufficient cooking: Chicken that is not cooked to a high enough internal temperature (minimum 75∘C75^{\circ}C throughout) may contain live Salmonella bacteria. Prevention: Cook chicken thoroughly until the juices run clear and the meat is no longer pink. Use a food thermometer to confirm the internal temperature has reached 75∘C75^{\circ}C for at least 30 seconds.

  3. Poor personal hygiene: The food handler could transfer bacteria from their hands, clothing, or hair to the food. Prevention: Wash hands thoroughly with soap and warm water before and after handling raw chicken. Wear clean protective clothing and hair nets. Avoid handling food when ill.

(Alternative: improper storage temperature -- chicken stored above 5∘C5^{\circ}C allows bacterial multiplication; use-by date expired.)

Integration Test 2: Genetic Engineering -- Insulin Production​

Question

Human insulin is produced commercially using genetically modified Escherichia coli bacteria.

(a) The human insulin gene is too large to be inserted directly as a single gene. Explain how scientists overcame this problem using two shorter DNA sequences (A chain and B chain genes) to produce functional insulin. [4 marks]

(b) After the recombinant bacteria have been cultured in a fermenter, the insulin must be extracted and purified. Describe the steps involved in extracting and purifying the insulin from the bacterial culture. [4 marks]

(c) Discuss two ethical considerations associated with the use of genetic engineering to produce medicines. [4 marks]

Worked Solution

(a) Insulin consists of two polypeptide chains: the A chain and the B chain, linked by disulfide bonds. Scientists overcame the size problem by:

  1. Synthesising two separate genes: One gene coding for the A chain and one gene coding for the B chain of insulin were chemically synthesised (artificially created) based on the known amino acid sequence of human insulin and the genetic code.

  2. Inserting each gene into separate plasmids: Each gene was inserted into a separate plasmid vector and each recombinant plasmid was used to transform separate cultures of E. coli bacteria.

  3. Separate expression: Each bacterial culture produced one of the insulin chains: one culture produced the A chain, and the other produced the B chain.

  4. Combining the chains: The A and B chains were extracted from the respective bacterial cultures. The two chains were then chemically joined together (linked by disulfide bonds in a controlled chemical reaction) to form the complete, functional insulin molecule.

(b) Extraction and purification of insulin:

  1. Harvesting: The bacterial culture is removed from the fermenter. Centrifugation is used to separate the bacterial cells (pellet) from the culture medium (supernatant).

  2. Cell disruption: The bacterial cells are broken open (lysed) to release their contents, including the insulin chains. Methods include enzymatic lysis (using lysozyme to break down the cell wall), high-pressure homogenisation, or sonication.

  3. Separation: The cell lysate is centrifuged or filtered to remove cell debris. The insulin chains must then be separated from the mixture of bacterial proteins, DNA, and other cellular components. This is done using chromatography (e.g. reverse-phase HPLC -- high-performance liquid chromatography), which separates molecules based on their chemical properties.

  4. Combining chains: The purified A and B chains are mixed under controlled conditions that promote the formation of disulfide bonds between the chains, producing proinsulin or directly forming active insulin.

  5. Final purification: The insulin is further purified by additional chromatography steps to ensure high purity. It is then crystallised, formulated, and packaged for medical use.

(c) Two ethical considerations:

  1. Safety and long-term effects: There are concerns about the long-term safety of medicines produced by genetic engineering. Although recombinant insulin has been extensively tested and is considered safe, there is always a risk of unforeseen side effects or contamination. Rigorous clinical trials and ongoing monitoring are required.

  2. Animal welfare: Historically, insulin was extracted from the pancreas of pigs and cattle. Genetic engineering has reduced the reliance on animal sources, which is an ethical advantage. However, the development and testing of GM products may still involve animal testing, raising ethical concerns about the use of animals in research.

(Alternative: accessibility and cost -- GM medicines may be expensive and not accessible to all populations; playing God -- some people have religious or moral objections to modifying the genetic code of organisms; patenting of life -- companies patenting GM organisms raises concerns about corporate control of living resources.)