Q1. Can you list 10 recombinant proteins which are used in medical practice? Find out where they are used as therapeutics (use the internet).
Answer: The following recombinant proteins are widely used in medical practice as therapeutic agents:

1. Human Insulin (Humulin): Used to control blood sugar levels in patients with diabetes mellitus.
2. Human Growth Hormone (HGH): Used to treat growth hormone deficiency and dwarfism.
3. Blood Clotting Factor VIII: Administered to patients with haemophilia A to aid blood clotting.
4. DNAse I (Dornase alfa): Helps break down thick mucus in patients suffering from cystic fibrosis.
5. Erythropoietin (EPO): Used to treat anaemia, especially in patients with chronic kidney disease.
6. Interferon-alpha (α-IFN): Used in the treatment of hepatitis C and certain cancers.
7. Tissue Plasminogen Activator (tPA): Acts as a clot-buster during heart attacks and strokes.
8. Hepatitis B Vaccine: Prevents Hepatitis B virus infection.
9. Interleukin-2 (IL-2): Used in cancer immunotherapy to boost the immune system.
10. Granulocyte Colony-Stimulating Factor (G-CSF): Stimulates white blood cell production in cancer patients undergoing chemotherapy.

Q2. Make a chart (with diagrammatic representation) showing a restriction enzyme, the substrate DNA on which it acts, the site at which it cuts DNA and the product it produces.
Answer: Action of Restriction Enzyme (Example: EcoRI)

Q3. From what you have learnt, can you tell whether enzymes are bigger or DNA is bigger in molecular size? How did you know?
Answer: DNA molecules are much larger in molecular size than enzymes. This is because DNA is a very long polymer made up of millions to billions of nucleotide pairs. For example, human DNA contains about 3.3 × 10⁹ base pairs, which equals nearly 2.2 meters in length when stretched out. In contrast, enzymes are proteins made up of a few hundred to a few thousand amino acids, making them much smaller. Even large enzymes like restriction endonucleases are tiny compared to an entire DNA molecule or even a small DNA fragment used in genetic experiments.

Q4. What would be the molar concentration of human DNA in a human cell? Consult your teacher.
Answer:
Human DNA Content: The diploid DNA content in a human somatic cell is 6.6 × 10⁹ bp.
Mass of DNA: The approximate mass of one DNA base pair is 660 Daltons.
Total mass of diploid human DNA ≈ (6.6 × 10⁹ bp) × (660 Daltons/bp) ≈ 4.36 × 10¹² Daltons.
Molar Mass of DNA: Daltons / Avogadro’s number ≈ 7.24 × 10⁻¹² grams/mole.
Molar Concentration: Since there are two copies of the entire genome in a diploid somatic cell, the molar concentration would be roughly 7.24 × 10⁻¹² moles/cell.

Q5. Do eukaryotic cells have restriction endonucleases? Justify your answer.
Answer: No, eukaryotic cells generally do not have restriction endonucleases.
It is beacuse restriction endonucleases are typically found in bacteria (prokaryotes). They function to restrict the growth of bacteriophages by cutting the foreign viral DNA, acting as a bacterial defense mechanism. Eukaryotic cells possess other types of nucleases that repair DNA damage, but they do not have restriction enzymes that recognize specific palindromic sequences and cut them to produce sticky ends.

Q6. Besides better aeration and mixing properties, what other advantages do stirred tank bioreactors have over shake flasks?
Answer: Stirred-tank bioreactors offer several advantages over shake flasks:

1. Large-scale production: They can handle large volumes of culture (up to thousands of liters), unlike shake flasks that are limited to small-scale experiments.
2. Controlled environment: Parameters like temperature, pH, oxygen, and nutrient supply can be precisely monitored and adjusted for maximum yield.
3. Maintained sterility: Steam sterilisation and sterile air supply prevent contamination.
4. Continuous operation: They support continuous culture, keeping microorganisms in their most productive phase.
5. Easy monitoring: Sampling ports allow periodic withdrawal of samples for testing and quality control.

Q7. Collect 5 examples of palindromic DNA sequences by consulting your teacher. Better try to create a palindromic sequence by following base-pair rules.
Answer: Examples of Palindromic Sequences (5′ → 3′):

1. EcoRI site:
    5′-GAATTC-3′
    3′-CTTAAG-5′
2. 5′-GGTACC-3′
    3′-CCATGG-5′
3. 5′-TTCGAA-3′
    3′-AAGCTT-5′
4. 5′-AAGCTT-3′
    3′-TTCGAA-5′
5. 5′-GCGCGC-3′
    3′-CGCGCG-5′

Q8. Can you recall meiosis and indicate at what stage a recombinant DNA is made?
Answer: In the natural biological process of meiosis, recombinant DNA is made during Prophase I. This is when homologous chromosomes pair and exchange DNA segments through crossing over, creating new genetic combinations.

