Study Notes on Biotechnology Principles and Processes
- Biotechnology deals with bunch of techniques by using live organism or enzyme from organisms to produce products and process which is useful to human.
- Those process in which genetically modified organism to achieve the same on a larger scale.
- There are so many other process or techniques also include under biotechnology.
- The definition which is given by EFB( European federation of biotechnology is the integration of natural science and organisms ,cells, parts thereof and molecular analogous for products and services.
Principles of biotechnology
- The two core techniques are :
- Genetic engineering in which techniques to alter the chemistry of genetic material. To introduce these into host organism so that change the phenotype of the host organism.
- The maintenance of sterile ambience in chemical engineering process to enable growth of only the desired microbes for the manufacture of bio technological products like antibiotics ,vaccine , enzyme.
- The technique of genetic engineering which also include creation of recombinant DNA by using gene cloning for gene transfer.
- Piece of DNA would not be able to multiply itself in the progeny cells of the organism but when it gets integrated into the genome of the recipient it may multiply and be inherited along with the host DNA.
- This happens because alien piece of DNA become part of a chromosome which has the ability to replicate.
- In chromosome there is a specific DNA sequence known as origin of replication which is responsible for initiating replication.
- This process is also known as cloning are making multiple identical copies of any template DNA.
- Construction of the first recombinant DNA emerged from the possibility of linking a gene encoding antibiotic resistance with the native plasmid.
- Cutting of DNA at specific location became possible with the discovery of the so-called molecular scissors restriction enzyme.
- The plasmid DNA act as vector to transfer the piece of DNA attached to it.
- Plasmid can be used as a vector to deliver alien piece of DNA into the host organism.
- The linking antibiotics resistance gene with the plasmid vector become possible with the enzyme DNA ligase.
- It cut the DNA and join their ends which max on you combination of circular autonomously replicating DNA created in vitro and known as recombinant DNA.
- The ability to multiply copies of antibiotic resistance gene in e coli was called cloning of antibiotic resistance gene in E.coli
Tools of recombinant DNA technology
- Genetic engineering or recombinant DNA technology can be accomplished only if we have key tools like restriction enzyme polymerase enzyme ,ligases ,vectors and the host organism.
- It cut the DNA a molecule at a particular point by recognising a specific sequence of 6 base pairs in which the specific base sequence is known as recognization sequence.
- Restriction enzyme belong to a larger class of enzyme known as nucleases. Which is of two types exonuclease and endonuclease
- Exonuclease remove nucleotide from the end of the DNA and endonuclease make specific cut within the DNA.
- Once restriction enzyme finds 8 specific recognisation sequence it will bind to the DNA and cut each of the two strands of the double helix at specific points in their sugar phosphate backbone.
- Each restriction endonuclease contain a specific palindromic nucleotide sequence in the DNA.
- Restriction enzyme cut the strand of DNA a little away from the centre of the palindrome sides
- Restriction endonuclease are used in genetic engineering to form recombinant molecule of DNA which is composed of DNA from different source.
- Cut by the same restriction enzyme the resultant DNA fragment have the same kind of sticky and these can be joined together by using DNA ligase.
separation and isolation of DNA fragments:
- Cutting DNA by restriction endonuclease result in the fragment of DNA.
- This fragment can be separated by a technique known as gel electrophoresis.
- As we know DNA fragments are negatively charged molecule that can be separated by forcing them to move toward the an odd under an electric field through a medium or Matrix.
- The separated DNA fragment can be seen only after sending the DNA with compound known as ethidium bromide followed by exposure to UV radiation.
- The separated band of DNA are cut from the cell also extracted from gel piece known as elution.
- This is DNA fragment purified in this way which is used in constructing recombinant DNA by joining them with cloning vector.
The following steps are required for facilate cloning into vector.
Origin of replication: In this sequence from where replication stars also this sequence responsible for controlling the copy number of the link DNA so that if one wants to recover many copies of the target DNA it should be cloned into a vector whose origins support high copy number.
- In the addition of the vector requires a selectable marker which gonna help in identifying and eliminating non transformants and selectively permitted the growth of the transformants.
- Transformation is basically a process through which a piece of DNA is introduced in the host bacterium.
- In order to link the alien DNA the vector need to have very few preferable single recognisation sides for the commonly used restriction enzyme.
- Ligation of alien DNA is carried out a restriction site present in one of the two antibiotic resistance genes.
Vectors for cloning genes in plants and animal:
- The art of delivering genes by pathogen in their eukaryotic host has generated knowledge to transform these tools of pathogen into useful vector for delivering genes of interest to human.
- By micro injection process or jhingan process we can also introduce Allen DNA into host cell.
Process of recombinant DNA technology:
- It involves several steps:
Isolation of genetic material material
- Genes are located on long molecule of DNA intervened with the protein such as histones. The RNA can be removed by treatment with ribonuclease square protein can be removed by treatment with protease.
- Other molecule can be removed by appropriate treatment and purified DNA ultimately precipitate out after the addition of chilled ethanol.
Cutting of DNA at specific locations:
- After having the cutoff source DNA as well as the vector DNA with the specific restriction enzyme the cutout gene of interest from the source DNA and the cut vector with space are mixed and like this is added. As result in the preparation of recombinant DNA.
Amplification of gene of interest using PCR:
- PCR stands for polymerase chain reaction so in this reaction multiple copies of the gene synthesized in vitro using two sets of primer and the enzyme DNA polymerase.
- The enzyme extend the primer using the nucleotide provide in the reaction and the genomic DNA as template.
Insertion of recombinant DNA into the host cell
- Recipient cell after making them competent to receive take-up. DNA present in its surrounding so that if a recombinant DNA bearing gene for resistance to an antibiotic is transformed into equal cells the host cell become transform into ampicillin resistant cell.
Obtaining the foreign gene product
- After clone the gene of interest and having optimise the condition to induce the expression of the target protein one has to consider producing it on a large scale.
- If any protein and coding gene is expressed in a heterologous host it is known as recombinant protein.
- When we insert a piece of alien DNA into cloning vector and transfer it into a bacterial plant or animal cell then the alien DNA get multiplied.
- In almost all recombinant technology the ultimate aim is to produce a desirable protein.•small volume culture can not yield appreciable quantities of product to produced in a large quantity the development of bioreactors square large volume of culture can we proceed was required.
- A street tank reactor is usually cylindrical or without purpose to facilitate the mixing of the reactor contacts.
- The stealer facilitates even mixing and oxygen availability throughout the bioreactor.
- Bioreactor has an educator system and oxygen delivery system and, a form control system a temperature, control system pH, control system and sampling ports so that small volume of the culture can be withdrawal periodically.
- After the biosynthetic stage the product has to be subjected through a series a process before it is ready for marketing as a finished product.
- The process includes separation and purification which are collectively referred to as downstream processing.
- The product has to be formulated with suitable preservatives such formulation has to undergo through clinical trials as in case of drugs.