Biotechnology: Principles and Processes | Plus 2 Botany | Exam Capsule Notes (Web and PDF)

BIOTECHNOLOGY- PRINCIPLES & PROCESSES: 

CHAPTER AT A GLANCE

Biotechnology: The technique of using live organisms or their enzymes for products & 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.

PRINCIPLES OF BIOTECHNOLOGY

Core techniques:

• Genetic engineering: Genetic material (DNA & RNA) is altered and introduced into host organisms.
• Bioprocess engineering: Maintenance of sterile ambience for growing desired microbe/eukaryotic cell for the manufacture of antibiotics, vaccines, enzymes etc.

Basic steps in genetically modifying an organism:

1. Identification of DNA with desirable genes.
2. Introduction of the identified DNA into the host using a vector DNA such as plasmid.
3. Maintenance of introduced DNA in host and transfer of the DNA to its progeny.

Recombinant DNA technology: Joining & inserting foreign DNA into a host to produce new genetic combinations.

Stanley Cohen & Herbert Boyer produced first recombinant DNA (rDNA). They isolated an antibiotic resistance gene from plasmid of Salmonella typhimurium and transferred into E. coli.

TOOLS OF RECOMBINANT DNA TECHNOLOGY

1. Restriction Enzymes (‘molecular scissors’)

• The enzymes that cut DNA at specific sites into fragments.
• They belong to a class of enzymes called nucleases.

Types of Restriction enzymes:

• Exonucleases: Remove nucleotides from the ends of DNA.
• Endonucleases: Cut at specific sites within DNA. E.g. EcoRI, Hind II (First restriction endonuclease). They recognize a palindromic nucleotide sequences (it reads the same on two strands in 5' → 3' & 3' → 5').

Palindromic sequence for EcoRI: 

5' — GAATTC — 3' 

3' — CTTAAG — 5' 

Restriction enzymes cut the strand at palindrome sites leaving single stranded overhanging stretches (sticky ends). This facilitates action of the enzyme DNA ligase.

2. Cloning Vector

It is a DNA molecule that can carry a foreign DNA segment and replicate inside host cells. E.g. Plasmids, bacteriophages.

Plasmids: Autonomously replicating circular extra-chromosomal DNA of bacteria.

Features required for cloning into a vector:

a. Origin of replication (ori): The sequence where replication starts. Alien piece of DNA linked to ori can replicate within the host cells. It also controls copy number of linked DNA.

b. Selectable marker (marker gene): It is a gene to select transformants and eliminate non-transformants. If a foreign DNA is introduced into host bacterium, it is called transformation. Such bacterium is transformant. If transformation does not occur, it is non-transformant. Selectable markers of E. coli include antibiotics resistance genes. Normal E. coli cells have no resistance against these antibiotics.

c. Cloning sites: Recognition sites for the restriction enzymes to link the alien DNA.

Ligation of alien DNA is carried out at a restriction site in one of the two antibiotic resistance genes. E.g. In vector pBR322, foreign DNA is ligated at Bam H I site of tetracycline resistance gene to form recombinant plasmid. If ligation does not occur, it is called non-recombinant plasmid.

When a foreign DNA is inserted within a gene of bacteria, that gene is inactivated. It is called insertional inactivation. Here, the recombinant plasmids lose tetracycline resistance due to insertion of foreign DNA.

Plasmids + E. coli cells → 3 types of cells:

• Non-transformants: No plasmid. Not resistant to tetracycline & ampicillin.
• Transformants with non-recombinant plasmid: Resistant to tetracycline & ampicillin.
• Transformants with recombinant plasmid: Resistant only to ampicillin.

Selection of Recombinant plasmids from non-recombinants:

Method 1: By plating transformants on ampicillin medium. Then transformants are transferred on tetracycline medium. The recombinants grow in ampicillin medium but not on tetracycline medium. But, non-recombinants grow on medium containing both the antibiotics. It is a difficult method.

Method 2: Recombinant DNA is inserted within the coding sequence (gene) of b-galactosidase enzyme. So, the gene is inactivated. Such colonies do not produce colour in presence of chromogenic substrate. These are recombinant colonies. If the plasmid has no insert, it gives blue colour.

d. Vectors for cloning genes in plants & animals:

• Tumor inducing (Ti) plasmid of Agrobacterium tumefaciens: Used to deliver genes of interest into plants.
• Retroviruses: Used to deliver genes into animal cells.

3. Competent Host (For Transformation with Recombinant DNA)

Since DNA is a hydrophilic molecule, it cannot pass through cell membranes. So bacterial cells are made ‘competent’ to take up alien DNA or plasmid. 

It is done as follows:

Treat bacterial cells with a divalent cation (e.g. calcium) → DNA enters bacterium through pores in cell wall. → Incubate the cells with recombinant DNA on ice → Place briefly at 420C (heat shock) → Put them back on ice → Bacteria take up recombinant DNA.

Other methods to introduce alien DNA into host:

• Micro-injection: Recombinant DNA is directly injected into the nucleus of an animal cell.
• Biolistics (gene gun): Plant cells are bombarded with high velocity micro-particles of gold or tungsten coated with DNA.
• ‘Disarmed pathogen’ vectors: They infect the cell and transfer the recombinant DNA into the host.

PROCESSES OF RECOMBINANT DNA TECHNOLOGY

1. Isolation of the Genetic Material (DNA):

Treat the cells/tissue with suitable enzymes (e.g. lysozyme for bacteria, cellulase for plants, chitinase for fungus) → Cell is broken releasing DNA & other macromolecules → Molecules other than DNA are removed with suitable enzymes (E.g. ribonuclease to remove RNA, protease to remove proteins) → chilled ethanol is added → purified DNA precipitates.

2. Cutting of DNA at Specific Locations:

Purified DNA + restriction enzyme → DNA digests → Agarose gel electrophoresis (for both source DNA & vector DNA) → negatively charged DNA moves towards the anode → DNA fragments are separated based on size (smaller sized fragment moves farther).

DNA bands can be seen in bright orange colour when they are stained with ethidium bromide and exposed to UV radiation.

DNA bands are cut out from agarose gel (elution).

Cut-out gene of interest + cut vector + ligase → recombinant DNA.

3. Amplification of Gene of Interest using PCR (Polymerase Chain Reaction):

PCR: Synthesis of multiple copies of the gene of interest.

Steps of PCR:

• Denaturation: Heating of target DNA at 940 C to separate the strands (templates for DNA synthesis).
• Annealing: Joining of the two primers (at 520 C) at the 3’ end of the DNA templates.
• Extension: Addition of nucleotides to the primer using Taq polymerase (a thermostable DNA polymerase isolated from a bacterium, Thermus aquaticus).

4. Insertion of Recombinant DNA into Host Cell:

The ligated DNA is introduced into host cells.

If a recombinant DNA having ampicillin resistant gene is transferred into E. coli cells, the host cells become ampicillin-resistant cells.

If the transformed cells are spread on agar plates containing ampicillin, only transformants will grow. Untransformed recipient cells will die.

5. Obtaining the Foreign Gene Product:

Host cells with foreign genes can be used to extract the desired recombinant protein and purify it.

Large quantity of products can be produced using Bioreactors (the vessels in which raw materials are converted to specific products, enzymes etc., using microbial plant, animal or human cells.

Most commonly used bioreactors are of stirring type (stirred-tank reactor).

6. Downstream Processing:

- A series of processes such as separation and purification of products after the biosynthetic stage. 

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