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Unit - V, Chapter - 11, Biotechnology: Principles and Processes

    The processes of preparing curd, wine and sura (a drink of gods) are not new to us. The fine idly and dosa in our kitchen and baking of bread and cake are also wonderful works of microorganisms releasing CO2 during fermentation. In the past these products were produced even without the knowledge of biotechnology. But today we have evolved in such a way that the tools and processes behind these activities are discovered and employed in large scale in more scientific and technical manner. Needless to say "Biotechnology" is as old as human evolution. However it is understood and applied at molecular level now. Now let us peep into few interesting aspects of this so called old-new science. 

      According to European Federation of Biotechnology (EFB) "Biotechnology is a science which utilizes properties of cells, cell constituents or microorganisms at industrial level for generating useful products essential to human welfare." The two techniques that gave rise to biotechnology are genetic engineering and tissue culture. 
Some of the important discoveries that led to development of this science are:
         1. Herbert Boyer: Discovered restriction enzymes that cut DNA strands in a particular fashion.
         2. Stanley Cohen: Developed the method of removing and reinserting plasmids into cells.
         3. Stanley Cohen and Herbert Boyer constructed the first rDNA using a plasmid from Salmonella typhimurium.
Tools of rDNA technology:
                   Tools of recombinant DNA technology:
                   1) Enzymes 
                   2) Vectors 
                   3) Competent hosts

1. Enzymes: Enzymes are the real tools of rDNA technology without which this science would not have evolved. Three kinds of enzymes are necessary for rDNA technology. They are Restriction enzymes endonucleases and exonucleases, polymerases and ligases.
Restriction enzymes:
     Restriction enzymes are often referred as molecular scissors. Hind-II was the first restriction endonuclease discovered. It cuts the DNA at specific sites with palindromes having 6 base pairs. Nearly 900 restriction enzymes are isolated from 230 strains of bacteria. The restriction endonucleases are the enzymes that cut the DNA at specific recognition sequences called palindromic sequences. A palindrome is a sequence of nitrogen bases which read the same on the two strands in opposite directions.
e.g.: 5’----GAATTC-----3’ (→)
        3’----CTTAAG----- 5’ (←)
e.g.: EcoRI is a restriction endonuclease enzyme obtained from Escherichia coli bacterium. It cuts the DNA between G and A recognizing a palindrome with 6 nucleotides (GAATTC).

The restriction enzymes cut the two strands of DNA at different location (staggered cut) to produce sticky ends or cohesive ends. DNA ligase can act only when DNA has sticky ends. The exonucleases help to remove nucleotides from the ends of DNA.
      Restriction enzymes are named by using the first letter from the genus and second two letters from the species of the organism from which they were isolated. For e.g.: EcoRI enzyme is derived from Escherichia coli RY13. ‘E’ stands for Escherichia and ‘co’ stands for coli, ‘R’ is the name of the strain and roman number ‘I’ indicate the order in which the enzymes were isolated.
Polymerases:
        Polymerases like DNA polymerase and RNA polymerase help to synthesize DNA and RNA strands from a template. Taq polymerase is a thermostable enzyme used for amplification of rDNA in PCR.
Ligases:
          Ligase enzymes help in joining the sticky ends or cohesive ends of cut genes of interest and the vectors.

2. Vectors 
         The DNA used as a carrier for transferring a segment of foreign DNA into a host is called vector. Vectors used for multiplying the foreign DNA sequences are called cloning vectors.
e.g.: Plasmids, bacteriophages, cosmids. T1 plasmid from Agrobacterium tumifaciens is the best vector used in plants. Retroviruses (disarmed) are used in animal cells. Plasmids are extra chromosomal circular DNA molecules found in bacteria. As they are easy to isolate and reintroduce into host cells they are considered to be most suitable as vectors. pBR 322 and pUC 19, 101 are the popularly used artificial plasmids. 
A cloning vector should have the following properties:
         Ori (origin of replication) with high copy number. 
         Should have selectable markers
e.g.: antibiotic resistance markers. Genes encoding resistance to antibiotics like ampicillin, chloramphenicol, tetracycline etc., 

        One or few recognition sites for cloning.
        Low molecular weight.

                              Plasmid

3. Competent hosts:
        DNA cannot pass through the cell walls and membranes easily as it is hydrophilic.

       The host cell can take the plasmid (rDNA) only when it is made competent.
       The host cells are made competent by treating with divalent cation like calcium which allows the DNA to pass through the pores in the cell walls.
Processes of Recombinant DNA technology
            Recombinant DNA technology involves a number of processes like Isolation of DNA, Fragmentation and isolation of DNA by restriction endonucleases, Ligation of DNA with vector and its multiplication, Insertion of rDNA into the host, Selection of transformed host cells, Culturing the host cells in a medium and extraction of the desired product.
1. Isolation of DNA
          To obtain pure form of DNA the cells should be treated with various substances. The cell wall is digested with lysozymes (bacteria), cellulase (Plant), chitinase (fungi) etc., The cell membranes are digested by using powered detergents (detergent lysis). Ribonucleases are used to remove RNA and proteases to remove proteins. Other molecules are removed by suitable treatments. Finally purified DNA is obtained by precipitating it with chilled ethanol. The fine threads of DNA can be separated by spooling.

