Biotechnology : Principles and Processes

The following are 10 recombinant proteins used in medical practice:

1. Recombinant Human Insulin (Humulin) – Used in the treatment of diabetes mellitus (Type 1 and Type 2).
2. Recombinant Human Growth Hormone (Somatotropin) – Used to treat growth hormone deficiency and dwarfism.
3. Recombinant Erythropoietin (EPO) – Used to treat anaemia, especially in patients with chronic kidney disease or undergoing chemotherapy.
4. Recombinant Tissue Plasminogen Activator (tPA / Alteplase) – Used as a thrombolytic agent in the treatment of heart attacks and strokes.
5. Recombinant Factor VIII – Used in the treatment of Haemophilia A (clotting factor deficiency).
6. Recombinant Factor IX – Used in the treatment of Haemophilia B.
7. Recombinant Hepatitis B Vaccine (HBsAg) – Used for immunisation against Hepatitis B virus infection.
8. Recombinant Interferon-alpha (IFN-α) – Used in the treatment of viral infections (e.g., Hepatitis C) and certain cancers.
9. Recombinant Interleukin-2 (IL-2 / Aldesleukin) – Used in the treatment of metastatic renal cell carcinoma and melanoma.
10. Recombinant DNase (Dornase alfa) – Used in the treatment of cystic fibrosis to reduce mucus viscosity in the lungs.

Note: All these proteins are produced by inserting the human gene encoding the protein into a suitable host organism (bacteria, yeast, or mammalian cells) using recombinant DNA technology, allowing large-scale production.

Example: Restriction Enzyme EcoRI

Given:
- Restriction Enzyme: *EcoRI* (isolated from *Escherichia coli*)
- Recognition/Substrate sequence (palindromic): 5′–GAATTC–3′ / 3′–CTTAAG–5′
- Type of cut: Staggered (produces sticky ends)

Diagrammatic Representation:

Substrate DNA (before cutting):
$$5'\text{---G} \downarrow \text{AATTC---}3'$$
$$3'\text{---CTTAA} \uparrow \text{G---}5'$$

The arrows (↓ and ↑) indicate the cut sites on each strand.

Products (after cutting):

Fragment 1:
$$5'\text{---G} \quad\quad\quad 3'$$
$$3'\text{---CTTAA} \quad 5'$$

Fragment 2:
$$5'\quad \text{AATTC---}3'$$
$$3'\quad\quad\quad \text{G---}5'$$

Summary Table:

| Feature | Detail |
|---|---|
| Enzyme | EcoRI |
| Source organism | *Escherichia coli* |
| Recognition sequence | 5′-GAATTC-3′ (palindrome) |
| Cut position | Between G and A on both strands |
| Type of ends produced | Sticky ends (cohesive ends) with 5′ overhang: 5′-AATT-3′ |

Conclusion: EcoRI recognises the palindromic sequence 5′-GAATTC-3′ and cuts between G and A on both strands in a staggered manner, producing fragments with 4-nucleotide 5′ overhangs called sticky ends.

Answer: DNA is bigger in molecular size than enzymes (proteins).

Reasoning:

1. Enzymes are proteins made up of amino acid chains. Even large enzymes typically consist of a few hundred to a few thousand amino acids, giving them a molecular weight in the range of a few thousand to a few hundred thousand Daltons (kDa).

2. DNA is a polynucleotide chain. The human genome, for example, consists of approximately $3 \times 10^9$ base pairs distributed across 23 chromosomes. Even a single chromosome contains DNA with a molecular weight in the range of $10^{11}$ Daltons or more.

3. Evidence from the chapter: Restriction endonucleases (enzymes) recognise and cut specific short sequences (4–8 base pairs) on the DNA molecule. The fact that a relatively small enzyme molecule can act on a specific site of a very long DNA molecule indicates that DNA is far larger than the enzyme.

4. Gel electrophoresis also confirms this: DNA fragments (even small ones of a few hundred base pairs) migrate much more slowly than protein molecules of comparable mass, and the intact genomic DNA barely enters the gel — indicating its enormous size.

Conclusion: DNA is much bigger in molecular size compared to enzymes. This is known because restriction enzymes act on specific short recognition sequences within the much larger DNA molecule, and from our knowledge of the structure and composition of both macromolecules.

