CHAPTER 9 - BIOTECHNOLOGY : PRINCIPLES AND PROCESSES

 

Biotechnology

  • Biotechnology deals with techniques of using live organisms or enzymes from organisms to produce products and processes useful to humans.
  • The European Federation of Biotechnology (EFB) has given a definition of biotechnology that encompasses both traditional view and modern molecular biotechnology. The definition given by EFB is as follows:
    • "The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services".

Principles of Biotechnology

  • There are two core techniques that enabled modern biotechnology. They are as below:
    • (i) 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.
    • (ii) Bioprocess engineering -
      • Maintenance of sterile (microbial contamination-free) ambience in chemical engineering processes to enable growth of only the desired microbe/eukaryotic cell in large quantities for the manufacture of biotechnological products like antibiotics, vaccines, enzymes, etc.

Basic Steps in Biotechnology

  • Here are three basic steps in biotechnology -
    • (i) identification of DNA with desirable genes;
    • (ii) introduction of the identified DNA into the host;
    • (iii) maintenance of introduced DNA in the host and transfer of the DNA to its progeny.

Tools of recombinant DNA technology

  • Genetic engineering or recombinant DNA technology have the key tools, i.e.,
    • restriction enzymes
    • polymerase enzymes
    • ligases
    • vectors
    • the host organism

Restriction enzymes

  • The enzymes which are used for cutting of DNA at specific locations are called restriction enzymes.
  • They restrict the growth of bacteriophage in Escherichia coli.
  • Restriction enzymes belong to a larger class of enzymes called nucleases.
  • These are of two kinds;
    • Exonucleases - This remove nucleotides from the ends of the DNA.
    • Endonucleases - This make cuts at specific positions within the DNA.

Separation and isolation of DNA fragments

  • The cutting of DNA by restriction endonucleases results in the fragments of DNA.
  • These fragments can be separated by a technique known as gel electrophoresis.
  • Since DNA fragments are negatively charged molecules they can be separated by forcing them to move towards the anode under an electric field through a medium/matrix.
    • The most commonly used matrix is agarose which is a natural polymer extracted from sea weeds.
  • The DNA fragments separate (resolve) according to their size through sieving effect provided by the agarose gel. Hence, the smaller the fragment size, the farther it moves.
  • The separated DNA fragments can be visualised only after staining the DNA with a compound known as ethidium bromide followed by exposure to UV radiation.
  • Bright orange coloured bands of DNA are seen in a ethidium bromide stained gel exposed to UV light.
  • The separated bands of DNA are cut out from the agarose gel and extracted from the gel piece.
    • This step is known as elution.
  • The DNA fragments purified in this way are used in constructing recombinant DNA by joining them with cloning vectors.