2. Fragmentation of DNA by restriction endonucleases and its isolation: 
           The purified DNA is incubated with a suitable restriction endonuclease enzyme at optimal conditions. The enzyme cuts the DNA at regular sites (digestion). Agarose gel electrophoresis is used to separate the DNA fragments. The separated DNA fragments are visualized by staining with ethidium bromide and exposing to UV radiation. Then the separate bands of DNA are cut (elution). The process is repeated with vector DNA using the same restriction enzyme.
3. Ligation of DNA with vector and its amplification:
           The cut gene of interest and the cut vector which have sticky ends are mixed with ligase enzyme. Ligation of gene of interest with cut vector results in formation of a recombinant DNA or chimaeric DNA. To obtain multiple copies of gene of interest PCR (Polymerase Chain Reaction) is employed. Taq polymerase from Thermus aquaticus bacterium is used in PCR.
4. Insertion of rDNA into the host cell or organism:
           Recombinant DNA is inserted into host cells through the following methods:
          Ice Incubation and heat shock (42º) in bacterial cells. Micro injection is used in animal cells. Gene gun method or biolistic method using gold or tungsten in plant cells. Disarmed pathogen vectors are also used for this purpose.

5. Selection of transformed host cells:
          The transformed host cells may be selected by using selectable markers, insertional activation or colony hybridization using probes.
6. Culturing the host cells in a medium and extraction of the desired product:
          The host cells are grown in a culture medium on a small scale in the laboratory. The cells are then multiplied in a continuous culture system in which the used medium is removed and fresh medium is added continuously. This culture produces large biomass of the host cells from which the desired protein is extracted. To convert the raw materials into specific products biologically bioreactors (stirring type) are used. After separation and purification of the product (downstream processing) the product is ready for marketing. Clinical trials or quality control tests are performed before releasing the product for use.
Additional reading:
1. Agarose gel electrophoresis:

          Agarose gel electrophoresis is used to check the progression of restriction enzyme digestion (cutting DNA with restriction enzymes). When DNA is treated with restriction enzymes the fragments are formed in different sizes.

    
The DNA fragments are negatively charged and move towards anode under electric field through a medium. Hence agarose (a natural polymer extracted from sea weeds) is used as a matrix. Due to the sieving effect of agarose gel the DNA fragments move towards the anode with differential speeds depending on the size of the fragments. The smaller fragments move faster and larger ones move slowly.

The separated DNA fragments are visualized by staining with ethidium bromide and exposing to UV light. The separated bands which appear bright orange coloured are cut from the agarose gel and extracted. This extraction of DNA from the agarose gel is called elution. Pure DNA fragments are obtained and used for constructing rDNA.
2. Polymerase Chain Reaction (PCR):
           Polymerase Chain Reaction is used to synthesize multiple copies of gene of interest (DNA). It uses two sets of primers and enzyme DNA polymerase. Amplification of DNA is done by using a thermostable DNA polymerase enzyme called Taq polymerase isolated from a bacterium Thermus aquaticus. This enzyme can withstand high temperature during denaturation. This process completes in 4 steps:

               1) Denaturation of DNA to separate the two strands.
               2) Primer annealing 
               3) Extension of primers 
               4) Amplification
3. Bio reactor 
             A bio-reactor is a large vessel which is used for biological conversion of raw material into specific products like enzymes, hormones antibiotics etc., It is used to produce recombinant proteins in large scale. It provides optimal conditions of temperature, pH, substrate, salts, vitamins, oxygen etc., to achieve the desired product. Continuous culture system is used where the medium is drained out from one side and fresh medium is added on the other site to maintain the cells in active physiological stage. A stirring type bio reactor is a vessel which is cylindrical and has a curved base to facilitate mixing of contents. It has an agitator system, an oxygen delivery system, a foam control system, a temperature control system, pH control system and sampling ports. A stirrer facilitates even mixing and oxygen supply.

Posted Date : 03-11-2020

గమనిక : ప్రతిభ.ఈనాడు.నెట్‌లో కనిపించే వ్యాపార ప్రకటనలు వివిధ దేశాల్లోని వ్యాపారులు, సంస్థల నుంచి వస్తాయి. మరి కొన్ని ప్రకటనలు పాఠకుల అభిరుచి మేరకు కృత్రిమ మేధస్సు సాంకేతికత సాయంతో ప్రదర్శితమవుతుంటాయి. ఆ ప్రకటనల్లోని ఉత్పత్తులను లేదా సేవలను పాఠకులు స్వయంగా విచారించుకొని, జాగ్రత్తగా పరిశీలించి కొనుక్కోవాలి లేదా వినియోగించుకోవాలి. వాటి నాణ్యత లేదా లోపాలతో ఈనాడు యాజమాన్యానికి ఎలాంటి సంబంధం లేదు. ఈ విషయంలో ఉత్తర ప్రత్యుత్తరాలకు, ఈ-మెయిల్స్ కి, ఇంకా ఇతర రూపాల్లో సమాచార మార్పిడికి తావు లేదు. ఫిర్యాదులు స్వీకరించడం కుదరదు. పాఠకులు గమనించి, సహకరించాలని మనవి.

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