Given/Known facts:
- Each human diploid cell contains approximately $6 \times 10^{-12}$ g (6 picograms) of DNA.
- The average molecular weight of a base pair of DNA $\approx 660$ g/mol.
- The human haploid genome has $\approx 3 \times 10^9$ base pairs, so the diploid genome has $\approx 6 \times 10^9$ base pairs.

Step 1: Calculate the molecular weight of human diploid DNA:
$$M_w = 6 \times 10^9 \text{ bp} \times 660 \text{ g/mol per bp}$$
$$M_w = 3.96 \times 10^{12} \text{ g/mol} \approx 4 \times 10^{12} \text{ g/mol}$$

Step 2: Calculate the number of moles of DNA in one cell:
$$\text{Mass of DNA per cell} = 6 \times 10^{-12} \text{ g}$$
$$\text{Moles} = \frac{\text{mass}}{M_w} = \frac{6 \times 10^{-12}}{4 \times 10^{12}} = 1.5 \times 10^{-24} \text{ mol}$$

Step 3: Calculate molar concentration (assuming cell volume $\approx 1$ pL $= 10^{-12}$ L):
$$C = \frac{1.5 \times 10^{-24} \text{ mol}}{10^{-12} \text{ L}} = 1.5 \times 10^{-12} \text{ mol/L}$$

Conclusion: The molar concentration of human DNA in a human cell is approximately $\mathbf{1.5 \times 10^{-12}}$ mol/L (1.5 picomolar). This is an extremely small molar concentration, reflecting the enormous molecular weight of the DNA molecule, even though the mass of DNA per cell is significant at the cellular level.

Answer: No, eukaryotic cells do not have restriction endonucleases.

Justification:

1. Origin of restriction endonucleases: Restriction endonucleases are naturally found only in prokaryotes (bacteria). They form part of the bacterial restriction-modification system, which acts as a primitive immune defence mechanism against foreign DNA (e.g., bacteriophage DNA).

2. Function in prokaryotes: In bacteria, restriction enzymes cut foreign (viral) DNA at specific palindromic sequences, thereby destroying it. The bacterium's own DNA is protected from self-digestion by methylation of the same recognition sequences (modification system).

3. Eukaryotes do not need this system: Eukaryotic cells have evolved different and more complex immune and cellular defence mechanisms (e.g., RNA interference, immune system). They do not rely on restriction-modification systems to protect against foreign DNA.

4. No evidence of restriction enzymes in eukaryotes: There is no known naturally occurring restriction endonuclease in eukaryotic cells. The restriction enzymes used in recombinant DNA technology are all isolated from bacterial sources (e.g., *EcoRI* from *E. coli*, *HindIII* from *Haemophilus influenzae*, *BamHI* from *Bacillus amyloliquefaciens*).

Conclusion: Eukaryotic cells do not possess restriction endonucleases because these enzymes are a prokaryotic adaptation for defence against foreign DNA, and eukaryotes have evolved entirely different mechanisms for cellular defence and DNA maintenance.

Given: Stirred tank bioreactors vs. shake flasks in large-scale production of biotechnological products.

Advantages of Stirred Tank Bioreactors over Shake Flasks (besides aeration and mixing):

1. Large volume capacity: Bioreactors can hold volumes ranging from a few litres to thousands of litres (up to $10^5$ litres), enabling large-scale industrial production, whereas shake flasks are limited to small volumes (typically up to a few litres).

2. Monitoring and control of physicochemical parameters: Bioreactors are equipped with sensors and control systems to continuously monitor and regulate:
- Temperature
- pH
- Dissolved oxygen levels
- Foam formation
- Substrate concentration
This ensures optimal conditions for microbial/cell growth and product formation.

3. Foam control: Bioreactors have foam breakers/antifoam systems to prevent excessive foaming, which can inhibit growth and product yield.

4. Aseptic conditions: Bioreactors are designed to maintain strict sterility over long periods, reducing the risk of contamination during large-scale fermentation.

5. Continuous or fed-batch operation: Bioreactors can be operated in batch, fed-batch, or continuous mode, allowing flexibility in production strategies and higher productivity.

