Chapter 9: Biotechnology - Principles and Processes

Introduction

Biotechnology deals with techniques of using live organisms or enzymes from organisms to produce products and processes useful to humans. The European Federation of Biotechnology (EFB) defines biotechnology as the integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services.

Principles of Biotechnology

Two core techniques that enabled the birth of modern biotechnology are:

1. Genetic Engineering: Techniques to alter the chemistry of genetic material (DNA and RNA), to introduce these into host organisms and thus change the phenotype of the host organism.
2. Bioprocess Engineering: Maintenance of sterile (microbial contamination-free) ambience in chemical engineering processes to enable growth of only the desired microbe/eukaryotic cell in large quantities for the manufacture of biotechnological products like antibiotics, vaccines, enzymes, etc.

The conceptual development of genetic engineering focuses on the creation of recombinant DNA (rDNA). The first recombinant DNA was constructed by Stanley Cohen and Herbert Boyer in 1972 by linking a gene encoding antibiotic resistance with a native plasmid of Salmonella typhimurium.

Tools of Recombinant DNA Technology

Three key biological tools are required:

1. Restriction Enzymes

These enzymes act as “molecular scissors” to cut DNA at specific locations. They belong to a larger class of enzymes called nucleases, which are of two kinds:

• Exonucleases: Remove nucleotides from the ends of the DNA.
• Endonucleases: Make cuts at specific positions within the DNA. Each restriction endonuclease recognizes a specific palindromic nucleotide sequence in the DNA (e.g., EcoRI recognizes 5'-GAATTC-3'). They cut the strand away from the center of palindrome sites, leaving single-stranded overhanging stretches called sticky ends. These sticky ends facilitate the action of the enzyme DNA ligase, which joins foreign DNA with the vector DNA.

2. Cloning Vectors

Vectors act as “molecular vehicles” to carry foreign DNA into a host cell. Plasmids and bacteriophages are commonly used vectors. To function optimally, a cloning vector requires:

• Origin of replication (\(ori\)): A sequence where replication starts. Every piece of DNA linked here replicates alongside the host DNA.
• Selectable marker: A gene (e.g., \(amp^R\), \(tet^R\) coding for ampicillin and tetracycline resistance respectively in E. coli) that helps identify and select transformants and eliminate non-transformants. An alternative method is insertional inactivation (e.g., interrupting the \(\beta\)-galactosidase gene resulting in white vs. blue colonies).
• Cloning sites: Specific recognition sites for the commonly used restriction enzymes to link the alien DNA. Often, unique recognition sites exist to prevent the vector from being fragmented.

3. Competent Host (For Transformation with Recombinant DNA)

Since DNA is a hydrophilic molecule, it cannot pass through cell membranes. Host cells (e.g., E. coli) must be made “competent” to take up DNA. Methods include:

• Treating them with a specific concentration of a divalent cation, such as calcium, and using a heat shock procedure (incubating on ice, placing briefly at 42°C, and putting back on ice).
• Micro-injection: Recombinant DNA is directly injected into the nucleus of an animal cell.
• Biolistics or gene gun: Plant cells are bombarded with high-velocity micro-particles of gold or tungsten coated with DNA.

Processes of Recombinant DNA Technology

Genetic engineering involves several steps:

Isolation of Genetic Material (DNA)

To cut the DNA with restriction enzymes, it needs to be pure and free from other macromolecules. Cells are treated with specific enzymes like lysozyme (bacteria), cellulase (plant cells), and chitinase (fungus) to break cell walls. RNA is removed by ribonuclease, and proteins by proteases. Finally, purified DNA precipitates out after adding chilled ethanol.

Cutting of DNA at Specific Locations

Restriction enzyme digestions are performed by incubating purified DNA molecules with the restriction enzyme. Agarose gel electrophoresis is used to check the progression of digestion. DNA fragments move towards the anode according to size; smaller fragments move farther. Recombinant DNA is formed by joining the cut ‘gene of interest’ and the cut vector with DNA ligase.

Amplification of Gene of Interest using PCR

Polymerase Chain Reaction (PCR) allows synthesizing multiple copies of the gene of interest in vitro. A PCR cycle involves:

1. Denaturation: Heating to separate DNA strands.
2. Annealing: Lowering the temperature to allow specific oligonucleotide primers to bind to the complementary DNA sequences.
3. Extension: The thermostable enzyme Taq polymerase (isolated from Thermus aquaticus) elongates the primers, synthesizing a new strand. After 30 cycles, DNA can be amplified 1 billion times.

Insertion of Recombinant DNA into Host Cell/Organism

Transforming competent host cells using the methods mentioned earlier. If a recombinant DNA bearing an ampicillin resistance gene is transferred into E. coli, only transformants will grow on agar plates containing ampicillin.

Obtaining the Foreign Gene Product

When alien DNA multiplies inside the host organism, its ultimate aim is generally the expression of a recombinant protein. Optimal conditions (pH, temperature, oxygen) must be provided in large-scale vessels called bioreactors (down to 100–1000 liters) to produce significant quantities. Commonly used are stirred-tank bioreactors that provide aeration and mixing.

Downstream Processing

After biosynthetic production in the bioreactor, the product undergoes separation and purification processes collectively called downstream processing. The product is then formulated with suitable preservatives and undergoes strict clinical quality control testing.

Competency Based Questions

Q1. In a PCR reaction utilizing an initial mixture of 5 double-stranded target DNA molecules, calculate the theoretical optimal number of target double-stranded DNA molecules present immediately upon the completion of 12 consecutive thermal cycles.

(A) 60
(B) \(12^5\)
(C) \(5 \times 2^{12}\)
(D) \(10^{12}\)

Q2. During gel electrophoresis, a mixture containing a 5 kb linear plasmid, a 3 kb circular vector, and an 800 bp inserted gene fragment is loaded into a single agarose well. Arrange these three distinct DNA species strictly according to the relative physical distance they migrate from the negative cathode towards the positive anode after 45 minutes of constant voltage run.

Q3. To insert a eukaryotic gene coding for human insulin directly into the pBR322 bacterial cloning vector, a scientist intentionally uses the specific restriction endonuclease BamHI, cutting the plasmid precisely within the sequence assigned to the tetracycline resistance gene (\(tet^R\)). Predict exactly what will phenotypically occur to successfully transformed E. coli cells plated differentially on antibiotic media.

Q4. A student extracts DNA from a leaf sample and accidentally forgets to add chilled ethanol at the final step of the isolation protocol. What critical physical property of the final extraction is lost, and what happens to the DNA?

Q5. Contrast the distinct functions of the origin of replication (\(ori\)) sequence and a selectable marker gene embedded within a functional cloning vector architecture.