Biotechnology - Principles and processes

Biotechnology deals with techniques of using live organisms or enzymes from organisms to produce products and
processes useful to humans. In this sense, making curd, bread or wine, which are all microbe-mediated processes,
could also be thought as a form of biotechnology.

Definition according to The European Federation of Biotechnology (EFB):

‘The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and
services’.

Principles of Biotechnology:

Among many, the two core techniques that enabled birth of modern biotechnology are :

a. 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.
b. 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.

Let us know focus on the genetic engineering which is mainly responsible for new world biotechnology.

Genetic engineering allows the isolation and introduction of only the desired genes into the organism without
introducing the undesirable genes.

The steps involved in genetic engineering are:

1.Development of recombinant DNA (rDNA).

2.Cloning of desired gene

3.Transfer of the cloned gene into suitable host organism.

 Origin of replication (ori): A specific DNA sequence in the chromosome that can initiate DNA replication. The
foreign DNA introduced into the host genome has to be linked to the origin of replication in the host
chromosome for the gene to be able to multiply. If the foreign gene is not linked to the ori sequence it may
not be able to multiply.
 Cloning: The process of making multiple identical copies of a template DNA
 Plasmid: A circular extra-chromosomal material that is capable of autonomous replication. Plasmids are used
as vectors for cloning and expression. Foreign gene is introduced into a plasmid and the plasmid is allowed
to multiply. This causes the multiplication of the desired gene.
 Antibiotic resistance gene: The gene in certain microorganisms that bestows on them the ability to grow in
the presence of the specific antibiotic as the gene gives them resistance. These genes are present on
plasmids. These are used as indicators of cloning and transformation.
 Restriction Enzymes: They are enzymes that can cut DNA at specific fragments. They are also called as
“molecular scissors”. The sequences at which they cut the DNA are specific for the restriction enzyme. They
allow the desired gene to be cut and be introduced in specific locations in the vector or host DNA.
 Vectors: These are plasmids that are used to multiply and transfer the desired gene from one organism to
the next.
 Ligase: Enzymes that are responsible for the joining of the desired gene fragment with the host DNA. Ligases
function by getting DNA fragments to stick together.
 The basic steps in genetic modification of an organism:
▪ Identification of desired DNA fragment
▪ Introduction of desired DNA fragment into suitable host
▪ Maintaining foreign DNA in the host and its transfer to the progeny
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Tools for genetic engineering (Recombinant DNA Technology):

1. Enzymes
Restriction Endonuclease cut the DNA at specific points and scan the length of
the DNA. This specific site of cutting is called the
restriction site.
Restriction Exonuclease drive away the nucleotides from the ends of the
strands
Polymerase help to synthesize
Ligase help to join
2. Vectors
They are the carrier and integrator of desired genes into the host organism. The vectors are built with an origin
of replication. These replications start from a sequence of nucleotides.
3. Host
The desired DNA with the help of the enzymes and through vectors comes here. Biolistics, alternate cooling
and heating, use of calcium ions etc are some ways to insert the desired DNA into the host.

1. Restriction Enzymes:
 Restriction enzyme is a protein (nuclease) that recognizes a specific, short nucleotide sequence of DNA
(known as restriction site or target sequence or recognition sequence) and then cuts the DNA only at
that specific site.
 They have the capacity to recognize specific base sequences on DNA and then to cut each strand at a
given place. Hence, they are also called as ‘molecular scissors’.
 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.
 Restriction enzymes belong to a larger class of enzymes called nucleases. These are of two kinds;
exonucleases and endonucleases.
 Exonucleases remove nucleotides from the ends of the DNA whereas, endonucleases make cuts at
specific positions within the DNA.

Source of Restriction Enzymes:-

 The natural source of restriction endonucleases are bacterial cells.

 These enzymes are called restriction enzymes because they restrict infection of bacteria by certain viruses
(i.e., bacteriophages), by degrading the viral DNA without affecting the bacterial DNA. Thus, their function in
the bacterial cell is to destroy foreign DNA that might enter the cell.
 The restriction enzyme recognizes the foreign DNA and cuts it at several sites along the molecule.
 Each bacterium has its own unique restriction enzymes and each enzyme recognizes only one type of
sequence.