6. Sampling ports: Allow periodic withdrawal of samples for analysis without disturbing the culture or compromising sterility.

7. Scalability: The design of stirred tank bioreactors allows easy scale-up from laboratory to industrial scale.

Conclusion: Stirred tank bioreactors offer superior control, scalability, sterility, and operational flexibility compared to shake flasks, making them essential for industrial-scale biotechnological production.

Definition: A palindromic sequence in DNA is a sequence of base pairs that reads the same on both strands in the 5′→3′ direction.

5 Examples of Palindromic DNA Sequences (Recognition sites of restriction enzymes):

| Restriction Enzyme | Palindromic Recognition Sequence |
|---|---|
| EcoRI | 5′–G↓AATTC–3′ / 3′–CTTAA↑G–5′ |
| BamHI | 5′–G↓GATCC–3′ / 3′–CCTAG↑G–5′ |
| HindIII | 5′–A↓AGCTT–3′ / 3′–TTCGA↑A–5′ |
| SalI | 5′–G↓TCGAC–3′ / 3′–CAGCT↑G–5′ |
| PstI | 5′–CTGCA↓G–3′ / 3′–G↑ACGTC–5′ |

Creating a palindromic sequence (following base-pair rules):

Let us construct a 6-base palindrome:
- Choose the first three bases: 5′–GAA–3′
- The complementary strand (3′→5′) reads: 3′–CTT–5′, i.e., 5′–TTC–3′
- For palindrome: the second half of the top strand must be the complement of the first half read in reverse.
- Complement of GAA (read 3′→5′) = CTT, so the top strand becomes: 5′–GAATTC–3′
- Bottom strand: 3′–CTTAAG–5′, i.e., 5′–GAATTC–3′ ✓

$$5'-\text{G-A-A-T-T-C}-3'$$
$$3'-\text{C-T-T-A-A-G}-5'$$

Both strands read GAATTC in the 5′→3′ direction — confirming it is a palindrome. (This is the recognition sequence of EcoRI.)

Concept: In the context of meiosis, recombinant DNA (genetic recombination) is produced during crossing over.

Stage: Recombinant DNA is made during Prophase I of Meiosis I, specifically at the Pachytene stage.

Explanation:

1. During Prophase I, homologous chromosomes pair up in a process called synapsis, forming bivalents (tetrads).

2. At the Pachytene stage, the paired homologous chromosomes (held together by the synaptonemal complex) undergo crossing over — a process in which non-sister chromatids of homologous chromosomes exchange segments of DNA.

3. The physical exchange of DNA segments between non-sister chromatids results in recombinant chromosomes containing new combinations of alleles — this is the natural formation of recombinant DNA.

4. The points of exchange are visible as chiasmata (singular: chiasma) during the Diplotene stage of Prophase I.

Summary:
$$\text{Meiosis I} \rightarrow \text{Prophase I} \rightarrow \text{Pachytene stage} \rightarrow \text{Crossing over} \rightarrow \text{Recombinant DNA formed}$$

Conclusion: In meiosis, recombinant DNA is naturally produced during the Pachytene stage of Prophase I through the process of crossing over between non-sister chromatids of homologous chromosomes.

Concept: Reporter enzymes (reporter genes) are used to visually or biochemically identify whether a host cell has been successfully transformed with foreign DNA.

How a Reporter Enzyme Monitors Transformation:

1. Reporter gene cloning: A reporter gene encoding an easily detectable enzyme (e.g., β-galactosidase encoded by the *lacZ* gene, or Green Fluorescent Protein – GFP, or luciferase) is cloned into the vector along with the gene of interest.

2. **Insertion inactivation principle (example with *lacZ*):**
- The *lacZ* gene is present in the vector (e.g., pUC19 plasmid).
- The foreign DNA (gene of interest) is inserted within the *lacZ* gene at the multiple cloning site (MCS).
- This insertion disrupts the *lacZ* gene → the transformed cells cannot produce functional β-galactosidase.

3. Detection using chromogenic substrate:
- When the growth medium contains the substrate X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside):
- Non-transformed cells or cells with intact *lacZ* (no insert): β-galactosidase cleaves X-gal → blue colonies.
- Transformed cells with recombinant DNA (insert disrupts *lacZ*): no functional β-galactosidase → white colonies.

