CBSE Important Questions Class 12 Biology Chapter 9 – Biotechnology: Principles and Processes

Class 12 Biology Chapter 9 Biotechnology: Principles and Processes is the ninth chapter of the NCERT Class 12 Biology textbook. It explains the two core techniques of modern biotechnology, genetic engineering and bioprocess engineering, and the tools of recombinant DNA technology: restriction enzymes, DNA ligase, cloning vectors such as the plasmid pBR322, competent host cells, and the steps of recombinant DNA technology including DNA isolation, PCR, transformation, selection, bioreactors and downstream processing.

These CBSE important questions for Class 12 Biology Chapter 9 cover the pBR322 transformation experiment and insertional inactivation, the difference between genetic engineering and bioprocess engineering, the nature of a plasmid, Taq polymerase from Thermus aquaticus, DNA ligase, restriction enzymes and chitinase, isolating DNA from plant cells, the labelled steps of recombinant DNA technology and the types of bioreactors. The set has multiple-choice, short-answer and long-answer questions with answers for board exam revision.

Q1. A researcher is studying the process of transformation in bacteria using the following experimental data:

Experiment Material Used Observation
1 Competent E. coli cells + plasmid pBR322 Colonies grew on ampicillin plates
2 Competent E. coli cells without plasmid No growth on ampicillin plates
3 Competent E. coli cells + plasmid pBR322 (ampR, tetR) Colonies grew on ampicillin but not on tetracycline plates

Analyse the results of all three experiments and provide a detailed scientific explanation for the observation in Experiment 3. Why did the transformed cells fail to grow on the tetracycline medium?

Answer: 1. Experiment 1: The growth of colonies on ampicillin plates shows that the competent E. coli cells successfully underwent transformation. They took up the pBR322 plasmid and expressed the ampicillin resistance gene (ampR), which acts as a selectable marker.
2. Experiment 2: This acts as a negative control. The absence of growth confirms that the host E. coli cells are not naturally resistant to ampicillin and need the plasmid to survive in its presence.
3. Experiment 3: The cells grew on ampicillin, confirming they are transformants (the ampR gene is intact). However, their failure to grow on tetracycline shows that the tetracycline resistance gene (tetR) has been inactivated. This is due to insertional inactivation: a piece of foreign DNA has been inserted into a restriction site located within the tetR gene, which breaks its coding sequence so that no functional tetracycline-resistance protein is made. Such cells are recombinants: they have lost resistance to one antibiotic while keeping resistance to the other.

Q2. The large-scale production of recombinant therapeutic proteins, such as human insulin, relies on the integration of two critical fields: genetic engineering and bioprocess engineering. Differentiate between these two fields by outlining the key steps involved in each process, starting from the isolation of the desired gene to the final purified product.

Answer: Genetic engineering and bioprocess engineering are two distinct but complementary fields in biotechnology.
Genetic engineering: This field works at the molecular level to create a recombinant organism capable of producing the desired product. The key steps are:
1. Isolation of the gene of interest (e.g. the human insulin gene) from the source DNA.
2. Construction of recombinant DNA (rDNA) by inserting the isolated gene into a suitable vector, such as a plasmid.
3. Introduction of the rDNA into a suitable host organism, like Escherichia coli, which can now produce the protein.
Bioprocess engineering: This field focuses on the large-scale production and purification of the product made by the genetically engineered organism. The key steps are:
1. Upstream processing: The recombinant host cells are grown in large quantities in bioreactors under sterile and optimised conditions (pH, temperature, nutrients, oxygen).
2. Downstream processing: After fermentation, the desired protein is separated from the host cells and culture medium and purified (e.g. by chromatography) to obtain a clinically safe and effective product.
In short, genetic engineering creates the producer organism, while bioprocess engineering handles the mass production and purification of the final product.

Q3. A plasmid is
(a) self replicating circular DNA.
(b) non-replicating circular DNA.
(c) self replicating double stranded RNA.
(d) non-replicating double stranded DNA.

Answer: (a) self replicating circular DNA.
A plasmid is an extra-chromosomal genetic element found in bacteria. It is a circular, double-stranded DNA molecule that carries its own origin of replication, so it can replicate independently of the chromosomal DNA.

Q4. How and why is the bacterium Thermus aquaticus employed in recombinant DNA technology? Explain.

