Biotechnology Principles and Processes Class 12 | Term 2 | Biotechnology

Biotechnology Principles and Processes Class 12, Biology

Introduction

Biotechnology deals with techniques of using live organisms or their enzymes for products and processes useful to humans.

Process/Techniques Included under Biotechnology

1. Microbe-mediated process(curd, wine etc.)
2. In vitro fertilization
3. Synthesis and using of a gene
4. Preparation of DNA vaccine
5. Correcting a defective gene

The European Federation of Biotechnology (EFB) defines biotechnology as the integration of natural science and organisms , cells , parts thereof , and molecular analogues for products and services.

PRINCIPLES OF BIOTECHNOLOGY

Modern biotechnology is based on two core techniques namely,

a. Genetic engineering

b. Maintenance of sterile ambience

Genetic Engineering

It is the technique in which the genetic material i.e. DNA and RNA is chemically altered and introduced into host organisms to change the phenotype .

Conceptual Development of the Principles of Genetic Engineering

(i) Genetic engineering is the technique to alter the chemistry of genetic material (DNA/RNA), to introduce these into another organisms and thus, change the phenotype of the host organism.
(ii) Adequate maintenance of sterile conditions to support growth of only the desired microbes/eukaryotic cells in large quantities for the manufacture of biotechnological products like antibiotics, vaccines, enzymes, etc.

The techniques of genetic engineering include the following:
(i) Creation of recombinant DNA by combining desired genes.
(ii) Gene transfer.
(iii) Maintenance of DNA in host and gene cloning.
The basic steps in genetic engineering can be summarised as:
(i) Identification of DNA with desirable genes.
(ii) Introduction of the identified DNA into the host.
(iii) Maintenance of introduced DNA in the host hrtd transfer of the DNA to its progeny.

Tools of Recombinant DNA Technology

The tools of recombinant DNA technology include;

1. Restriction Enzymes
2. Polymerase Enzymes
3. Ligases
4. Vector
5. Host Organisms

Restriction Enzymes ( Molecular Scissors)

• In 1963, two enzymes responsible for restricting growth of bacteriophage in E.coli were isolated.
• One of these added methyl groups to DNA while the other cut DNA.
• The first endonuclease is Hind II
• It always cuts DNA molecules at a particular point by recognizing a specific sequence of six bas pairs known as the recognition sequence for Hind II.
• Today more than 900 restriction enzymes have been isolated from over 230 strains of bacteria.

Naming of the Restriction Enzymes

• First letter indicates genus and the second two letters indicate species of the prokaryotic cell form which they were isolated.
• For example : EcoRI comes from E.coli RY13 (R=the strain while the Roman numbers indicate the order in which the enzymes were isolated from that strain of bacteria).
• Restriction enzymes belong to a class of enzymes called nucleases.

Types of Nucleases

1. Endonucleases – they cut at specific positions within the DNA.
2. Exonucleases- they nucleotides form the ends of the DNA.

Restriction Enzymes or ‘Molecular Scissors’ are used for cutting DNA.

(i) Two enzymes from E. coli that were responsible for restricting the growth of bacteriophage were isolated in 1963, one of them added methyl group to DNA and the other cut DNA into segments. The later was called restriction endonuclease.

(ii) The first restriction endonuclease Hind II was isolated by Smith Wilcox and Kelley (1968). They found that it always cut DNA molecules at a particular point by recognising a specific sequence of six base pairs known as recognition sequence.

(iii) Besides Hind II, more than 900 restriction enzymes have been isolated now, from over 230 strains of bacteria, each of which recognise different recognition sequences.

(iv) Naming of Restriction Enzymes

(a) The first letter is derived from the genus name and the next two letters from the species name of the prokaryotic cell from which enzymes are extracted.
(b) The Roman numbers after name show the order in which the enzymes were isolated from the bacterial strain.

For example : Eco RI comes from Escherichia coli RY13 and Eco RII comes from E. coli R 245, etc.
(v) Restriction enzymes belong to a class of enzymes called nucleases.

