Department of Biology, SKCH PU College, Bangalore 1
Chapter:09
SRI KUMARAN CHILDREN’S HOME
COMPOSITE PU COLLEGE
II PUC
STUDY MATERIAL
UNIT IX: BIOTECHNOLOGY
CHAPTER 09: BIOTECHNOLOGY –
PRINCIPLES AND PROCESSES
DEPARTMENT OF BIOLOGY
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Chapter: 09
SRI KUMARAN CHILDREN’S HOME
COMPOSITE PU COLLEGE
II PUC
STUDY MATERIAL
UNIT IX: BIOTECHNOLOGY
CHAPTER 09 : BIOTECHNOLOGY –
PRINCIPLES AND PROCESSES
DEPARTMENT OF BIOLOGY
PRINCIPLES AND PROCESSES
The term biotechnology was coined by Károly Ereky in the year 1919. Stanley Cohen and Herbert Boyer were the first scientists to transplant genes from one living organism to another, a fundamental discovery for genetic engineering. Many products have been developed on the basis of their work, including human growth hormone and hepatitis B vaccine. Meaning of biotechnology is using principles of physics and engineering techniques on living cells to produce required products and processes for human usage. Ancient practices like preparation of curds, wine and bread are also microbe mediated processes and are part of biotechnology, now it has reached a new height. Some of the examples are in vitro fertilization leading to test tube baby, synthetic gene and its usage, production of vaccine, gene therapy, production of transgenic plants and transgenic animals, production of insulin and growth hormone………etc. The European Federation of Biotechnology [EFB] has given a definition of biotechnology - “The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services”. PRINCIPLES OF BIOTECHNOLOGY It includes two core techniques - i. Genetic engineering and ii. Bioprocess engineering. Genetic engineering: It is a manipulation of genes under laboratory conditions through gene slicing and gene splicing with the help of Restriction endonuclease and ligase to obtain required product using vector and host organism. It includes techniques to alter the chemistry of genetic material like DNA and RNA, to introduce these into host organisms and thus change the phenotype of the host organism. Bioprocess engineering: To produce rDNA or its products in bulk requires sterile conditions or microbial contamination free area, maintenance of sterile ambience in chemical engineering processes to enable growth of only the desired microbe or eukaryotic cell in large quantities for the manufacture of biotechnological products like antibiotics, vaccines, hormones, and enzymes. Conceptual development of the principles of genetic engineering
- Each restriction endonuclease recognises specific sequences of nucleotides in the DNA while cutting, the unique sequence is a recognition site for that enzyme.
- Recognition site is made up of either four or six nucleotides in each strand, and is always in palindromic sequence , the sequences of nucleotides when identical in any one direction of the DNA strands then the sequence is called palindromic sequence. Example 5’ - GAATTC - 3’ 3’ - CTTAAG - 5’.
- Nomenclature of restriction enzymes is based on some of the rules, for example EcoRI is isolated from the bacteria Escherichia coli RY 13. In naming they use the first letter from the genus (E), next two letters from the species (co), and the letter ‘R’ is from the strain RY 13.
- The site in which RENase acts/cuts the DNA is called restriction site. The enzymes cut the strand of DNA little away from the centre of the palindromic sites, but between the same two bases on the opposite strands. Example; EcoRI recognise 5’ - GAATTC - 3’ 3’ - CTTAAG - 5’ The cut ends of the DNA are either sticky ends or blunt ends, in sticky ends the cut ends are carried with unpaired nucleotides, in blunt end the cut ends of the DNA carried with paired nucleotides.
LIGASE ENZYME
It is popularly called molecular glue or molecular sticher, because it is
involved in joining of cut ends of the DNA.
Restriction endonuclease and ligase enzymes are extensively used in rDNA
technique to produce rDNA through gene slicing/cutting of DNA and gene
splicing/joining of DNA fragments.
GEL ELECTROPHORESIS Separation and isolation of DNA fragments: After the isolation of DNA from the cell, it is treated with restriction endonuclease enzymes to cut the DNA into DNA fragments, these fragments are subjected to gel electrophoresis to separate and select the desired gene. The gel used to migrate the DNA fragments is agarose gel, it is used as a matrix, it is a natural polymer extracted from sea weeds. The gel slab is fitted with electrodes, separation of DNA fragments is based on positive and negative charges, when the DNA fragments are subjected to gel electrophoresis, DNA fragments migrate towards the anode under an electric field through the matrix, because DNA fragments are negatively charged, the fragments separate according to their size through sieving effect of the agarose gel. DNA fragments can be visualised by using an orange coloured stain ethidium bromide, followed by exposure to UV radiation. After identifying the orange coloured bands(DNA), these fragments are isolated from the agarose gel, and are used in rDNA production. The technique of separation of DNA fragments from the agarose gel is called elution
Gene amplification through PCR technique PCR – Polymerase Chain Reaction, is a technique used to multiply/amplify the desired gene in vitro (out side the body, under laboratory conditions) using an enzyme taq polymerase (thermostable enzyme isolated from Thermus aquaticus bacteria) and two sets of primers (small chemically synthesized oligonucleotides that are complementary to the sequences of DNA/ strands of desired gene). Involves three steps I. Denaturation: Separation of the two strands of the desired gene/DNA by using high temperature of about 92 degree to 96 degree centigrade. II. Annealing: Primers are introduced to assemble in 5’ - > 3’ direction. III. Extension: Taq polymerase enzyme extends the primers using the nucleotides(dNTPs – deoxy nucleotide triphosphates) provided in the reaction and the desired gene/DNA as template. To amplify the same gene, the process is repeated many times, the segment of DNA can be amplified up to one billion copies.