Answer: In the polymerase chain reaction (PCR), a segment of DNA is amplified. During the denaturation step of PCR, the temperature is very high (about 94–95°C) to separate the two DNA strands. The DNA polymerases used earlier could not withstand this high temperature and had to be added fresh after every cycle. Hence, a thermostable DNA polymerase (Taq polymerase) isolated from the bacterium Thermus aquaticus is used. It remains active during the high-temperature denaturation of double-stranded DNA, so the primers can be extended at 72°C in each cycle and the DNA can be amplified repeatedly (25–35 cycles) without adding new enzyme.

Q5. Write a short note on DNA ligase.

Answer: DNA ligase is an enzyme that joins double-stranded DNA fragments. In vivo, DNA ligase is essential for DNA repair and DNA replication (it joins the Okazaki fragments). In vitro, it is used in recombinant DNA technology to seal the nicks between the vector and the foreign DNA, forming the recombinant DNA molecule. DNA ligase forms covalent phosphodiester bonds between the 3’ hydroxyl end of one nucleotide and the 5’ phosphate end of another, using energy from ATP.

Q6. The given image depicts the sealing of the bacterial plasmid and the gene of interest together with the help of an enzyme. Name the enzyme.

Cut plasmid plus gene of interest joined to form a recombinant plasmid

(a) Ligase
(b) Amylase
(c) Endonuclease
(d) Exonuclease

Answer: (a) Ligase
The plasmid vector and the gene of interest are both cut by the same restriction endonuclease, which produces complementary sticky ends. The sticky ends base-pair with each other, and DNA ligase then forms phosphodiester bonds at the nicks, sealing the sugar-phosphate backbone to create a stable recombinant DNA molecule.

Q7. Which of the following enzymes allows the linking of an antibiotic resistance gene to the plasmid vector?
(a) Endonuclease
(b) DNA polymerase
(c) DNA primase
(d) DNA ligase

Answer: (d) DNA ligase
A restriction endonuclease cuts the plasmid and produces sticky ends. The antibiotic resistance gene with matching sticky ends pairs with the plasmid ends, and DNA ligase joins the 3’-OH and 5’-phosphate ends by forming phosphodiester bonds. So DNA ligase links the gene to the plasmid vector.

Q8. Explain briefly:
PCR
Restriction enzymes and DNA
Chitinase

Answer: PCR: PCR (Polymerase Chain Reaction) is a method developed by Kary Mullis in the 1980s to make many copies of a specific DNA segment. It is based on the ability of DNA polymerase to synthesise a new strand of DNA complementary to the template strand. Since DNA polymerase can add a nucleotide only to a pre-existing 3’-OH group, it needs a primer (a small stretch of DNA) to which it can add the first nucleotide. This lets the scientist choose the specific region of the template to be amplified. A PCR reaction needs a DNA template, DNA polymerase, primers and dNTPs. It has three steps, repeated for about 30–40 cycles: (i) denaturation at about 94°C to separate the DNA strands, (ii) annealing, in which the primers bind to the denatured DNA by base pairing, and (iii) extension at 72°C, in which dNTPs are added to the growing chain using the template. Taq polymerase from Thermus aquaticus is used because it is thermostable and tolerates the high temperature of the denaturation step. At the end, billions of copies of the sequence are obtained.
Restriction enzymes and DNA: Restriction enzymes are enzymes present in bacteria that stop the growth of bacteriophages inside the host bacteria, acting as the bacteria's self-defence. They recognise specific sequences in the DNA, called restriction (recognition) sequences, and cut the DNA within or around them. They inspect the DNA, and once the restriction sequence is found, they bind to the DNA and cut each of the two strands at a specific point in the sugar-phosphate backbone. The enzymes are named from the source organism: the first letter from the genus and the next two from the species of the prokaryote, e.g. EcoRI comes from Escherichia coli RY13 (R from the strain, and the Roman numeral shows the order in which the enzyme was isolated from that strain). Restriction enzymes are widely used in genetic engineering to make recombinant DNA molecules from DNA of different sources.
Chitinase: Chitinases are enzymes that break the glycosidic bonds in chitin. Chitin forms the cell walls of fungi and the exoskeleton of some animals such as arthropods. These organisms use chitinase to reshape their own cell walls or exoskeletons. In genetic engineering, chitinase is widely used to digest fungal cell walls during DNA isolation.

