BIOTECHNOLOGY : PRINCIPLES AND PROCESSES

1. Biotechnology deals with techniques of using live organisms or enzymes from organisms to
produce products and processes useful to humans.
2. In this sense, making curd, bread or wine, which are all microbe-mediated processes, could
also be thought as a form of biotechnology
3. in vitro fertilisation leading to a ‘test-tube’ baby, synthesising a gene and using it,
developing a DNA vaccine or correcting a defective gene, are all part of biotechnology
4. The European Federation of Biotechnology (EFB) has given a definition of biotechnology
that encompasses both traditional view and modern molecular biotechnology.
5. 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’.
6. 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.
7. Asexual reproduction preserves the genetic information, while sexual reproduction permits
variation.
8. The techniques of genetic engineering which include creation of recombinant DNA, use of
gene cloning and gene transfer, overcome this limitation and allows us to isolate and
introduce only one or a set of desirable genes without introducing undesirable genes into
the target organism.
9. origin of replication, which is responsible for initiating replication
10. Therefore, for the multiplication of any alien piece of DNA in an organism it needs to be a
part of a chromosome(s) which has a specific sequence known as ‘origin of replication’.
11. he construction of the first recombinant DNA emerged from the possibility of linking a
gene encoding antibiotic resistance with a native plasmid (autonomously replicating circular
extra-chromosomal DNA) of Salmonella typhimurium.
12. Stanley Cohen and Herbert Boyer accomplished this in 1972 by isolating the antibiotic
resistance gene by cutting out a piece of DNA from a plasmid which was responsible for
conferring antibiotic resistance.
13. The cutting of DNA at specific locations became possible with the discovery of the so-
called‘molecular scissors’– restriction enzymes.
14. These plasmid DNA act as vectors to transfer the piece of DNA attached to it.
15. The linking of antibiotic resistance gene with the plasmid vector became possible with the
enzyme DNA ligase, which acts on cut DNA molecules and joins their ends.
16. This makes a new combination of circular autonomously replicating DNA created in vitro and
is known as recombinant DNA.
17. The ability to multiply copies of antibiotic resistance gene in E. coli was called cloning of
antibiotic resistance gene in E. coli.
18. You can hence infer that there are three basic steps in genetically modifying an organism —
(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.
19. recombinant DNA technology can be accomplished only if we have the key tools, i.e.,
restriction enzymes, polymerase enzymes, ligases, vectors and the host organism.
20. In the year 1963, the two enzymes responsible for restricting the growth of bacteriophage
in Escherichia coli were isolated One of these added methyl groups to DNA, while the other
cut DNA. The later was called restriction endonuclease.
21. The first restriction endonuclease–Hind II, whose functioning depended on a specific DNA
nucleotide sequence was isolated and characterised five years later.
22. It was found that Hind II always cut DNA molecules at a particular point by recognising a
specific sequence of six base pairs.
23. Besides Hind II, today we know more than 900 restriction enzymes that have been isolated
from over 230 strains of bacteria each of which recognise different recognition sequences.
24. The convention for naming these enzymes is the first letter of the name comes from the
genus and the second two letters come from the species of the prokaryotic cell from which
they were isolated, e.g., EcoRI comes from Escherichia coli RY 13.
25. Roman numbers following the names indicate the order in which the enzymes were isolated
from that strain of bacteria.
26. Restriction enzymes belong to a larger class of enzymes called nucleases. These are of two
kinds; exonucleases and endonucleases.
27. Exonucleases remove nucleotides from the ends of the DNA whereas, endonucleases make
cuts at specific positions within the DNA.
28. Each restriction endonuclease recognises a specific palindromic nucleotide sequences in the
DNA.

29.
30. Restriction enzymes cut the strand of DNA a little away from the centre of the palindrome
sites, but between the same two bases on the opposite strands.
31. Restriction endonucleases are used in genetic engineering to form ‘recombinant’ molecules
of DNA, which are composed of DNA from different sources/genomes.
32. When cut by the same restriction enzyme, the resultant DNA fragments have the same kind
of ‘sticky-ends’ and, these can be joined together (end-to-end) using DNA ligases

33.
34. You may have realised that normally, unless one cuts the vector and the source DNA with
the same restriction enzyme, the recombinant vector molecule cannot be created.
35. 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.
36. 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.
37. Nowadays the most commonly used matrix is agarose which is a natural polymer extracted
from sea weeds.
38. The DNA fragments separate (resolve) according to their size through sieving effect
provided by the agarose gel
39. 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 (you cannot see
pure DNA fragments in the visible light and without staining).
40. You can see bright orange coloured bands of DNA in a ethidium bromide stained gel exposed
to UV light
41. The separated bands of DNA are cut out from the agarose gel and extracted from the gel
piece. This step is known as elution.
42. The DNA fragments purified in this way are used in constructing recombinant DNA by
joining them with cloning vectors.
43. You know that plasmids and bacteriophages have the ability to replicate within bacterial
cells independent of the control of chromosomal DNA.
44. Some plasmids may have only one or two copies per cell whereas others may have 15-100
copies per cell. Their numbers can go even higher.
45. 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.
46. Selectable marker : In addition to ‘ori’, the vector requires a selectable marker, which helps
in identifying and eliminating nontransformants and selectively permitting the growth of the
transformants.
47. Transformation is a procedure through which a piece of DNA is introduced in a host
bacterium (you will study the process in subsequent section).
48. The normal E. coli cells do not carry resistance against any of these antibiotics.
49. 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.
50. The ligation of alien DNA is carried out at a restriction site present in one of the two
antibiotic resistance genes.
51. For example, you can ligate a foreign DNA at the BamH I site of tetracycline resistance
gene in the vector pBR322.
52. 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.
53. Selection of recombinants due to inactivation of antibiotics is a cumbersome procedure
because it requires simultaneous plating on two plates having different antibiotics.
54. This results into inactivation of the enzyme, which is referred to as insertional inactivation.
55. 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.
56. retroviruses in animals have the ability to transform normal cells into cancerous cells
57. 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
58. Since DNA is a hydrophilic molecule, it cannot pass through cell membranes.
59. 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.
60. In a method known as micro-injection, recombinant DNA is directly injected into the nucleus
of an animal cell.
61. 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.
62. And the last method uses ‘disarmed pathogen’ vectors, which when allowed to infect the
cell, transfer the recombinant DNA into the host.
63. Recall that nucleic acid is the genetic material of all organisms without exception.

64.
65. 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
66. This can be achieved by treating the bacterial cells/plant or animal tissue with enzymes
such as lysozyme (bacteria), cellulase (plant cells), chitinase (fungus).
67. You know that genes are located on long molecules of DNA interwined with proteins such as
histones.
68. PCR stands for Polymerase Chain Reaction.

69.
70. The enzyme extends the primers using the nucleotides provided in the reaction and the
genomic DNA as template.
71. 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.
72. 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
73. 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.
74. In almost all recombinant technologies, the ultimate aim is to produce a desirable protein.
75. The foreign gene gets expressed under appropriate conditions.
76. 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.
77. If any protein encoding gene is expressed in a heterologous host, it is called a recombinant
protein.
78. 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.
79. 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.
80. A bioreactor provides the optimal conditions for achieving the desired product by providing
optimum growth conditions (temperature, pH, substrate, salts, vitamins, oxygen).

81.
82. The stirrer facilitates even mixing and oxygen availability throughout the bioreactor.
83. The processes include separation and purification, which are collectively referred to as
downstream processing.
84. The downstream processing and quality control testing vary from product to product.

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