4. Blue-White Screening: This technique allows easy visual identification of recombinant (transformed) colonies — white colonies contain the foreign DNA insert.

5. In addition to selectable marker: The selectable marker (e.g., antibiotic resistance gene) confirms that the cell has taken up the vector. The reporter enzyme (*lacZ*/β-galactosidase) further confirms that the foreign DNA has been inserted into the vector (recombinant), not just the empty vector.

Conclusion: A reporter enzyme like β-galactosidase, when its gene is disrupted by insertion of foreign DNA, allows visual screening of transformed cells through blue-white colony selection, providing an additional and confirmatory method beyond the selectable marker to identify successful transformation with recombinant DNA.

(a) Origin of Replication (ori):

- The origin of replication (ori) is a specific sequence of nucleotides in a DNA molecule from which DNA replication is initiated.
- It is a necessary component of any vector (plasmid or viral) used in recombinant DNA technology.
- When a foreign DNA fragment is linked to the ori-containing vector, it can replicate within the host cell using the host's replication machinery.
- The ori sequence also controls the copy number of the plasmid — i.e., how many copies of the recombinant DNA are produced per cell.
- Example: The plasmid pBR322 contains an ori sequence that allows it to replicate autonomously in *E. coli*.

(b) Bioreactors:

- A bioreactor is a vessel or container in which raw materials are biologically converted into specific products using microorganisms, plant, animal, or human cells, or their enzymes, under optimally controlled conditions.
- Bioreactors provide the optimal conditions required for the desired product by controlling: temperature, pH, substrate concentration, dissolved oxygen, vitamins, and salts.
- The most commonly used type is the stirred tank bioreactor, which is cylindrical with a curved base to facilitate mixing. It has:
- A stirrer/agitator for mixing
- An oxygen delivery system
- A foam control system
- A temperature control system
- A pH control system
- Sampling ports
- Bioreactors can have volumes ranging from a few litres to $10^5$ litres, enabling large-scale production of biotechnological products such as enzymes, antibiotics, vaccines, and recombinant proteins.

(c) Downstream Processing:

- After the biosynthetic stage (fermentation/production) in a bioreactor, the product must be subjected to a series of processes before it is ready for marketing as a finished product. These processes are collectively called downstream processing.
- It includes:
1. Separation of the product from the culture medium or cells (e.g., centrifugation, filtration).
2. Purification of the product to remove contaminants (e.g., chromatography, precipitation).
3. Formulation with suitable preservatives and stabilisers.
4. Quality control testing — each batch must be tested for purity, potency, and safety.
5. Clinical trials (in the case of drugs/therapeutics).
- Downstream processing and quality control vary from product to product depending on its nature and intended use.

(a) PCR (Polymerase Chain Reaction):

- PCR is an in vitro technique used to amplify a specific segment of DNA (gene of interest) to produce millions of copies in a short time without using a living cell.
- Principle: It is based on the property of DNA polymerase to synthesise a new strand of DNA complementary to the template strand.
- Components required: Template DNA, two specific primers (short oligonucleotide sequences complementary to the flanking regions of the target DNA), thermostable DNA polymerase (e.g., *Taq* polymerase from *Thermus aquaticus*), dNTPs (deoxyribonucleoside triphosphates), and buffer.
- Steps (repeated in cycles, typically 30–40 cycles):
1. Denaturation (~94°C): The double-stranded DNA is heated to separate the two strands.
2. Annealing (~50–65°C): Primers bind (anneal) to their complementary sequences on each template strand.
3. Extension (~72°C): *Taq* polymerase extends the primers, synthesising new complementary DNA strands.
- Each cycle doubles the amount of DNA, so after $n$ cycles, $2^n$ copies are produced.
- Applications: Diagnosis of genetic diseases, forensic science, detection of pathogens, gene cloning.

