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Introduction :

  • Biotechnology deals with the techniques of using living organisms or enzymes or enzymes from organisms to produce products useful to humans.
  • The term ‘Biotechnology’ was given by Karl Ereky (1919).
  • According to European federation of biotechnology (EFB),biotechnology is the integration of natural science and organism, cells, parts thereof, and molecular analogues for products and services.

Principles of Biotechnology 

The two core techniques that developed moderns technology are :

  1. i) Genetics 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. ii) Sterilisation method to maintain growth and manipulation of only the desired microbes or cells in large quantities, for the manufacture of biotechnology products like antibiotics, vaccines, enzymes, etc.

The basic steps in genetic engineering include :

(i)  Identification of DNA with desirable genes.

(ii) introduction of the DNA into host to form recombinant DNA (rDNA)

(iii) maintenance of DNA in host and gene cloning.

(iv) Gene transfer.

In 1972 , stanley cohen and herbert boyer constructed the first recombinant DNA.

Steps carried out in constructing first recombination DNA.

(i) A gene encoding antibiotic resistance in the native plasmid of salmonella typhi murium v. was identified, plasmid is an autonomously replicating circular extra-choroosmal DNA.

(ii) the desired DNA was cut at specific locations by restriction enzymes.

(iii) the cut DNA was linked to plasmid DNA and transferred to E.coli for gene multiplication.

Important tools of recombinant DNA technology are – 

  • Restriction enzymes-

  Restriction enzymes are called as molecular scissors because these enzymes cut DNA at specific sites.

  • Cloning vector –

   Plasmids and bacteriophages have the ability to replicate within bacterial cells independent of the control of chromosomal DNA.

  • Competent host –

The host should be competent enough to take up the foreign DNA.

  • Bioreactors –

Bioreactor is the cylindrical vessel in which biological processes is carried out on a large scale.



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 defence mechanism called the ‘restriction modification system.

The first restriction endonuclease , Hindi II.

The restriction enzymes cut DNA at specific base sequence, and these specific base sequence is known as the recognition sequence.

The convention for naming restriction enzymes –

  • The first letter of the name comes from the genus.
  • The second two letters come from the species of the prokaryotic from which they were isolated, e.g,, EcoRI comes from Escherichia coli RY 13.
  • In EcoRI, the letter ‘R’ is derived from the name of stain.
  • Roman numbers following the names indicate the order in which the enzymes were isolated from that stain of bacteria.

These are of two kinds:-

  1. Exonucleases 

  2. Endonnucleases 

  • Exonucleases remove nucleotides from the ends of the DNA whereas, endonucleases make cuts at specific positions within the DNA.
  • Each restriction endonuclease recognizes a specific palindromic nucleotide sequences in the DNA.
  • The palindrome in DNA is a sequence of base pairs that reads same on the two stands when orientation of reading is kept the same.

Example- the following sequences reads the same on the two strands in ‘5’ a ‘3’ direction, this is also true if read in the ‘3’ a ‘5’ direction.

5’_ _ GAATTC _  _ 3’

3’_ _ CTTAAG_   _ 5’

Restriction enzymes cut the strand of DNA a little away from the center of the palindrome sites, but between the same two bases on the opposite which leaves a single stranded portions at the ends and the overhanging stretches called sticky ends on each stand.

When cut by the same restriction enzyme , the resultant DNA fragments have the same kind of ‘sticky-ends’ these can be joined together using DNA.

What is Gel Electrophoresis ?

Gel electrophoresis is a technique commonly used in laboratories to separate charged molecules like DNA, RNA and proteins according to their size.

  • Firstly, the sample DNA is cut into fragments by restriction endonucleases.
  • The DNA fragments beings 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 the sea weeds.
  • The DNA fragments separates out ( revolve) according to their size because of the sieving property of agarose gel, hence, smaller the fragments size, the farther it will move.
  • The separated DNA fragments are visualized 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.

Cloning Vectors 

  • A  cloning vector is a small piece of DNA, taken from any organism into which a foreign DNA fragment can be inserted for cloning purposes.
  • Plasmids and bacteriophages have the ability to replicate within bacterial cells independent of the control of chromosomal DNA 

Vectors may be

  • a) Plasmids:- These are autonomously replicating circular extra -chromosomal DNA .
  • b) Bacteriophages:- These are viruses infecting bacteria.
  • The best known vector is the plasmid vector .
  • pBR322 is the first artificial cloning vector developed in 1977 by Bolivar and Rodriguez from E. coli plasmid.

The following are the features that are required to facilitate cloning into vector are :

  • Origin of replication (ori)
  • Selectable marker 
  • Cloning sites 

Origin Of Replication (ori):

This is the sequence

from where replication starts and any piece of DNA when linked to this sequence can be made to replicate within  the host cells 

It also controls the copy no. of the linked DNA.

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 .
  • The genes encoding resistance to antibiotics such ampicillin chloramphenicol tetracycline or kanamycin etc, re useful selectable marker for E.coli normal E coli cells do not carry  resistance against any of these antibiotics.
  • The normal E. coli cells do not carry resistance against any of these antibiotics.

Cloning sites

To link aliene 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.

Vector For Cloning Genes In Plants and Animals.

