Biotechnology : Principles and ProcessesClass 12 Biology NCERT Solutions

1. Human Insulin (Humulin): Used for the treatment of diabetes mellitus to control blood sugar levels.
2. Human Growth Hormone (Somatotropin): Used to treat pituitary dwarfism in children and growth hormone deficiency in adults.
3. Erythropoietin: Used to stimulate the formation of red blood cells in patients suffering from anemia, especially during kidney failure or chemotherapy.
4. Tissue Plasminogen Activator (tPA): Used as a 'clot buster' to dissolve blood clots in patients who have had a heart attack or stroke.
5. Interferon-$\alpha$: Used for the treatment of certain viral infections like hepatitis C and some types of cancer like hairy-cell leukemia.
6. Blood Clotting Factor VIII: Used for the treatment of Hemophilia A, a genetic disorder that impairs blood clotting.
7. Blood Clotting Factor IX: Used for the treatment of Hemophilia B.
8. Hepatitis B Vaccine: A recombinant protein (HBsAg) produced in yeast, used for immunization to prevent Hepatitis B infection.
9. Interleukin-2: Used in cancer immunotherapy to stimulate the activity of immune cells (T-cells) to fight cancer.
10. DNase I (Pulmozyme): Used in the treatment of cystic fibrosis to break down DNA in the thick mucus in the lungs, making it easier to clear.

              $3'—\text{CTTAA}$                  $3'—\text{G}$ |

1. Composition and Structure: DNA is a macromolecule composed of millions or even billions of nucleotide pairs that make up a chromosome. Enzymes are proteins, which are polymers of amino acids, typically consisting of a few hundred to a few thousand amino acids.
2. Scale of Interaction: The chapter explains that a restriction enzyme 'inspects' the length of a DNA sequence to find its specific recognition site. This implies that the enzyme is a smaller entity that moves along a much larger DNA molecule. The enzyme acts on a very small portion (e.g., a 6 base-pair sequence) of the entire DNA molecule.
3. Molecular Weight: The molecular weight of a single human chromosome can be in the range of billions of daltons, whereas the molecular weight of a typical enzyme like EcoRI is in the range of thousands of daltons. Therefore, a DNA molecule is vastly larger and more massive than an enzyme molecule.

1. Amount of DNA: A diploid human cell contains approximately $6.4 \times 10^9$ base pairs (bp).
2. Molar Mass of a Base Pair: The average molecular weight of a base pair is approximately 650 g/mol.
3. Total Mass of DNA: The total mass of DNA in a single cell can be calculated: Mass = (Number of bp) $\times$ (Avg. mass per bp) / (Avogadro's number) Mass $\approx$ ($6.4 \times 10^9$ bp) $\times$ (650 g/mol/bp) / ($6.022 \times 10^{23}$ molecules/mol) $\approx 6.9 \times 10^{-12}$ g.
4. Volume of the Nucleus: The DNA is contained within the nucleus. A typical human nucleus has a diameter of about 6 µm. Assuming it is a sphere: Radius (r) = 3 µm = $3 \times 10^{-4}$ cm Volume (V) = $\frac{4}{3} \pi r^3 = \frac{4}{3} \pi (3 \times 10^{-4} \text{ cm})^3 \approx 1.13 \times 10^{-10} \text{ cm}^3 = 1.13 \times 10^{-13}$ L.
5. Molar Concentration: Molarity is moles per liter. In this context, we can consider the entire genome (all 46 chromosomes) as a set. The number of moles of this 'set' in one cell is 1 / Avogadro's number. Moles = $1 / (6.022 \times 10^{23}) \approx 1.66 \times 10^{-24}$ mol. Molar Concentration = Moles / Volume = ($1.66 \times 10^{-24}$ mol) / ($1.13 \times 10^{-13}$ L) $\approx 1.47 \times 10^{-11}$ M.