Cloning Vectors

  • Making multiple identical copies vecor plasmid is termed as cloning vectors.
  • If we are able to link an alien piece of DNA with bacteriophage or plasmid DNA, we can multiply its numbers equal to the copy number of the plasmid or bacteriophage.
  • Vectors used at present, are engineered in such a way that they help easy linking of foreign DNA and selection of recombinants from non-recombinants.
  • The following are the features that are 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 cells.
      • This sequence is also responsible for controlling the copy number of the linked DNA.
      • So, if one wants to recover many copies of the target DNA it should be cloned in a vector whose origin support high copy number.
    • Selectable marker -
      • In addition to ‘ori’, the vector requires a selectable marker, which helps in identifying and eliminating non-transformants and selectively permitting the growth of the transformants.
      • Transformation is a procedure through which a piece of DNA is introduced in a host bacterium.
      • Normally, the genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, tetracycline or kanamycin, etc., are considered useful selectable markers for E. coli.
      • The normal E. coli cells do not carry resistance against any of these antibiotics.
    • Cloning sites -
      • In order to link the alien DNA, the vector needs to have very few, preferably single, recognition sites for the commonly used restriction enzymes.
      • Presence of more than one recognition sites within the vector will generate several fragments, which will complicate the gene cloning.
      • The ligation of alien DNA is carried out at a restriction site present in one of the two antibiotic resistance genes.
      • e.g.
        • Ligate a foreign DNA at the BamH I site of tetracycline resistance gene in the vector pBR322.
        • The recombinant plasmids will lose tetracycline resistance due to insertion of foreign DNA but can still be selected out from non-recombinant ones by plating the transformants on tetracycline containing medium.
        • The transformants growing on ampicillin containing medium are then transferred on a medium containing tetracycline.
        • The recombinants will grow in ampicillin containing medium but not on that containing tetracycline.
        • But, non- recombinants will grow on the medium containing both the antibiotics. In this case, one antibiotic resistance gene helps in selecting the transformants, whereas the other antibiotic resistance gene gets ‘inactivated due to insertion’ of alien DNA, and helps in selection of recombinants.
      • Selection of recombinants due to inactivation of antibiotics is a cumbersome procedure because it requires simultaneous plating on two plates having different antibiotics.
      • Therefore, alternative selectable markers have been developed which differentiate recombinants from non-recombinants on the basis of their ability to produce colour in the presence of a chromogenic substrate.
        • In this, a recombinant DNA is inserted within the coding sequence of an enzyme, β-galactosidase.
        • This results into inactivation of the gene for synthesis of this enzyme, which is referred to as insertional inactivation.
        • The presence of a chromogenic substrate gives blue coloured colonies if the plasmid in the bacteria does not have an insert.
        • Presence of insert results into insertional inactivation of the β-galactosidase gene and the colonies do not produce any colour, these are identified as recombinant colonies.
    • Vectors for cloning genes in plants and animals -
      • Agrobacterium tumifaciens, a pathogen of several dicot plants is able to deliver a piece of DNA known as ‘T-DNA’ to transform normal plant cells into a tumor and direct these tumor cells to produce the chemicals required by the pathogen.
        • The tumor inducing (Ti) plasmid of Agrobacterium tumifaciens has now been modified into a cloning vector which is no more pathogenic to the plants but is still able to use the mechanisms to deliver genes of our interest into a variety of plants.
      • Similarly, retroviruses in animals have the ability to transform normal cells into cancerous cells.
        • Retroviruses have also been disarmed and are now used to deliver desirable genes into animal cells.
      • So, once a gene or a DNA fragment has been ligated into a suitable vector it is transferred into a bacterial, plant or animal host (where it multiplies).

Competent Host (For Transformation with Recombinant DNA)

  • Since DNA is a hydrophilic molecule, it cannot pass through cell membranes.
  • In order to force bacteria to take up the plasmid, the bacterial cells must first be made ‘competent’ to take up DNA.
  • This is done by following methods -
    • Treatment with a divalent cation -
      • This is done by treating them with a specific concentration of a divalent cation, such as calcium, which increases the efficiency with which DNA enters the bacterium through pores in its cell wall. Recombinant DNA can then be forced into such cells by incubating the cells with recombinant DNA on ice, followed by placing them briefly at 420C (heat shock), and then putting them back on ice. This enables the bacteria to take up the recombinant DNA.
    • Micro-injection -
      • Other way to introduce alien DNA into host cells is known as micro-injection, recombinant DNA is directly injected into the nucleus of an animal cell.
    • Biolistics or gene gun -
      • In another method, suitable for plants, cells are bombarded with high velocity micro-particles of gold or tungsten coated with DNA in a method known as biolistics or gene gun.
    • Transformation
      • And the last method uses ‘disarmed pathogen’ vectors, which when allowed to infect the cell, transfer the recombinant DNA into the host.

Processes Of Recombinant DNA Technology

  • Recombinant DNA technology involves following steps in specific sequence such as
    • isolation of DNA
    • fragmentation of DNA by restriction endonucleases
    • isolation of a desired DNA fragment
    • ligation of the DNA fragment into a vector
    • transferring the recombinant DNA into the host
    • culturing the host cells in a medium at large scale and
    • extraction of the desired product

Isolation of the Genetic Material (DNA)

  • In order to cut the DNA with restriction enzymes, it needs to be in pure form, free from other macro-molecules.
  • Since the DNA is enclosed within the membranes, we have to break the cell open to release DNA along with other macromolecules such as RNA, proteins, polysaccharides and also lipids.
  • This can be achieved by treating the bacterial cells/plant or animal tissue with enzymes such as lysozyme (bacteria), cellulase (plant cells), chitinase (fungus).
  • Genes are located on long molecules of DNA interwined with proteins such as histones.
  • The RNA can be removed by treatment with ribonuclease whereas proteins can be removed by treatment with protease.
  • Other molecules can be removed by appropriate treatments and purified DNA ultimately precipitates out after the addition of chilled ethanol.
  • This can be seen as collection of fine threads in the suspension.

