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Vaccine

Biology

BIOTECHNOLOGY: PRINCIPLES AND PROCESSES

Biotechnology deals with microorganisms, plant or animal cells or their enzymes to produce the useful products for human’s welfare.

Intro

Biotechnology

Biotechnology deals with microorganisms, plant or animal cells or their enzymes to produce

The useful products for human’s welfare.

  • The term "Biotechnology" was given by Karl Ereky (1919).

  • According to European Federation of Biotechnology (EFB), biotechnology is the integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services.

 

Principles of Biotechnology

The two core techniques that developed modern biotechnology are:

  1. Genetic engineering which is modification of chemical nature of DNA/RNA and their introduction into another host organism to change the phenotypic characters of the host.

  2. Sterilization methods to maintain growth and manipulation of only the desired microbes or cells in large quantities, for the manufacture of biotechnological products like antibiotics, vaccines, enzymes, etc.

 

The basic steps in genetic engineering include:

  1. Identification of DNA with desirable genes.

  2. Introduction of the DNA into host to form recombinant DNA (rDNA).

  3. Maintenance of introduced DNA in host and gene cloning.

  4. Transfer of the DNA to its progeny.

 

In 1972, Stanley Cohen and Herbert Boyer constructed the first recombinant DNA.

 

Steps carried out in constructing first recombinant DNA:

  1. A gene encoding antibiotic resistance in the native plasmid of Salmonella typhimurium V. was identified. Plasmid is an autonomously replicating circular extra-chromosomal DNA.

  2. The desired DNA was cut at specific locations by restriction enzymes.

  3. The cut DNA was linked to plasmid DNA and transferred to E. coli for gene multiplication.

tools

Tools of Recombinant DNA Technology

The key tools required for the recombinant DNA technology are:

  1. Restriction enzymes

  2. Ligases

  3. Host organism/cell

  4. Polymerase enzymes

  5. Vectors

 

Restriction Enzymes

  • The restriction enzymes are called "molecular scissors" and are responsible for cutting DNA.

  • They are present in bacteria to provide a type of defense mechanism called the "restriction-modification system".

  • The first restriction endonuclease, Hind II, was isolated by Smith, Wilcox and Kelley (1968) from Haemophilus influenzae bacterium. It was used to cut DNA molecules at a particular point by recognising a specific sequence of six base pairs, known as the recognition sequence.

 

Naming of Restriction Enzymes

The first letter is derived from the genus name and the next two letters from the species name of the prokaryotic cell from which they were isolated. The roman numbers, following the names indicate the order in which the enzymes were isolated from the bacterial strain.

For example:

EcoRI is derived from Escherichia coli RY 13, the letter ‘R’ derived from the name of strain.

Hind II from HaemophiIus influenzae Rd, BamH I from Bacillus amyloliquefaciens H, EcoR II from E. coli R245, etc.

 

Restriction enzymes belong to a class of enzymes called nucleases and are of two kinds:

  1. Exonucleases – Remove nucleotides from the end of the DNA.

  2. Endonucleases – It makes cut at specific positions within the DNA.

 

The recognition sequences of endonucleases are palindromic, i.e., the sequence of base pairs that reads the same on both the DNA strands, when orientation of reading is kept same.

 

Mechanism of action of endonucleases

  • Every endonuclease inspects the entire DNA sequence for the palindromic recognition sequences.

  • On finding the palindrome, the endonuclease binds to the DNA.

  • It cuts the opposite strands of DNA in the sugar-phosphate backbone; a little away from the centre of the palindrome sites but between the same bases on both strands.

  • This results in the formation of single stranded overhanging stretches at the end of each strand called sticky ends.

  • The sticky ends facilitate the action of the enzyme DNA ligase by readily forming hydrogen bonds with complementary strands.

  • In genetic engineering, DNA from different sources are cut with the same restriction enzymes so that both DNA fragments have same kind of sticky ends.

  • These sticky ends are complementary to each other and thus can be joined by DNA ligase (end-to-end).

gel

Separation and Isolation of DNA Fragments (Gel Electrophoresis)

  • Gel electrophoresis is a technique for separating DNA fragments based on their size and net electric charges.

  • Firstly, the sample DNA is cut into fragments by restriction endonucleases.

  • The DNA fragments being negatively charged can be separated by forcing them to move towards the anode under an electric field through a medium/matrix.

  • Commonly used matrix is agarose, which is a natural linear polymer of D-galactose and 3, 6-anhydro- L -galactose which is extracted from sea weeds.

  • The DNA fragments separate-out according to their size because of the sieving property of agorose gel. Hence, smaller the fragment size, the farther (faster) it will move.

  • The separated DNA fragments are visualised after staining the DNA with ethidium bromide followed by exposure to UV radiation.

  • The DNA fragments are seen as orange coloured bands.

  • The separated bands of DNA are cut out and extracted from the gel piece. This step is called elution.