(b) Restriction Enzymes and DNA:

- Restriction enzymes (restriction endonucleases) are molecular scissors that cut DNA at specific recognition sequences.
- They were first isolated from bacteria, where they serve as a defence mechanism against foreign (viral) DNA.
- They recognise short, specific palindromic sequences (4–8 bp) in double-stranded DNA and cleave both strands.
- Types of cuts:
- Sticky ends (cohesive ends): Staggered cuts producing single-stranded overhangs (e.g., EcoRI produces 5′-AATT overhangs).
- Blunt ends: Straight cuts at the same position on both strands (e.g., SmaI).
- Sticky ends are particularly useful in recombinant DNA technology because they can hydrogen-bond with complementary sticky ends of another DNA fragment, facilitating ligation by DNA ligase.
- Naming convention: First letter from genus, next two from species, strain designation, and order of discovery (e.g., *Eco*RI from *Escherichia coli* strain R, first enzyme).

(c) Chitinase:

- Chitinase is an enzyme that breaks down chitin, a polysaccharide that forms the cell wall of fungi.
- Role in biotechnology: To isolate DNA from fungal cells, the fungal cell wall (made of chitin) must first be broken down. Chitinase is used to digest the chitin in the fungal cell wall, allowing the release of the cellular contents including DNA.
- This is analogous to the use of lysozyme to break the peptidoglycan cell wall of bacteria, or cellulase to break the cellulose cell wall of plant cells, for DNA isolation.
- Source: Chitinase is produced by various bacteria, fungi, and plants.
- Other applications: Chitinase is also used in biocontrol of fungal pathogens in agriculture and in the production of chitooligosaccharides for pharmaceutical use.

(a) Plasmid DNA vs. Chromosomal DNA:

| Feature | Plasmid DNA | Chromosomal DNA |
|---|---|---|
| Location | Cytoplasm (extrachromosomal) | Nucleoid region (prokaryotes) / nucleus (eukaryotes) |
| Size | Small (1–200 kb) | Very large (millions to billions of bp) |
| Shape | Circular, double-stranded | Linear (eukaryotes); circular (prokaryotes) |
| Association with proteins | Not associated with histones | Associated with histone proteins (eukaryotes) |
| Replication | Replicates autonomously, independent of chromosomal replication | Replicates along with the main chromosome |
| Genes encoded | Accessory genes (e.g., antibiotic resistance, toxin genes) | Essential genes for cell survival and function |
| Copy number | Multiple copies per cell possible | Usually one copy per cell (haploid) |
| Separation by gel electrophoresis | Migrates faster (smaller size); shows supercoiled, relaxed, and linear forms | Barely enters the gel due to enormous size |

(b) RNA vs. DNA:

| Feature | DNA | RNA |
|---|---|---|
| Full name | Deoxyribonucleic acid | Ribonucleic acid |
| Sugar | Deoxyribose (lacks –OH at 2′ carbon) | Ribose (has –OH at 2′ carbon) |
| Bases | Adenine, Guanine, Cytosine, Thymine | Adenine, Guanine, Cytosine, Uracil |
| Strands | Usually double-stranded | Usually single-stranded |
| Stability | More stable (no 2′–OH) | Less stable (2′–OH makes it susceptible to hydrolysis) |
| Location | Mainly in nucleus; also mitochondria/chloroplasts | Nucleus and cytoplasm |
| Function | Stores and transmits genetic information | Involved in protein synthesis (mRNA, tRNA, rRNA) |
| Chemical test | Diphenylamine test (blue colour) | Orcinol test (green colour) |

(c) Exonuclease vs. Endonuclease:

| Feature | Exonuclease | Endonuclease |
|---|---|---|
| Definition | Enzyme that cleaves nucleotides one at a time from the ends (5′ or 3′) of a DNA/RNA strand | Enzyme that cleaves the phosphodiester bonds within (internally) a DNA/RNA strand |
| Site of action | Terminal ends of the polynucleotide chain | Internal phosphodiester bonds |
| Products | Individual nucleotides (mononucleotides) | Oligonucleotide fragments of varying lengths |
| Specificity | Less sequence-specific | Restriction endonucleases are highly sequence-specific (palindromic sequences) |
| Example | Exonuclease III (degrades from 3′ end) | EcoRI, BamHI, HindIII (restriction endonucleases) |
| Use in biotechnology | Used to create single-stranded DNA; remove unwanted ends | Used to cut DNA at specific sites for cloning and recombinant DNA technology |