Cutting of DNA at Specific Locations

  • Restriction enzyme digestions are performed by incubating purified DNA molecules with the restriction enzyme, at the optimal conditions for that specific enzyme.
  • Agarose gel electrophoresis is employed to check the progression of a restriction enzyme digestion.
  • DNA is a negatively charged molecule, hence it moves towards the positive electrode (anode).
  • The process is repeated with the vector DNA also.
  • The joining of DNA involves several processes.
  • After having cut the source DNA as well as the vector DNA with a specific restriction enzyme, the cut out ‘gene of interest’ from the source DNA and the cut vector with space are mixed and ligase is added.
  • This results in the preparation of recombinant DNA.

Amplification of Gene of Interest using PCR

  • PCR stands for Polymerase Chain Reaction.
  • In this reaction, multiple copies of the gene (or DNA) of interest is synthesised in vitro using two sets of primers (small chemically synthesised oligonucleotides that are complementary to the regions of DNA) and the enzyme DNA polymerase.
  • The enzyme extends the primers using the nucleotides provided in the reaction and the genomic DNA as template.
  • If the process of replication of DNA is repeated many times, the segment of DNA can be amplified to approximately billion times, i.e., 1 billion copies are made.
  • Such repeated amplification is achieved by the use of a thermostable DNA polymerase (isolated from a bacterium, Thermus aquaticus), which remain active during the high temperature induced denaturation of double stranded DNA.
  • The amplified fragment if desired can now be used to ligate with a vector for further cloning.

Insertion of Recombinant DNA into the Host Cell/Organism

  • There are several methods of introducing the ligated DNA into recipient cells.
  • Recipient cells after making them ‘competent’ to receive, take up DNA present in its surrounding.
  • So, if a recombinant DNA bearing gene for resistance to an antibiotic (e.g., ampicillin) is transferred into E. coli cells, the host cells become transformed into ampicillin-resistant cells.
  • If we spread the transformed cells on agar plates containing ampicillin, only transformants will grow, untransformed recipient cells will die.
  • Since, due to ampicillin resistance gene, one is able to select a transformed cell in the presence of ampicillin.
  • The ampicillin resistance gene in this case is called a selectable marker.

Obtaining the Foreign Gene Product

  • When you insert a piece of alien DNA into a cloning vector and transfer it into a bacterial, plant or animal cell, the alien DNA gets multiplied.
  • In almost all recombinant technologies, the ultimate aim is to produce a desirable protein.
  • Hence, there is a need for the recombinant DNA to be expressed.
  • The foreign gene gets expressed under appropriate conditions.
  • The expression of foreign genes in host cells involve understanding many technical details.
  • After having cloned the gene of interest and having optimised the conditions to induce the expression of the target protein, one has to consider producing it on a large scale.
  • If any protein encoding gene is expressed in a heterologous host, it is called a recombinant protein.
  • The cells harbouring cloned genes of interest may be grown on a small scale in the laboratory.
  • The cultures may be used for extracting the desired protein and then purifying it by using different separation techniques.
  • The cells can also be multiplied in a continuous culture system wherein the used medium is drained out from one side while fresh medium is added from the other to maintain the cells in their physiologically most active log/exponential phase.
  • This type of culturing method produces a larger biomass leading to higher yields of desired protein.
  • Small volume cultures cannot yield appreciable quantities of products.
  • To produce in large quantities, the development of bioreactors, where large volumes (100-1000 litres) of culture can be processed, was required.
  • Thus, bioreactors can be thought of as vessels in which raw materials are biologically converted into specific products, individual enzymes, etc., using microbial plant, animal or human cells.
  • A bioreactor provides the optimal conditions for achieving the desired product by providing optimum growth conditions (temperature, pH, substrate, salts, vitamins, oxygen).
  • The most commonly used bioreactors are of stirring type
  • A stirred-tank reactor is usually cylindrical or with a curved base to facilitate the mixing of the reactor contents.
  • The stirrer facilitates even mixing and oxygen availability throughout the bioreactor.
  • Alternatively air can be bubbled through the reactor.
  • The bioreactor has an agitator system, an oxygen delivery system and a foam control system, a temperature control system, pH control system and sampling ports so that small volumes of the culture can be withdrawn periodically.