then they use tetracycline antibiotic in the nutrient medium, if the cloning site is BamHI then they use ampicillin nutrient medium. In general they make use of both the sites and both the nutrients, 50% of the plating is with the ampicillin nutrient medium and 50% of the plating is with tetracycline nutrient medium. This way transformants can be developed and non transformants are avoided. INSERTIONAL INACTIVATION It is one of the alternative to selection of recombinants due to inactivation of antibiotics, so it is one of the alternative selectable marker. It is called insertional inactivation because, by inserting the desired gene at the lac Z gene of the plasmid associated with the E.coli bacteria, the lac Z product beta galactosidase enzyme production get inactivated. When the E.coli bacteria are introduced in to chromogenic substrate, the lac Z gene associated with plasmid of E.coli synthesize beta galactosidase enzyme as a result the bacterial colony exhibit blue colouration. If that lac z
gene is used for cloning a desired gene then the bacteria is a transformant, and after introducing them into the chromogenic substrate, no colour is found at these colonies. So it helps to maintain transformants. Vectors for cloning genes in plants and animals In nature some of the pathogens are capable of gene cloning in plants and animals, one such example is Agrobacterium tumifaciens, it is pathogen for several dicot plants, it is able to transfer a piece of DNA known as ‘T - DNA’, to transform normal plant cells in to a tumor, direct the tumor cells to produce the chemicals required by the pathogen. In animals, retroviruses can inject the genetic material to transform normal cells into cancerous cells. After knowing the ability of these pathogens to deliver the genes in to the host body, these tools are transformed into useful vectors, the Ti plasmid/tumor inducing plasmid of Agrobacterium tumifaciens and disarmed pathogen of retroviruses. These vectors are used in genetic engineering, these vectors are no more harmful or pathogenic. Competent host Host is the organism that receives rDNA, and capable of producing recombinant product, in which desired product or protein can be isolated. After receiving the desired gene through the plasmid DNA, the organism is called transformant and the process of transfer is called transformation. Escherichia coli is used as a competent host, because it can be cultured using nutrient medium, easy to maintain under laboratory conditions, which do not reject the rDNA, faster rate of multiplication. DNA is a hydrophilic macromolecule, it cannot pass through cell membrane of the bacteria, to make the membrane porous, to make the bacteria competent to receive
- When ethanol is added then the DNA precipitates out, the purified DNA is collected and used for fragmentation. Cutting of DNA at specific locations The collected DNA is subjected to gel electrophoresis to obtain the desired gene, by the activity of restriction endonuclease enzyme the DNA get fragmented, the fragments are distributed on the gel slab, based on the size desired gene can be selected. Desired gene amplification through PCR technique Desired gene is subjected to PCR technique to obtain multiple copies of desired genes, it is an in vitro technique where taq polymerase enzyme and primers are used. Production of rDNA and transformation Using the desired gene and plasmid as a vector, rDNA is produced through gene slicing and splicing, transfer of rDNA to the host or E.coli is called transformation. The transformants are introduced into the nutrient medium, based on the antibiotics used in the medium, and the cloning site used for ligation. If the cloning site used is BamHI then the transformants are introduced into the nutrient medium with ampicillin antibiotic, if the cloning site is Pvu I then the transformants are introduced in to the nutrient medium with tetracycline antibiotics. Obtaining the foreign gene product The transformants in the nutrient medium undergo series of multiplication, where each progeny is carried with the rDNA and capable of producing a recombinant protein. The product is a protein, it is called recombinant protein because, the plasmid DNA and desired gene both produce the protein inside the body of host. If the transformants are grown in a small scale in the laboratory then the cultures are used for extraction and purification of desired protein.
If the transformants are grown in large scale for bulk production of gene product then they are introduced into the specially designed huge vessels called bioreactor. The vessel provides optimum conditions for exponential growth of transformants. Downstream processing Separation and purification of desired product/protein is called down stream processing. After series of processing the finished product is formulated with suitable preservatives, then it will undergo thorough clinical trials as in case of drugs. Strict quality control testing is done before releasing it for marketing. Bioreactor It is a specially designed vessel to carry out biochemical reactions in bulk. The capacity of the vessel varies from 100litres to 1000liters, it provides optimum conditions for biochemical reactions like, temperature, pH, substrate, salts, vitamins, and oxygen. The most commonly used bioreactors are stirred tank bioreactor and sparged stirred tank bioreactor. Simple stirred bioreactor(A): It is a cylindrical vessel with curved base to facilitate the mixing of the reactor contents. It has a stirrer to facilitates mixing and oxygen availability throughout the vessel. It has an agitator system, oxygen delivery system and a foam control system, a temperature control system, pH control system and sampling ports to with draw small volume of culture periodically. Sparged stirred bioreactor(B): In sparged stirred bioreactor sterile air bubbles are sparged to the reaction mixture.