Biotechnology: Principles and Processes Class 12 Notes

• Genetic engineering involves gene isolation, manipulation and transfer to create organisms with new traits (e.g. Bt cotton, insulin-producing E. coli).
• Process engineering ensures that recombinant organisms are grown under controlled conditions in bioreactors for maximum yield.
• Cloning: producing identical copies of a gene or an organism. Molecular cloning copies a gene; reproductive cloning produces identical organisms (e.g. Dolly the sheep).

• EcoRI: recognises the palindromic sequence 5'-GAATTC-3' and cuts between G and AATTC on each strand, producing sticky (cohesive) ends with single-stranded overhangs.
• Sticky ends: short single-stranded overhangs at the cut ends that can base-pair with complementary sticky ends from another DNA fragment cut by the same enzyme, facilitating ligation.
• Blunt ends: the enzyme cuts straight across both strands, producing no overhangs (e.g. SmaI cuts at CCCGGG). Blunt-end ligation is less efficient.

• Ti plasmid: from Agrobacterium tumefaciens, used to transfer genes into plant cells. The T-DNA region of the Ti plasmid integrates into the plant genome.
• Competent cells: host cells that can take up foreign DNA from the environment. E. coli cells are made competent by CaCl2 treatment (calcium ions neutralise the negative charges on DNA and cell membrane, allowing DNA entry).
• Other transformation methods: electroporation (brief electric pulse creates transient pores in the cell membrane), gene gun (biolistics — DNA-coated gold/tungsten particles are shot into plant cells), microinjection (direct injection of DNA into animal cells using a fine needle).

The overall workflow of recombinant DNA technology follows a logical sequence of steps:

• Isolation of DNA: cells are lysed with detergents and enzymes (lysozyme for bacteria, cellulase for plants) to release DNA. Proteins are removed with proteases and RNA with RNases. The purified DNA is precipitated with chilled ethanol.
• PCR (Polymerase Chain Reaction): amplifies a specific DNA segment in vitro through repeated cycles of three steps — (1) Denaturation at 94 deg C: the double-stranded DNA separates into single strands; (2) Annealing at 55-65 deg C: primers bind to complementary sequences flanking the target; (3) Extension at 72 deg C: Taq polymerase (thermostable DNA polymerase from Thermus aquaticus) synthesises new strands. After 30 cycles, the target sequence is amplified approximately 2^30 (about 1 billion) times.
• Gel electrophoresis: DNA fragments are separated by size on an agarose gel using an electric field. DNA is negatively charged (phosphate backbone) and moves toward the positive electrode (anode). Smaller fragments move faster. The gel is stained with ethidium bromide and visualised under UV light. Specific bands are cut out for further use.
• Ligation: the isolated gene of interest and the vector are cut with the same restriction enzyme to produce compatible sticky (or blunt) ends. DNA ligase joins them, creating a recombinant DNA molecule (recombinant vector).
• Transformation: the recombinant vector is introduced into a competent host cell (usually E. coli) by CaCl2 treatment or electroporation. The host cell replicates the recombinant DNA along with its own genome.

• Example: if the gene is inserted into the tetR gene of pBR322, recombinants are AmpR but TetS. They grow on ampicillin plates but not on tetracycline plates.
• Blue-white screening: uses the lacZ gene on the vector. The vector carries the lacZ gene encoding beta-galactosidase, and the host has a mutant lacZ. In the presence of X-gal, cells with non-recombinant vector produce blue colonies (functional lacZ). Recombinants (with foreign DNA inserted into lacZ) produce white colonies. This is a rapid visual screen.
• Antibiotic resistance method: cells are plated on media containing the antibiotic encoded by the intact selectable marker. Only transformed cells (with the vector) grow; non-transformed cells die.

• Simple stirred-tank bioreactor: has a motor-driven impeller for agitation, an air sparger for oxygen supply, pH and temperature control systems and ports for sampling.
• Airlift bioreactor: mixing and aeration are achieved by pumping air; gentler than stirred tanks, suitable for shear-sensitive cells.
• Batch vs continuous culture: in batch culture, all nutrients are added at the start and products are harvested at the end. In continuous culture, fresh medium is continuously added and product is withdrawn, maintaining cells in a log phase for maximum productivity.
• Downstream processing: after the product is formed in the bioreactor, it must be separated, purified and packaged. Steps include cell separation (centrifugation, filtration), extraction, chromatographic purification (ion exchange, affinity chromatography), formulation and quality control.