1. Origin and Function: As stated in the chapter, restriction endonucleases were isolated from prokaryotic organisms like bacteria (e.g., Escherichia coli). In bacteria, these enzymes are part of a defense mechanism called the restriction-modification system, which protects the bacterium from invading viruses (bacteriophages) by cutting the foreign viral DNA.
2. Self-DNA Protection: Bacteria protect their own DNA from being cut by their restriction enzymes by modifying it, usually through methylation at the recognition sites.
3. Eukaryotic Defense: Eukaryotic organisms have more complex and advanced defense mechanisms against viruses, such as the immune system in animals and RNA interference (RNAi) in many eukaryotes. They do not rely on a restriction-modification system for defense.
4. Absence of Evidence: Restriction endonucleases are characteristically found in prokaryotes, and there is no evidence of their natural presence or function within eukaryotic cells.

1. Process Control: Bioreactors are equipped with automated control systems to monitor and maintain optimal growth conditions. This includes sensors and regulators for temperature, pH, and dissolved oxygen, ensuring consistency and maximizing yield.
2. Large-Scale Production: Bioreactors are designed for large volumes (100-1000 litres or more), allowing for the production of commercially significant quantities of a product. Shake flasks are limited to small, laboratory-scale volumes.
3. Sterility and Aseptic Operation: Bioreactors are designed as closed systems that can be sterilized and maintained in a sterile (aseptic) condition for long periods, preventing contamination which is a major risk in large-scale cultures.
4. Continuous Culture System: They can be operated as a continuous culture system, where fresh medium is continuously added and used medium is drained out. This keeps the cells in the physiologically active log/exponential phase, leading to higher productivity over a longer time.
5. Sampling Ports: Bioreactors have sampling ports that allow for the sterile withdrawal of small volumes of the culture for periodic testing and quality control without contaminating the entire batch.
6. Foam Control: They often include a foam control system or foam breaker, which is essential as protein-rich culture media can produce significant foam during agitation and aeration, which can interfere with the process.

1. EcoRI: $5'—\text{GAATTC}—3'$ $3'—\text{CTTAAG}—5'$
2. BamHI: $5'—\text{GGATCC}—3'$ $3'—\text{CCTAGG}—5'$
3. HindIII: $5'—\text{AAGCTT}—3'$ $3'—\text{TTCGAA}—5'$
4. HaeIII: $5'—\text{GGCC}—3'$ $3'—\text{CCGG}—5'$
5. SalI: $5'—\text{GTCGAC}—3'$ $3'—\text{CAGCTG}—5'$

1. Vector Design: The cloning vector is engineered to contain a selectable marker (e.g., an ampicillin resistance gene, $amp^R$) and a reporter gene (e.g., lacZ, which codes for the enzyme $\beta$-galactosidase). The multiple cloning site (where foreign DNA is to be inserted) is located within the lacZ gene.
2. Ligation: When a foreign DNA fragment is successfully ligated into the cloning site, it disrupts the coding sequence of the lacZ gene, rendering it non-functional. This is called insertional inactivation.
3. Transformation and Selection: The mixture of plasmids (recombinant and non-recombinant) is introduced into host cells. The cells are then plated on a medium containing the antibiotic (e.g., ampicillin) and a chromogenic substrate for the reporter enzyme (e.g., X-gal for $\beta$-galactosidase).
4. Screening:
   • Transformants vs. Non-transformants: Only cells that have taken up a plasmid (either recombinant or non-recombinant) will survive because they have the ampicillin resistance gene. Non-transformed cells will die.
   • Recombinants vs. Non-recombinants:
     • Non-recombinant colonies (Blue): Cells that took up a plasmid without the foreign DNA insert have a functional lacZ gene. They produce $\beta$-galactosidase, which cleaves the X-gal substrate, resulting in blue-colored colonies.
     • Recombinant colonies (White): Cells that took up a plasmid with the foreign DNA insert have an inactivated lacZ gene. They do not produce functional $\beta$-galactosidase, so X-gal is not cleaved, and the colonies remain white.