Q9. A researcher is working with plant cells to isolate DNA. Explain the steps involved in breaking open the cell and removing the associated biomolecules, and justify why each step is necessary for obtaining pure DNA.

Answer: 1. Cell disruption: Plant cells have a rigid cell wall, so the enzyme cellulase (or mechanical grinding) is used to break the wall, followed by detergents to dissolve the plasma membrane. This releases the cellular contents, including DNA. (For bacteria, lysozyme is used, and for fungi, chitinase.)
2. Removal of proteins and RNA: Proteins are removed with proteases, and RNA is removed with ribonuclease (RNase). This is essential because proteins and RNA would interfere with later steps such as restriction digestion and ligation.
3. Precipitation of DNA: The DNA is precipitated by adding chilled ethanol after adding salts. The salts neutralise the negative charge on the DNA and ethanol lowers its solubility, so the DNA separates out as fine threads that can be spooled out.

Q10. Which of the following statements correctly differentiates the roles of genetic engineering and bioprocess engineering in the production of recombinant therapeutic proteins like insulin?
(a) Genetic engineering includes isolation of the gene of interest, construction of recombinant DNA, and its introduction into a suitable host; bioprocess engineering includes optimising fermentation conditions, scaling up culture, and downstream processing.
(b) Genetic engineering includes isolation of the gene and scaling up of recombinant protein production; bioprocess engineering includes designing vectors and selecting host organisms.
(c) Genetic engineering includes isolation of the gene and downstream processing; bioprocess engineering includes only transformation and expression of the gene in host cells.
(d) Genetic engineering and bioprocess engineering both involve the same steps, as there is no distinction between laboratory-level manipulation and industrial-scale production.

Answer: (a) Genetic engineering includes isolation of the gene of interest, construction of recombinant DNA, and its introduction into a suitable host; bioprocess engineering includes optimising fermentation conditions, scaling up culture, and downstream processing.
Genetic engineering works at the molecular level to make the recombinant organism (isolating the insulin gene, inserting it into a plasmid, and transforming E. coli). Bioprocess engineering then produces the protein on an industrial scale: optimising pH, temperature, aeration and nutrients, scaling up from laboratory fermenters to large bioreactors, and purifying the insulin by downstream processing.

Q11. In the given diagram showing the steps of recombinant DNA technology, identify the correct combination of enzymes and processes labelled A, B, C and D:

Steps of recombinant DNA technology with enzymes and processes labelled A, B, C and D

(a) A. Restriction endonuclease; B. DNA ligase; C. Polymerase; D. Transformation
(b) A. Restriction endonuclease; B. Restriction endonuclease; C. DNA ligase; D. Transformation
(c) A. Exonuclease; B. Restriction endonuclease; C. DNA ligase; D. Transduction
(d) A. Restriction endonuclease; B. DNA ligase; C. Ligase; D. Translation

Answer: (b) A. Restriction endonuclease; B. Restriction endonuclease; C. DNA ligase; D. Transformation
At A, a restriction endonuclease cuts the foreign DNA at its recognition site. At B, the same restriction endonuclease cuts the vector plasmid to produce compatible ends. At C, DNA ligase joins the foreign DNA fragment into the opened plasmid to form the recombinant DNA molecule. At D, transformation introduces the recombinant DNA into competent E. coli cells, which then divide and clone the inserted gene.

Q12. Which statement correctly explains the difference between stirred-tank and sparged-tank bioreactors?
(a) Stirred-tank uses impellers for mixing and oxygen distribution, while sparged-tank introduces sterile air bubbles through a sparger for aeration.
(b) Both systems use identical aeration methods and are equally suitable for all cultures.
(c) Sparged-tank is better for shear-sensitive cells because bubbles are gentle.
(d) Stirred-tank provides oxygen only through sparging, not mechanical mixing.

Answer: (a) Stirred-tank uses impellers for mixing and oxygen distribution, while sparged-tank introduces sterile air bubbles through a sparger for aeration.
In a stirred-tank bioreactor, mechanical impellers mix the culture medium evenly and distribute oxygen throughout it. In a sparged-tank bioreactor, sterile air is bubbled through a sparger; the bubbles rise through the medium and dissolve oxygen without mechanical mixing.

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