Recognition Sites

 The DNA sequences recognized by restriction enzymes are called palindromes. Palindromes are the base
sequences that read the same on the two strands but in opposite directions.
 For example, if the sequence on one strand is GAATTC read in 5’→3′ direction, the sequence on the opposite
strand is CTTAAG read in the 3’→5′ direction, but when both strands are read in the 5’→ 3′ direction the
sequence is the same. The palindrome appears accordingly —

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5′ GAATTC 3′

3′ CTTAAG 5′

 In addition, there is a point of symmetry within the palindrome. In the example, this point is in the centre
between the AT/AT.
 The value of restriction enzymes is that they make cuts in the DNA molecule around this point of symmetry.
 Some enzymes cut straight across the molecule at the symmetrical axis producing blunt ends.
 Of more value, however, are the restriction enzymes that cut between the same two bases away from the
point of symmetry on two strands, thus, producing a staggering break.

Nature of cleavage by restriction endonucleases:

The nature of cleavage produced by a restriction endonuclease is of considerable importance.

1. Many restriction endonucleases cleave both strands of DNA simply at the same point within the recognition
sequence. As a result of this type of cleavage, the DNA fragments with blunt ends are generated. PvuII,
Haelll, Alul are the examples of restriction endonucleases producing blunt ends. Blunt ends may also be
referred to as protruding ends.
2. In the other style of cleavage by the restriction endonucleases, the two strands of DNA are cut at two
different points. Such cuts are termed as staggered cuts and this results into the generation of protruding
ends i.e., one strand of the double helix extends a few bases beyond the other strand. Such ends are, called
cohesive or sticky ends. These can be joined together (end to end) by DNA ligase.

Each restriction endonuclease recognises a specific palindromic nucleotide sequences in the DNA.

If we place the two DNA strands together in a straight line, they appear as palindromes (mirror images)

5'-...GAATTC...-3' 3'-...CTTAAG...-5'

A palindromic sequence is a nucleic acid sequence on double-stranded DNA or RNA, wherein reading 5'-end to 3'-
end forward on one strand matches the sequence reading 3'-end to 5'-end on the complementary strand with which
it forms a double helix.

The top strand reads 5'-GAATTC-3', while the bottom strand reads 3'-CTTAAG-5'. If the DNA strand is flipped over,
the sequences are exactly the same ( 5'GAATTC-3' and 3'-CTTAAG-5').

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Nomenclature of restriction enzymes:-

 Restriction enzymes are named based on the organism in which they were discovered. For example, the
enzyme Hind III was isolated from the bacterium Haemophilus influenzae, strain Rd.
 The first three letters (Hin) of the name are italicized because they abbreviate the genus (H) and species (in)
names of the organism. The fourth letter (d) typically comes from the bacterial strain designation.
 Typically, the Roman numerals (I, II, III, etc.) indicates the order in which restriction enzymes were
discovered in a particular strain.
 Example: the name of EcoRI restriction enzyme is derived as:

Abbreviation Corresponding full form

E Escherichia (genus)
Co coli (species)
R RY13 (strain)
I First identified in the bacterium

Gel Electrophoresis:

 The DNA fragments obtained after cutting with restriction enzymes are separated by using gel
electrophoresis. 
 Electric field is applied to the electrophoresis matrix (commonly agarose gel- obtained from seaweeds) and
negatively charged DNA fragments move towards the anode. 
 Fragments separate according to their size by the sieving properties of agarose gel. Smaller the fragment,
farther it moves. 
 Staining dyes such as ethidium bromide followed by exposure to UV radiations are used to visualise the DNA
fragments. 
 DNA fragments are visible as bright orange coloured bands in the agarose matrix. 
 These bands are cut from the agarose gel and extracted from the gel piece (elution). 
 DNA fragments are purified and these purified DNA fragments are used in constructing recombinant DNAs.