Q9. Can you think and answer how a reporter enzyme can be used to monitor transformation of host cells by foreign DNA in addition to a selectable marker?
Answer: A reporter enzyme is used to visually identify cells that have successfully taken up foreign DNA, alongside a selectable marker.
When the foreign DNA is inserted into the gene encoding the reporter enzyme (such as β-galactosidase), the enzyme becomes inactive and this process is called insertional inactivation.
The transformed cells are then grown on a medium containing a chromogenic substrate (like X-gal), which helps distinguish between recombinants and non-recombinants based on colour:

• Non-recombinant cells: The enzyme remains active and breaks down the substrate, forming blue colonies.
• Recombinant cells: The enzyme is inactivated due to the DNA insertion, so no colour forms and they appear white.

Hence, recombinant colonies can be easily identified by their white colour, making the reporter enzyme a visual indicator of successful transformation.

Q10. Describe briefly the following:
(a) Origin of replication
(b) Bioreactors
(c) Downstream processing
Answer: 

(a) Origin of replication (ori): The origin of replication (ori) is a specific DNA sequence in a chromosome or vector from where replication starts. For any alien piece of DNA to replicate and multiply in an organism, it must be part of a chromosome or plasmid containing this sequence. The ori also controls the copy number of the linked DNA.

(b) Bioreactors: Bioreactors are large vessels (100–1000 liters) in which raw materials are biologically converted into specific products (like proteins or enzymes) using microbial, plant, animal, or human cells. They provide optimal conditions (temperature, pH, substrate, oxygen, etc.) for product formation. The most common type is the stirred-tank reactor.

(c) Downstream processing: Downstream processing refers to all processes applied after the biosynthetic stage to make a product market-ready. These include separation and purification, formulation with preservatives, and quality control testing. The precise steps vary with the product.

Q11. Explain briefly:
(a) PCR
(b) Restriction enzymes and DNA
(c) Chitinase
Answer:

(a) PCR: PCR (Polymerase Chain Reaction) is a process to synthesise multiple copies of a DNA or gene in vitro. It uses two sets of primers and a thermostable DNA polymerase (Taq polymerase). The process involves repeated cycles of denaturation, primer annealing, and primer extension, amplifying the DNA segment to billions of copies.

(b) Restriction enzymes and DNA: Restriction enzymes (molecular scissors) are nucleases that inspect the length of DNA. Endonucleases cut DNA at specific positions by recognizing particular palindromic nucleotide sequences. They are essential tools in genetic engineering to cut DNA from different sources at the same site, the first step in creating recombinant DNA.

(c) Chitinase: Chitinase is an enzyme used to break open the cell wall of a fungus to release genetic material (DNA) along with other macromolecules. It is used during the initial step of isolating genetic material in recombinant DNA technology.

Q12. Discuss with your teacher and find out how to distinguish between:
(a) Plasmid DNA and Chromosomal DNA
(b) RNA and DNA
(c) Exonuclease and Endonuclease
Answer:

(a) Plasmid DNA vs Chromosomal DNA

1. Plasmid DNA is small, circular, and extra-chromosomal, whereas chromosomal DNA is the main, large, linear (or circular in prokaryotes) genetic material.
2. Plasmid DNA replicates independently, while chromosomal DNA replication is controlled and linked with cell division.
3. Plasmids are usually present in many copies per cell, whereas chromosomal DNA occurs in a fixed copy number.
4. Plasmid DNA often carries accessory genes (like antibiotic resistance), on the other hand, chromosomal DNA carries essential genes for survival.

(b) DNA vs RNA

1. DNA contains deoxyribose sugar, whereas RNA contains ribose sugar.
2. DNA has the base thymine, while RNA has uracil instead of thymine.
3. DNA is usually double-stranded, whereas RNA is mostly single-stranded.
4. DNA serves as the permanent genetic material, on the other hand, RNA functions in protein synthesis (mRNA, tRNA, rRNA).

(c) Exonuclease vs Endonuclease

1. Exonucleases remove nucleotides from the ends of DNA, whereas endonucleases cut DNA within the strand.
2. Exonucleases are less sequence-specific, while endonucleases act at specific palindromic sequences.
3. Exonucleases are used for DNA trimming and sequencing, on the other hand, endonucleases are essential for cutting vector and foreign DNA in recombinant DNA technology.