  • Vector for cloning genes in plants is Agrobacterium tumefaciens , a pathogen of server dicot plants which delivers a piece of DNA known as ‘T-DNA ‘to transform normal plant cells into a tumor and direct these tumour cells to produce the chemicals  required by the pathogen .
  • The tumor inducing (Ti) plasmid of agrobacterium tumefaciens has now been modified into a cloning vector.

Tumor formation by  agrobacterium tumefaciens

  • Vector for cloning genes in animals is retrovirus which transforms normal cells into cancerous cells.
  • Retroviruses have been disarmed and used to deliver desirable gene.

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

  1.  Inactivation of antibiotics

  • If foreign DNA lights at the BamHl  site of tetracycline resistance gene in the vector pBR322, The recombinant plasmid loses the tetracycline resistance due insertion of foregine DNA.
  • It can still be selected out from non-recombinant one by plating the transmission on ampicillin containing tetracycline.
  • The transformation growing on ampicillin medium are then transferred on to a medium containing tetracycline.
  • The recombination can grow in a ampicillin containing medium but not on the containing tetracycline where non- recombinants can grow on the medium containing  both the antibiotics and thus recombinant are selected. 
  1. 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 B- galactosidase which results into inactivation of the enzymes.  

Competent Host ( For Transformation with Recombinant DNA)

DNA is a hydrophilic molecule. cannot pass through cell membranes.

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

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

The cell is made competent by the following method:

  1. Chemical method 
  2. Physical method

Chemical method –

  • The cell is treated with specific concentration of a divalent cation such as calcium to increase pore size in cell wall.
  • The cel are incubated with recombinant DNA on ice followed by placing them briefly at 42 degree celsius and then putting its back on ice. This is called heat shock treatment.
  • The bacteria now take up the recombinant DNA. 

Physical methods –

  • The physical method include 
  • Micro-injection method : – Recombinant DNA is directly injected into the nucleus of an animal cell.
  • Biolistic gene gun method :- cell are bombarded with high velocity micro particle of gold or tungsten coted with DNA in plant.
  • Note: Disarmed pathogen vector are also used to transfer y DNA.

Process Of Recombination DNA Technology

    • Recombination DNA technology involves the following steps:

      1. Isolation of DNA.
      2. Fragmentation of DNA by restriction of endonucleases.
      3. Isolation of a desire 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 higher at a large scale.
      8. Extraction of the desired gene product.
      9. Downstream processing of the product as finished product ready for marketing

Isolation of the genetic material (DNA)

  1. RNA is removed by treatment with ribonuclease and proteins are removed by treatments with protease.
  2. After several treatments, the purified DNA is precipitated by adding chilled ethanol.
  3. The bacterial / plant / animal cell is broken down by enzymes to release DNA, along with RNA, proteins, and polysaccharides and lipids.
  4. Bacterial cell is treated with enzyme lysozyme.
  5. Plant cell is treated with enzyme cellulase.
  6. Fungal cell is treated with chitinase.

Cutting of DNA at specific locations:-

  • The DNA is using restriction enzymes.
  • The purified DNA is incubated, with the specific restriction editions optimum for the enzymes to act.

Isolation of desired DNA fragments:- 

  • Using agarose gel electrophoresis, the activity of the restriction enzymes an be checked.
  • Since the DNA is negatively charged, it moves towards the positive electrodes or anode and in the process, DNA fragments separate our based on their sizes.
  • The desired DNA fragments is eluted out.
    • Amplification of gene of interest using PCR :-

      • The polymerase chain reaction (PCR) is reaction in which amplification of specific DNA sequence is carried out in vitro.
      • This technique was developed by kary mullis in 1905 and for this he received nobel prize for chemistry in 1993.
      • Requirements od PCR

      1. DNA template : – The double stranded DNA that needs to be amplified.
      2. Primers :- small chemically synthesized oligonucleotides of about 10-18 nucleotides that are complementary to a aquaticus and vent polymerase (isolated from thermococcus litoralis).

       

    • PCR is carried out in the following three steps:

      Denaturation :-

      • The double stranded DNA is denatured by subjecting it to high temperature of 95 degree celsius for 15 seconds.
      • Each separated single stranded strand now acts as template for DNA synthesis.

        Annealing –

      • Two sets of primers are added which anneal to the ‘3’ end of each separated strand.
      • Primers act as initiators of replication.

      Extension :- 

      • DNA polymerase extends the primers by adding nucleotides complementary to the template provide 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.

      Ligation of DNA fragment into a vector 

      • The vector DNA and source DNA are 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.

 

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

 

Culturing the host cells

  • The transformed host cells are grown in appropriate nutrient medium at optimal conditions.
  • The DNA gets multiplied and expresses itself form desired product.

 

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 they expressed protein is extracted and purified by different separation techniques.
  • On large scale, the cell are grown in a continuous culture system in which fresh medium is added from one side to medium cells in exponential growth phase and desired protein is collected from other side.
  • In large scale method, larger biomass is produced which lads to high yield.

 

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.
  • Bioreactors 

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

    A bioreactor has the following components :- 

    1. An agitator system
    2. An oxygen delivery system
    3. Foam controls system
    4. pH control system
    5. Samping 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.