Cloning vectors:

 A vector is a DNA molecule that have the ability to replicate autonomously in the host cell and into which
DNA fragment to be cloned.
 Cloning vectors are small piece of DNA which have the ability and used to introduce foreign gene of interest
into the host cell.
 They can be stably maintained insides the host cell.
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  Cloning vector are generally used to obtain multiple copies of desired foreign gene.
 Example- Plasmid, Cosmid and Phages, BACs, YACs.
 These type of vectors generally contains selectable marker, origin of replication and a restriction site.
The following are the features that are required to facilitate cloning into a vector:
(i) 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.
(ii) Selectable marker :
 In addition to ‘ori’, the vector requires a selectable marker, which helps in help to select recombinants over non-
recombinants.
 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.
(iii) Cloning sites/Restriction site:
 These are 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.

How antibiotic resistance genes help in selecting recombinants?

 For example, you can ligate a foreign DNA at the BamH I site of tetracycline(tet R) 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.

Another method to find out the transformed cells is

insertional inactivation/ Blue White Screening:
 This is based on the 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.
 Beta-Galactosidase breaks galactose into lactose.
 If a gene is inserted into this region, β-galactosidase will
not be able to convert galactose into lactose. This results
into inactivation of the enzyme, which is referred to as
insertional inactivation.
 The presence of a chromogenic substrate gives blue
coloured colonies in non-transformed cells.
 Presence of gene of insert results into insertional
inactivation of the galactosidase and the colonies do not
produce any colour, these are known as Recombinant
Colonies.

Insertional inactivation: The inactivation of an enzyme due to

the insertion of gene of interest in the region of DNA coding for
the enzyme.

Vectors for cloning in plants:

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  Agrobacterium tumefaciens, a pathogen of several dicot plants is used as a vector for plants.
 It can 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.
 Gene of interest is inserted into T-DNA to transform plant cells with required gene.
 The tumor inducing (Ti) plasmid of Agrobacterium tumefaciens has now been modified into a cloning vector
which is no more pathogenic to the plants.
 Cytokinin and auxin coding genes in plasmid acts as growth regulator. Opine catabolism gene codes for
energy source. Right and left border are needed to transfer T-DNA into the required host plant cell

Other cloning vectors:

1. pBR322 is plasmid vector (A plasmid is a naturally occurring extrachromosomal double stranded DNA,
circular DNA). P- plasmid, B- Boliver, R- Rodriguez; 322- Differentiate it from the other plasmid produced in
the same laboratory E.g. – pBR325, pBR327, etc. It is 4363 base pair long. Cloning limit: 0.1-10 kb.
2. Bacterial artificial chromosomes (BACs) are simple plasmid which is designed to clone very large DNA
fragments ranging in size from 75 to 300 kb. It is artificially synthesized plasmid- e.g. pUvBBAC
3. Yeast artificial chromosome (YAC) – has yeast telomere (TEL) at each end and centromere sequence (CEN)
allowing regulated segregation during mitosis. Cloning limit: 100-1000 kb. E.g. pYAC3(artificial chromosome
having yeast centromere isolated from Saccharomyces cerevisiae and ligated to bacterial plasmid).
4. 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 allow bacterial cells to take
up the DNA, bacterial cell should be made competent. This is done by following methods-

1. Heat shock:
 Treating the cells 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.
2. Microinjection- recombinant DNA is directly injected into the nucleus of an animal cell.
3. Biolistics/Gene Gun- suitable for plants, cells are bombarded with high velocity micro-particles of gold or
tungsten coated with DNA.
4. And the last method uses ‘disarmed pathogen’ vectors, which when allowed to infect the cell, transfer the
recombinant DNA into the host.

