Unit 9 : Biotechnology; Principles and Processes

Principles of Biotechnology

Biotechnology deals with techniques of using live organisms or enzymes from organisms to products and processes useful to humans. 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.

Core Techniques of Modern Biotechnology

Modern biotechnology largely rests on two core techniques:

• 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.
• Bioprocess Engineering: Maintenance of sterile ambient conditions in chemical engineering processes to enable growth of only the desired microbe/human cell in large quantities for the manufacture of biotechnological products like antibiotics, vaccines, enzymes, etc.

Conceptual Development of Principles of Genetic Engineering

To understand why genetic engineering is crucial, consider sexual reproduction which introduces variations and mixing of genetic information. Traditional hybridization procedures often lead to inclusion and multiplication of undesirable genes along with desired genes.

Genetic engineering overcomes this limitation by allowing us to isolate and introduce only one or a few desirable genes without introducing undesirable genes into the target organism.

The creation of recombinant DNA, use of gene cloning, and gene transfer allow us to bypass sexual reproduction constraints and direct the genetic makeup precisely.

Steps in Generating a Genetically Modified Organism (GMO)

1. Identification of DNA with desirable genes.
2. Introduction of the identified DNA into the host.
3. Maintenance of introduced DNA in the host and transfer of the DNA to its progeny.

Tools of Recombinant DNA Technology

Recombinant DNA technology requires specific tools to accomplish the task of manipulating genetic material. The primary tools include restriction enzymes, cloning vectors, and competent hosts.

1. Restriction Enzymes (Molecular Scissors)

Restriction enzymes belong to a larger class of enzymes called nucleases. They are of two kinds: exonucleases (remove nucleotides from the ends of DNA) and endonucleases (make cuts at specific positions within the DNA).

Restriction endonucleases inspect the length of a DNA sequence, bind to specific recognition sequences, and cut each of the two strands of the double helix at specific points in their sugar-phosphate backbones.

Palindromic Nucleotide Sequences: Restriction enzymes recognize specific palindromic sequences in DNA. A palindrome in DNA is a sequence of base pairs that reads same on the two strands when orientation of reading is kept the same.

Example of a palindromic sequence recognized by EcoRI:

5' - GAATTC - 3'
3' - CTTAAG - 5'

Action of Restriction Enzyme: Restriction enzymes cut the strand a little away from the centre of the palindrome sites, between the same two bases on the opposite strands. This leaves single-stranded stretches at the ends called sticky ends. These sticky ends form hydrogen bonds with their complementary cut counterparts, facilitating action by the enzyme DNA ligase.

2. Cloning Vectors

Vectors are DNA molecules used as vehicles to artificially carry foreign genetic material into another cell, where it can be replicated and expressed. Plasmids (extra-chromosomal autonomous replicating circular DNA in bacteria) and bacteriophages are common vectors.

Features Required to Facilitate Cloning into a Vector

• Origin of replication (ori): This is a sequence from where replication starts, and any piece of DNA when linked to this sequence can be made to replicate within the host cell. It is also responsible for controlling the copy number of the linked DNA.
• Selectable marker: The vector requires a selectable marker, which helps in identifying and eliminating non-transformants and selectively permitting the growth of the transformants. Genes encoding resistance to antibiotics such as ampicillin, tetracycline, kanamycin, or chloramphenicol are useful selectable markers for E. coli.
• Cloning sites: In order to link the alien DNA, the vector needs to have single or very few recognition sites for the commonly used restriction enzymes. Ligation of alien DNA is carried out at a restriction site present in one of the two antibiotic resistance genes.
• Vectors for plant and animal gene transfer: Tumour inducing (Ti) plasmid of Agrobacterium tumefaciens is used as a cloning vector for plants, and retroviruses are used for animals after transforming them into non-pathogenic forms.

3. Competent Host (For Transformation with Recombinant DNA)

Since DNA is a hydrophilic molecule, it cannot pass through cell membranes. Therefore, the bacterial host must be made competent to take up DNA.