Example: A researcher wants to clone a gene into pBR322. The gene has EcoRI sites at both ends. After cutting both the gene and pBR322 with EcoRI, the fragments are ligated with DNA ligase. The recombinant plasmid is introduced into E. coli by CaCl2 treatment. How would you select for cells containing the recombinant plasmid?

Solution: EcoRI sites on pBR322 are located within the tetR gene. Insertion of the foreign gene disrupts tetR (insertional inactivation) but leaves ampR intact. Plate the bacteria on ampicillin-containing medium — only cells that took up the plasmid (recombinant or non-recombinant) will grow. Replica-plate onto tetracycline medium. Cells that grow on ampicillin but NOT on tetracycline are the recombinants (AmpR, TetS). Verify by further screening.

Example: Explain why the polymerase used in PCR must be thermostable, and name the organism from which it is isolated.

Solution: PCR involves repeated cycles of denaturation at 94 deg C, which would denature (unfold) a normal DNA polymerase. The polymerase must withstand this high temperature without losing activity across 30 or more cycles. Taq polymerase, isolated from the thermophilic bacterium Thermus aquaticus (found in hot springs), is thermostable and functions optimally at 72 deg C, making it ideal for PCR.

Exercises (NCERT)

1. 1
   What are the two core techniques of modern biotechnology?
2. 2
   Name the enzyme used to cut DNA at specific sites.
3. 3
   What are restriction endonucleases?
4. 4
   What is a palindromic sequence?
5. 5
   Name the enzyme used to join DNA fragments.
6. 6
   What is a cloning vector? Give an example.
7. 7
   Name the features that make a good vector.
8. 8
   What is the role of ori in a vector?
9. 9
   What is a selectable marker? Give an example.
10. 10
    What is a bioreactor? Name the most common type.
11. 11
    What is downstream processing?
12. 12
    How is a competent host cell prepared?
13. 13
    What is transformation?
14. 14
    How are recombinant cells selected?
15. 15
    What is insertional inactivation?
16. 16
    Why is a stirrer used in a bioreactor?
17. 17
    What are the steps of recombinant DNA technology?

Answers at a glance

1. 1
   The two core techniques are genetic engineering and maintaining a sterile environment for microbial growth.
2. 2
   Restriction enzymes (restriction endonucleases) cut DNA at specific recognition sites.
3. 3
   Restriction endonucleases are enzymes that recognise specific palindromic sequences and cut DNA.
4. 4
   A palindromic sequence is a base sequence read the same on both strands in the 5'→3' direction.
5. 5
   DNA ligase joins DNA fragments.
6. 6
   A cloning vector is a carrier that can replicate in a host; e.g. pBR322.
7. 7
   A good vector has an ori, a selectable marker, cloning sites and a small size.
8. 8
   ori is the origin of replication and controls the copy number.
9. 9
   A selectable marker aids identification of recombinants; e.g. ampicillin resistance.
10. 10
    A bioreactor is a vessel for large-scale culture; the most common is the stirred-tank bioreactor.
11. 11
    Downstream processing is the separation and purification of the product after biosynthesis.
12. 12
    Competent host cells are prepared by treatment with divalent cations (e.g. CaCl2) or by electroporation.
13. 13
    Transformation is the uptake of foreign DNA by a host cell.
14. 14
    Recombinant cells are selected using selectable markers (antibiotic resistance and insertional inactivation).
15. 15
    Insertional inactivation is the loss of a marker gene's function when foreign DNA is inserted into it.
16. 16
    A stirrer ensures even mixing and aeration of the culture in a bioreactor.
17. 17
    DNA isolation, cutting, ligation into a vector, transformation, selection and downstream processing.

MCQ Questions

2 Mark Questions

1. 1
   Name the two core techniques of modern biotechnology.
2. 2
   What is a palindromic sequence?
3. 3
   Name the features that make a good vector.
4. 4
   What is insertional inactivation?
5. 5
   What is downstream processing?

3 Mark Questions

1. 1
   Describe the tools of recombinant DNA technology.
2. 2
   How is a competent host cell prepared?
3. 3
   Describe the selection of recombinant cells.
4. 4
   Describe the steps of recombinant DNA technology.
5. 5
   Describe the components of a bioreactor.

5 Mark Questions

1. 1
   Describe the process of recombinant DNA technology.
2. 2
   Describe the tools of recombinant DNA technology and their functions.
3. 3
   Explain transformation, selection and downstream processing.