Steps of Genetic Recombination Technology:-

a. Isolation of Genetic Material:-

 The first step in rDNA technology is to isolate the desired DNA in its pure form i.e. free from other
macromolecules.
 Since DNA exists within the cell membrane along with other macromolecules such as RNA,
polysaccharides, proteins, and lipids, it must be separated and purified which involves enzymes such
as lysozymes, cellulase, chitinase, ribonuclease, proteases etc.
 Other macromolecules are removable with other enzymes or treatments. Ultimately, the addition of
ethanol causes the DNA to precipitate out as fine threads. This is then spooled out to give purified
DNA.
b. Restriction Enzyme Digestion:-
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  Restriction enzymes act as molecular scissors that cut DNA at specific locations. These reactions are
called ‘restriction enzyme digestions’.
 They involve the incubation of the purified DNA with the selected restriction enzyme, at conditions
optimal for that specific enzyme.
c. Amplification Using PCR
 Polymerase Chain Reaction or PCR is a method of making multiple copies of a DNA sequence using
the enzyme – DNA polymerase in vitro.
 It helps to amplify a single copy or a few copies
of DNA into thousands to millions of copies.
 Two sets of primers (chemically synthesised
oligonucleotide stretches that are
complementary to a region of DNA), enzyme
DNA polymerase,and deoxynucleotides are
added.
 PCR consists of 3 steps:
 Denaturation − Double helical DNA is
denatured by providing high
temperature 95-1100C.
 Primer annealing- The oligo
nucleotides hybridise with the single
stranded DNA sequences.
 Extension − DNA polymerase (Taq polymerase) extends the primers using the nucleotides
provided in the reaction and the genomic DNA as the template.
 DNA polymerase does not get degraded in such high temperatures since the DNA polymerase used in this
reaction is thermostable .It is isolated from the bacteria, Thermus aquaticus (Taq).
 This cycle is repeated several times to generate up to 1 billion identical copies of the DNA.
d. Ligation of DNA Molecules
 The purified DNA and the vector of interest are cut with the same restriction enzyme.
 This gives us the cut fragment of DNA and the cut vector, that is now open.
 The process of joining these two pieces together using the enzyme ‘DNA ligase’ is ‘ligation’.
 The resulting DNA molecule is a hybrid of two DNA molecules – the interest molecule and the vector.
In the terminology of genetics this intermixing of different DNA strands is called recombination.
 Hence, this new hybrid DNA molecule is also called a recombinant DNA molecule and the technology
is referred to as the recombinant DNA technology.
e. Insertion of Recombinant DNA Into Host
 In this step, the recombinant DNA is introduced into a recipient host cell mostly, a bacterial cell. This
process is ‘Transformation’.
 Bacterial cells do not accept foreign DNA easily. Therefore, they are treated to make them
‘competent’ to accept new DNA. The processes used may be thermal shock, Ca++ ion treatment,
electroporation etc.
f. Isolation of Recombinant Cells
 The transformation process generates a mixed population of transformed and non-trans- formed
host cells.
 The selection process involves filtering the transformed host cells only.
 For isolation of recombinant cell from non-recombinant cell, marker gene of plasmid vector is
employed.

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  For examples, PBR322 plasmid vector contains different marker gene (Ampicillin resistant gene and
Tetracycline resistant gene. When pst1 RE is used it knock out Ampicillin resistant gene from the
plasmid, so that the recombinant cell become sensitive to Ampicillin.

Obtaining the Foreign Gene Product:

 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. 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.
 Bioresctors:
 To produce large quantities(100-1000 lits) of recombinant protein, large vessels known as bioreactors are
used.
 A bioreactor provides the optimal conditions for achieving the desired product by providing optimum
growth conditions (temperature, pH, substrate, salts, vitamins, oxygen).
 Basic parts of a bioreactor: 1. Agitator 2. Oxygen Control system 3. Foam control system 4. Temperature
control 5. pH control 6. Sampling port 7. Inlet 8. Outlet
 Bioreactors are mainly of two types: Stirred type and the sparger type-

Stirring type bioreactor Sparger type bioreactor

A stirrer is fixed to a bioreactor having a curved base to In this air is bubbled into the bioreactor from the base of
facilitate better mixing of the contents. It also improves the bioreactor. This bubbling of air results in mixing as
aeration of the medium well as aeration of the contents

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Downstream Processing:

 Biosynthesis of many compounds such as enzymes, alcohols, and antibiotics take place within the bioreactor.
 The products so obtained are crude and require separation, purification, and finishing, which is done under
downstream processing (DSP).
 DSP makes a crude bio product marketable.
 After proper separation and purification, preservatives are added and the finished product is made to undergo
clinical trials and quality checks before being sent to market.

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