Nucleases are of two types
Exonucleases : They remove nucleotides from the ends.
Endonucleases : They cut at specific positions within the DNA.

(a) Each restriction endonuclease recognises a specific palindromic nucleotide sequences in the DNA.
(b) Palindrome in DNA is a. sequence of base pairs that reads same on the two strands when orientation of reading is kept same.

For example : the following sequences reads the same on the two strands in 5′ -> 3′ direction as well as 3′ -> 5′ direction.
5′ — GAATTC — 3′
3′ — CTTAAG — 5′
(vi) Mechanism of Action of Restriction Enzymes

Separation and Isolation of DNA Fragments

1. DNA fragments formed by restriction endonucleases can be separated by a technique called gel electrophoresis.
2. DNA fragments are negatively charged and hence can be separated by moving them towards the anode under electric field through a medium/matrix such as agarose, a natural polymer extracted from sea weeds.
3. The DNA fragments separate according to their size through sieving effect provided by the agarose gel.
4. The smaller sized fragment move farther.
5. The separated DNA fragments can be visualized after staining the DNA with ethidium bromide followed by exposure to UV radiation . Bright orange coloured DNA bands can be seen .
6. The separated DNA bands are cut out from agarose gel and extracted from gel piece. this step is called elution.
7. These purified DNA fragments are used in constructing recombinant DNA by joining them with cloning vectors.

Cloning vectors are the DNA molecules that can carry a foreign DNA segment into the host cell.

(i) The vectors used in recombinant DNA technology can be:

(a) Plasmids Autonomously replicating circular extra-chromosomal DNA.
(b) Bacteriophages Viruses infecting bacteria.
(c) Cosmids Hybrid vectors derived from plasmids which contain cos site of X phage.

(ii) Copy number can be defined as the number of copies of vectors present in a cell.
(iii) Bacteriophages have high number per cell, so their copy number is also high in genome.
(iv) Plasmids have only one or two copies per cell.
(v) Copy number can vary from 1-100 or more than 100 copies per cell.
(vi) If an alien piece of DNA is linked with bacteriophage or plasmid DNA, its number can be multiplied equal to the copy number of the plasmid or bacteriophage.

vii) Features Required to Facilitate Cloning into Vector

1. Origin of replication (Ori)
2. Selectable marker
3. Cloning sites
4. Vectors for cloning genes in plants and animals.

(a) Origin of replication (Ori) is a sequence from where replication starts.

• Any piece of DNA when linked to this sequence can be made to replicate within the host cells.
The sequence is also responsible for controlling the copy number of the linked DNA.
(ii) Selectable marker helps in identifying and eliminating non-transform ants and selectively permitting the growth of the transform ants.
• Transformation is a process through which a piece of DNA is introduced in a host bacterium.
• The genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, tetracycline or kanamycin, etc, are some useful selectable markers for E. coli.

Ligation of Alien DNA is carried out at a restriction site present in one of the twoantibiotic resistance genes. Example is ligating a foreign DNA at the Bam HI site of tetracycline resistance gene in the vector pBR322.

• The recombinant plasmids will lose tetracycline resistance due to insertion of foreign DNA. But, it still can be selected out from non-recombinant ones by plating the transform ants on ampicillin containing medium.

• The transform ants 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.

The non-recombinants will grow on the medium containing both the antibiotics.
In this example, 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, so alternative selectable markers are 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 method, a recombinant DNA is inserted within the coding sequence of an enzyme J3-galactosidase.
-» This results into inactivation of the enzyme, P-galactosidase (insertional inactivation).
-> The bacterial colonies whose plasmids do not have an insert, produce blue colour, but others do not produce any colour, when grown on a chromogenic substrate.
(c) Cloning sites are required to link the alien DNA with the vector.
• The vector requires very few or single recognition sites for the commonly used restriction enzymes.
• The presence of more than one recognition sites within the vector will generate several fragments leading to complication in gene cloning.
(d) Vectors for cloning genes in plants and animals are many which are used to clone genes in plants and animals.
• In plants, the Tumour inducing (Ti) plasmid of Agrobacterium tumefaciens is used as a cloning vector.
-» Agrobacterium tumefaciens is a pathogen of several dicot plants.
-» It delivers a piece of DNA known as T-DNA in the Ti plasmid which 1 transforms normal plant cells into tumour cells to produce chemicals
required by pathogens.
• Retrovirus, adenovirus, papillomavirus are also now used as cloning vectors in animals because of their ability to transform normal cells into cancerous cells.