  • The purified DNA fragments are used to form recombinant DNA which can be joined with cloning vectors.

Gel Electrophoresis.png
cloning

Cloning Vectors

            The vectors are the DNA molecules that can carry a foreign DNA segment into the host cell.

Vectors may be:

  1. Plasmids: These are autonomously replicating circular extra-chromosomal DNA.

  2. Bacteriophages: These are viruses infecting bacteria.

 

Copy number: It is defined as the number of copies of vectors present in a cell. It varies from 15-100 copies per cell.

The best-known vector is the plasmid vector.

 

pBR322 is the first artificial cloning vector developed in 1977 by Boliver and Rodriguez from E. coli plasmid.

​​

pBR322.png

The following features are required to facilitate cloning into a vector

1. Origin of replication (ori)

  • This is a DNA sequence that is responsible for initiating replication. Any piece of DNA when linked to this sequence can replicate within the host cells.

  • ori also controls the copy numbers of the linked DNA.

 

2. Selectable marker

  • It helps to select the host cells which contain the vector (transformants) and eliminate the non-transformants.

  • Transformation is defined as the procedure by which a piece of DNA is introduced into a bacterial host.

  • Genes encoding resistance to antibiotics like tetracycline or chloramphenicol, ampicillin, kanamycin, are useful selectable markers for E. coli.

  • The normal E. coli cells do not carry resistance against any of these antibiotics.

 

3. Cloning sites

  • To link the alien DNA, the vectors require very few (mostly single) recognition sites for the restriction enzymes.

  • More than one recognition sites within the vector, can complicate the gene cloning as it will generate several fragments.

  • Ligation of alien DNA can be carried out at a restriction site present in one of the two antibiotic resistance genes.

 

4. Vectors for cloning genes in plants and animals

  • There are several vectors which are used for cloning genes in plants and animals.

  • In plants, the tumour inducing plasmid (Ti) of Agrobacterium tumefaciens is used as a cloning vector.

  • A. tumefaciens is a pathogen of several dicot plants.

  • It delivers a piece of DNA known as 'T-DNA' in the Ti plasmid which transforms normal plant cells into tumor cells to produce chemicals against pathogens.

  • Retrovirus, adenovirus, papillomaviruses are also now used as cloning vectors in animals because of their ability to transform normal cells into cancerous cells.

 

Selection of recombinants formed can be done by one of the following methods:

1. Inactivation of antibiotics

  • If a foreign DNA ligates at the BamHI site of tetracycline resistance gene in the vector pBR322, the recombinant plasmid loses the tetracycline resistance due to insertion of foreign DNA.

  • It can still be selected out from non-recombinant ones by plating the transformants on ampicillin containing medium.

  • The transformants growing on ampicillin containing medium are then transferred on to a medium containing tetracycline.

  • The recombinants can grow in ampicillin containing medium but not on that containing tetracycline whereas non-recombinants can grow on the medium containing both the antibiotics and thus recombinants are selected

 

2. Insertional inactivation

  • On the basis of colour production in the presence of chromogenic substrate, the recombinants and non-recombinants can also be differentiated.

  • Here, a recombinant DNA is inserted within the coding sequence of an enzyme         β-galactosidase, which results into inactivation of the enzyme.

  • The bacterial colonies having inserted plasmid, shows no colouration while those without inserted plasmid form blue colour colonies.

competent

Competent Host (For Transformation with Recombinant DNA)

  • DNA being a hydrophilic molecule, cannot pass through cell membranes.

  • Therefore, the bacteria should be made competent to accept the DNA molecules.

  • Competency is the ability of a cell to take up foreign DNA.

  • The cell is made competent by the following methods:

  1. Chemical method

  2. Physical method

 

a) Chemical method

  • The cell is treated with specific concentration of a divalent cation such as calcium to increase pore size in cell wall.

  • The cells are incubated with recombinant DNA on ice, followed by placing them briefly at 420C and then putting it back on ice. This is called heat shock treatment.

  • The bacteria now take up the recombinant DNA.

 

b) Physical methods

The physical methods include

  • Micro-injection method: Recombinant DNA is directly injected into the nucleus of an animal cell.

  • Biolistics or gene gun method: Cells are bombarded with high velocity micro-particles of gold or tungsten coated with DNA in plants. Disarmed pathogen vectors are also used to transfer rDNA.

Process of Recombinant DNA Technology

Recombinant DNA technology involves the following steps:

  1. Isolation of DNA.

  2. Fragmentation of DNA by restriction endonucleases.

  3. Isolation of a desired DNA fragment.

  4. Amplification of the gene of interest.

  5. Ligation of the DNA fragment into a vector.

  6. Insertion of recombinant DNA into the host.

  7. Culturing the host cells on a suitable medium at a large scale.

  8. Extraction of the desired gene product.

  9. Downstream processing of the products as finished product, ready for marketing.

 

1. Isolation of the genetic material (DNA)

  • RNA is removed by treatment with ribonuclease and proteins are removed by treatment with protease.