• Chemical Treatment: Treating bacterial cells with a specific concentration of a divalent cation, such as calcium, increases the efficiency with which DNA enters the bacterium through pores in its cell wall.
• Heat Shock Method: Recombinant DNA can then be forced into such cells by incubating the cells with recombinant DNA on ice, followed by placing them briefly at 42 degrees Celsius (heat shock), and then putting them back on ice. This enables bacteria to take up the recombinant DNA.
• Micro-injection: Recombinant DNA is directly injected into the nucleus of an animal cell.
• Biolistics or Gene Gun: Cells are bombarded with high velocity micro-particles of gold or tungsten coated with DNA in plants.
• Disarmed pathogen vectors: Pathogenic vectors are allowed to infect the cell, transferring the recombinant DNA safely.

Processes of Recombinant DNA Technology

Recombinant DNA technology involves several steps executed in a specific sequence.

1. Isolation of the Genetic Material (DNA)

Since DNA is enclosed within membranes, the cell must be broken open to release DNA along with other macromolecules like RNA, proteins, polysaccharides, and lipids. Bacterial cells, plant cells, or animal tissue are treated with enzymes such as lysozyme (bacteria), cellulase (plants), and chitinase (fungi). RNA can be removed with ribonuclease and proteins with protease. Other molecules are removed with appropriate treatments, and purified DNA precipitates out after the addition of chilled ethanol.

2. Cutting of DNA at Specific Locations

Purified DNA is incubated with restriction endonucleases at optimal conditions. The agarose gel electrophoresis is employed to check the progression of restriction enzyme digestion. DNA is negatively charged, so it moves towards the positive electrode (anode) under an electric field through an agarose matrix. The smaller the fragment size, the farther it moves. The separated DNA bands are stained with ethidium bromide and exposed to UV radiation to observe bright orange colored bands.

3. Amplification of Gene of Interest using PCR

Polymerase Chain Reaction (PCR) is a method to synthesize multiple copies of gene of interest in vitro using two sets of primers (small chemically synthesized oligonucleotides that are complementary to the regions of DNA) and the enzyme DNA polymerase.

• Denaturation: Double stranded DNA is thermally denatured at high temperature to separate strands.
• Annealing: Two sets of primers are allowed to bind to complementary regions on the single-stranded DNA templates at a lower temperature.
• Extension: Thermostable DNA polymerase (Taq polymerase, isolated from bacterium Thermus aquaticus) extends the primers using nucleotides provided in the reaction and the genomic DNA as template.

Repeating this process many times amplifies the DNA segment roughly a billion times.

4. Insertion of Recombinant DNA into the Host Cell or Organism

This step involves introducing the ligated DNA into a recipient host cell using transformation methods such as heat shock, microinjection, or gene guns, making the host a transformant.

5. Culturing the Host Cells on a Suitable Scale

The transformed host cells are grown in large quantities in a nutrient medium. The recombinant cell can multiply and produce the desired protein. If a protein is encoded by a heterologous gene expressed in a heterologous host, it is called a recombinant protein.

6. Downstream Processing

After the formation of the product, it has to be subjected through a series of processes before it is ready for marketing as a finished product. Downstream processing includes separation and purification of products. The product is formulated with suitable preservatives and undergoes strict quality control testing.

Bioreactors

To produce large quantities of products, bioreactors are used where large volumes (100-1000 liters) of culture can be processed. A bioreactor provides the optimal growth conditions by furnishing temperature, pH, substrate, salts, vitamins, and oxygen.

Exam-Oriented Notes and Common Mistakes

• Common Mistake: Confusing exonucleases with endonucleases. Remember, exonucleases cut DNA from the ends, whereas endonucleases cut at specific internal sites.
• Important Definition: Palindromic sequence - sequence of base pairs that reads same forward and backward on opposite strands with same reading polarity.
• Key Enzyme: Taq polymerase is heat-stable and isolated from Thermus aquaticus, allowing it to withstand the high denaturation temperatures of PCR.
• Important Observation: Selectable markers are essential to differentiate transformants from non-transformants.