Competent Host Organism (for transformation with recombinant DNA) is required because DNA being a hydrophilic molecule, cannot pass through cell membranes, Hence, the bacteria should be made competent to accept the DNA molecules,
(i) Competency is the ability of a cell to take up foreign DNA.
(ii) Methods to make a cell competent are as follow.

(a) Chemical method In this method, the cell is treated with a specific concentration of | a divalent cation such as calcium to increase pore size in cell wall.
The cells are then incubated with recombinant DNA on ice, followed by placing them briefly at 42°C and then putting it back on ice. This is called heat shock treatment.
• This enables the bacteria to take up the recombinant DNA.

(b) Physical methods In this method, a recombinant DNA is directly injected into the nucleus of an animal cell by microinjection method.
• In plants, cells are bombarded with high velocity microparticles of gold or tungsten coated with DNA called as biolistic or gene gun method.

(c) Disarmed pathogen vectors when allowed to infect the cell, transfer the recombinant
DNA into the host.

Different Methods to Introduce Alien/ Foreign DNA into Host Cells 

There are three methods to introduce alien DNA into host cells namely;

1. Micro Injection
2. Biolistics or Gene Gun
3. Disarmed Pathogen

Micro Injection-in this method the recombinant DNA is directly injected into the nucleus of an animal cell.

Biolistics (Gene Gun)- in this method, the cells are bombarded with high velocity micro- particles of gold or tungsten coated with DNA. This method is suitable for plants.

‘Disarmed Pathogen’​​ Vectors-when these vectors infect the cell, it transfers the recombinant DNA into the host.

What is Recombinant DNA technology?

Recombinant DNA technology is also known as Genetic Engineering. It is the process of joining together two DNA molecules from two different organisms. This is known as the recombinant DNA.

The steps involved in the processes of Recombinant DNA technology  are:

1. Isolation of DNA
2. DNA fragmentation using restriction endonucleases
3. Ligation of the desired DNA fragment into the vector
4. Transfer of the recombinant DNA into the host
5. Culture of the transformed cells in a nutrient medium.
6. Extraction of the desired product.

Steps in PCR technique

Step 1: Denaturation

As in DNA replication, the two strands in the DNA double helix need to be separated.

The separation happens by raising the temperature of the mixture, causing the hydrogen bonds between the complementary DNA strands to break. This process is called denaturation.

Step 2: Annealing

Primers bind to the target DNA sequences and initiate polymerisation. This can only occur once the temperature of the solution has been lowered. One primer binds to each strand.

Step 3: Extension

New strands of DNA are made using the original strands as templates. A DNA polymerase enzyme joins free DNA nucleotides together. This enzyme is often Taq polymerase, an enzyme originally isolated from a thermophilic bacteria called Thermus aquaticus. The order in which the free nucleotides are added is determined by the sequence of nucleotides in the original (template) DNA strand.

The result of one cycle of PCR is two double-stranded sequences of target DNA, each containing one newly made strand and one original strand.

The cycle is repeated many times (usually 20–30) as most processes using PCR need large quantities of DNA. It only takes 2–3 hours to get a billion or so copies.

Continuous Culture System  

A continuous culture system is maintained in a bioreactor by continuously and regularly feeding with culture medium steadily and by providing optimum growth conditions like pH, temperature, vitamins, salts, oxygen.

Bioreactors

Bioreactor is the cylindrical vessel in which biological processes is carried out on a large scale. A bioreactor provides the optimal conditions for achieving the desired product by providing optimum growth conditions such as temperature, pH, substrate, salts, vitamins, oxygen.

Stirred Tank Reactor