  • After several treatments, the purified DNA is precipitated by adding chilled ethanol.

  • The bacterial/plant/animal cell is broken down by enzymes to release DNA, along with RNA, proteins, polysaccharides and lipids.

  • Bacterial cell is treated with enzyme lysozyme.

  • Plant cell is treated with enzyme cellulase.

  • Fungal cell is treated with chitinase.

 

2. Cutting of DNA at specific locations

  • The DNA is cut using restriction enzymes.

  • The purified DNA is incubated, with the specific restriction enzyme at conditions optimum for the enzyme to act.

 

3. Isolation of desired DNA fragment

  • Using agarose gel electrophoresis, the activity of the restriction enzymes can be checked.

  • Since the DNA is negatively charged, it moves towards the positive electrode or anode and in the process, DNA fragments separate out based on their sizes.

  • The desired DNA fragment is eluted out.

 

4. Amplification of gene of interest using PCR

  • The Polymerase Chain Reaction (PCR) is a reaction in which amplification of specific DNA sequences is carried out in vitro.

  • This technique was developed by Kary Mullis in 1985, and for this he received Nobel Prize for Chemistry in 1993.

 

Requirements for PCR:

  1. DNA template: "The double-stranded DNA that needs to be amplified.

  2. Primers: Small chemically synthesised oligonucleotides of about 10-18 nucleotides that are complementary to a region of template DNA.

  3. Enzyme: Two commonly used enzymes are Taq polymerase (isolated from thermophilic bacterium, Thermus aquaticus) and Vent polymerase (isolated from Thermococcus litoralis).

 

PCR is carried out in the following three steps:

 

1. Denaturation

  • The double-stranded DNA is denatured by subjecting it to high temperature of 950C for 15 seconds. Each separated single stranded strand now acts as template for DNA synthesis.

 

2. Annealing

  • TWO sets of primers are added which anneal to the 3' end of each separated strand.

  • Primers act as initiators of replication.

 

3. Extension

  • DNA polymerase extends the primers by adding nucleotides complementary to the template provided in the reaction.

  • A thermostable DNA polymerase (Taq polymerase) is used in the reaction which can tolerate the high temperature of the reaction.

  • All these steps are repeated many times to obtain several copies of desired DNA.

 

5. Ligation of DNA fragment into a vector

  • The vector DNA and source DNA are cut with the same endonuclease to obtain sticky ends.

  • These are then ligated by mixing vector DNA, gene of interest and enzyme DNA ligase to form a recombinant DNA.

 

6. Insertion of recombinant DNA into the host cell/organism

  • Introduction of ligated DNA into recipient cells occurs by several methods, before which the recipient cells are made competent to receive the DNA.

  • If recombinant DNA carrying antibiotic resistance (e.g., ampicillin) is transferred into E. coli cells, the host cell is transformed into ampicillin-resistant cells.

  • The ampicillin resistant gene in this case is called a selectable marker.

  • On growing transformed cells on agar plates containing ampicillin, only transformants will grow and others will die.

 

7. Culturing the host cells

  • The transformed host cells are grown in appropriate nutrient medium at optimal conditions.

  • The DNA gets multiplied and expresses itself to form desired product.

 

8. Extraction of desired gene product

  • When a protein encoding gene is expressed in a heterologous host, it is called a recombinant protein.

  • The cells having genes of interest can be grown on a small scale or on a large scale.

  • On small scale, the cells are grown on cultures in laboratory and then the expressed protein is extracted and purified by different separation techniques.

  • On large scale, the cells are grown in a continuous culture system in which fresh medium is added from one side to maintain cells in exponential growth phase and the desired protein is collected from the other side.

  • In large scale method, larger biomass is produced which leads to high yield.

 

9. Downstream processing

  • All the processes to which a product is subjected to before being marketed as a finished product are called downstream processing.

  • It includes:

  1. Separation of the product from the reactor.

  2. Purification of the product.

  3. Formulation of the product with suitable preservatives.

  4. Quality control testing and clinical trials in case of drugs.

rDNA

Bioreactors

  • Bioreactors are vessels of large volumes (100-1000 litres) in which raw materials are biologically converted into specific products.

  • It provides all the optimal conditions for achieving the desired product by providing optimal growth conditions like temperature, pH, substrate, salt, vitamins and oxygen.

  • Stirred-tank bioreactors are commonly used bioreactors.

  • These are cylindrical with curved base to facilitate proper mixing of the contents.

  • Bioreactor has the following components:

  1. An agitator system

  2. An oxygen delivery system

  3. Foam control system

  4. Temperature control system

  5. pH control system

  6. Sampling ports to withdraw cultures periodically.

  • The stirrer mixes the contents and makes oxygen available throughout the bioreactor.

  • Sparged stirred-tank reactor is a stirred type reactor in which air is bubbled.

Bioreactor
Simple Bioreactor.png
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