Downstream Processing

  • After completion of the biosynthetic stage, the product has to be subjected through a series of processes before it is ready for marketing as a finished product.
  • The processes include 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 thorough clinical trials as in case of drugs.
  • Strict quality control testing for each product is also required.
  • The downstream processing and quality control testing vary from product to product.

One Mark Questions

Q1. Who firstly constructed an artificial recombinant DNA molecule?

Ans. Stanley Cohen and Herbert Boyer firstly constructed an artificial recombinant DNA molecule in 1972 by isolating the antibiotic resistance gene from a plasmid.

Q2. What are the other name of restriction enzymes?

Ans. Molecular scissors

Q3. What was the first restriction endonuclease?

Ans. The first restriction endonuclease–Hind II

Q4. What are palindrome?

Ans. The palindrome in DNA is a sequence of base pairs that reads same on the two strands when orientation of reading is kept the same.

Q5. What is cloning?

Ans. Cloning is making multiple identical copies of any template DNA.

CHAPTER 8 – MICROBES IN HUMAN WELFARE

CHAPTER 10 – BIOTECHNOLOGY AND ITS APPLICATIONS

Comments

Post a Comment

Popular posts from this blog

CHAPTER 6 - EVOLUTION

  Evolutionary Biology is the biology in which we study about history of life forms on earth. Various theory has been proposed about origin of life forms. Theories about origin of life forms Various theories has been given in support how first life appear on earth. They are as below - Some scientists believe that it came from outside. Early Greek thinkers thought units of life called spores were transferred to different planets including earth. ‘Panspermia’ is still a favourite idea for some astronomers. For a long time it was also believed that life came out of decaying and rotting matter like straw, mud, etc. This was the theory of spontaneous generation . Louis Pasteur by careful experimentation demonstrated that life comes only from pre-existing life. He showed that in pre-sterilised flasks, life did not come from killed yeast while in another flask open to air, new living organisms arose from ‘killed yeast’. Oparin of Russia and Haldane of England pro...
Read more

CHAPTER 7 - HUMAN HEALTH AND DISEASE

  Health Health is defined as a state of complete physical, mental and social well-being. Factors Affecting Health Health is affected by – Genetic disorders – deficiencies with which a child is born and deficiencies/defects which the child inherits from parents from birth; Infections Life style including food and water we take, rest and exercise we give to our bodies, habits that we have or lack etc. Disease When the functioning of one or more organs or systems of the body is adversely affected, this state is called disease. Types of disease - Infectious Non-infectious Pathogens A wide range of organisms belonging to bacteria, viruses, fungi, protozoans, helminths, etc., causing diseases in human are called pathogens . Sometimes, pathogens are carried on/in other's organisms body, these pathogens transmitting agents/organisms are called vectors . Common Disease in Humans Typhoid Salmonella typhi is a pa...
Read more

Rest work

   Population Interactions For any species, the minimal requirement is one more species on which it can feed. Even a plant species, which makes its own food, cannot survive alone; it needs soil microbes to break down the organic matter in soil and return the inorganic nutrients for absorption. In nature, animals, plants and microbes interact in various ways to form a biological community. Interspecific interactions arise from the interaction of populations of two different species. They could be beneficial, detrimental or neutral (neither harm nor benefit) to one of the species or both. Assigning a ‘+’ sign for beneficial interaction, ‘-’ sign for detrimental and 0 for neutral interaction, look at all the possible outcomes of interspecific interactions. Species A Species B Name of Interaction + + Mutualism - - Competition + - Predation + - Parasitism + 0 Commensalism - 0 Amensalism Both the species benefit in mutualism and both lose in competition in their interactions with ea...
Read more