BIOTECHNOLOGY: PRINCIPLE AND PROCESSES

The technique of using life organisms or enzymes from organisms so as to produce products and processes that are useful to humans is called biotechnology. According to European Federation of biotechnology (EFB), Biotechnology is the integration of natural science and organisms, cells, parts thereof, and molecular analogs for products and services.

Principles of Biotechnology

Biotechnology has two main techniques:

Genetic Engineering: Genetic engineering is described as the alteration of an organism's genome (DNA and RNA). It entails the introduction of additional genes into host species in order to improve their function or characteristic, hence altering the host organism's phenotype.

Bioprocess engineering: Sterility is maintained in chemical engineering processes to allow only desirable bacteria to thrive for the production of biotechnological goods such as antibiotics, vaccines, enzymes, and so on.

The technique of genetic engineering includes:

o Creation of recombinant DNA

o Gene cloning

o Gene transfer

The fate of the piece of DNA transferred to alien organism:

the DNA transferred into the alien organism may not be able to multiply itself in the progeny cells of the organism but when it gets integrated into the genome of the recipient it may multiply and be inherited along with the host DNA This is because the alien piece of DNA has now become a part of a chromosome which has the ability to replicate in a chromosome.

There is a specific DNA sequence called the ‘origin of replication’ that is responsible for initiating the replication, so for the multiplication of any alien piece of DNA in an organism, it needs to be a part of a chromosome that has a specific sequence known as ‘origin of replication’.

So alien DNA is linked with the ‘origin of replication’ so that this alien piece of DNA can replicate and multiply itself in the host organism, it is also called as cloning.

Construction of an artificial rDNA molecule:

• The potential of connecting an antibiotic resistance gene with a native Plasmid of Salmonella typhimurium led to the creation of the first recombinant DNA.

• Stanley Cohen and Herbert Boyer extracted the antibiotic resistance gene by removing a fragment of DNA from a Salmonella typhimurium plasmid (autonomously reproducing circular extra-chromosomal DNA). The discovery of molecular scissors'–restriction enzymes– made it feasible to cut DNA at specified spots.

• The cut piece of DNA was linked with the plasmid DNA with the help of DNA ligase that acts on the cut ends of the DNA molecules and then joins their ends, this gives rise to rDNA.

• When this DNA is transferred into E.coli, it has the potential to replicate numerous times utilising the new host DNA polymerase enzyme. Cloning of antibiotic resistance gene in E.coli refers to the capacity to multiply copies of an antibiotic resistance gene in E.coli.

• These “recombinant DNA” (rDNA) molecules are then introduced into host cells, where they can be propagated and multiplied.

Tools of Recombinant DNA technology

The major tools that are used in rDNA technology are:

• Enzymes: the major enzymes used in rDNA technology are:

• Molecular scissors:

these are the restriction enzymes, that belong to the class Nucleases. They are of two types:

(i) Endonucleases: they remove nucleotides from somewhere within the DNA, it is very helpful in producing specific cuts in the DNA. The specific base sequence is of six base pairs.

Palindromes: Palindromes are group of letters that form the same words when read both forward and backward. E.g. “MALYALAM”.

(ii) Exonucleases: they remove nucleotides from the ends of the DNA. E.g. Hind II. Each restriction endonuclease is unique to a palindromic nucleotide sequence in the DNA. Today, we know more than 900 restriction enzymes that have been isolated from over 230 strains of bacteria each of which recognise different recognition sites.

Each restriction endonuclease functions by ‘inspecting’ the length of a DNA sequence. Once it finds its specific recognition sequence, it will bind to the DNA and cut each of the two strands of the double helix at specific points in their sugar -phosphate backbones. Restriction enzymes cut the strand of DNA a bit away from the palindrome site's centre between the identical two bases on opposing strands with sticky strand. The strands' stickiness facilitates the operation of the enzyme DNA ligase. Restriction endonucleases are utilised in genetic engineering to create recombinant DNA molecules made up of DNA from diverse sources or genomes. When the same restriction enzyme is used to cut the DNA, the resulting fragments contain the same type of Sticky ends, the stickiness of the ends facilitates the action of the enzyme DNA ligase.

Agarose gel Electrophoresis:

✓ the cutting of DNA by restriction endonucleases results in the fragments of DNA fragments can be separated by a technique known as gel electrophoresis.

✓ DNA is negatively charged and hence it can be separated by making them move towards the anode under an electric field through a matrix.

✓ the commonly used Matrix in DNA gel electrophoresis is agarose that is a natural polymer extracted from seaweeds.

✓ DNA fragments resolve according to the size through the sieving effect provided by the agarose gel. ✓ the DNA fragments that are separated can be seen after staining the DNA with ethidium Bromide and later by exposing it to ultraviolet radiation.

✓ the separated DNA fragments can be seen as orange colour bands that can be cut out from the gel and purified from the gel, this process is called DNA elution.

✓ the DNA fragments are used in constructing the Recombinant DNA by attaching them with cloning vectors.

o Polymerases:

These enzymes catalyse the synthesis of DNA molecules from nucleoside diphosphate and are essential for DNA replication, they usually work in groups to create two identical DNA duplexes from a single original DNA duplex, during this process DNA polymerase reads the existing DNA strands to create two news strands that match the existing one.

o Ligases:

these enzymes catalyse the joining of two large molecules of DNA by forming a chemical bond.

• Vectors:

Plasmids and Bacteriophages are two typical vectors for cloning. They have the ability to multiply within bacterial cells independently of chromosomal DNA regulation. Bacteriophages have very high copy numbers of their genome within bacterial cells due to their large frequency per cell. For example, 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. 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.

Similarly, retroviruses in animals have the ability to transform normal cells into cancerous cells. They have also been disarmed and are now used to deliver desirable genes into animal cells.

The main features a vector should have for gene cloning are:

(i) Origin of replication (ori): When any fragment of DNA is joined to this sequence, it may be made to reproduce within the host cells. This segment is in charge of regulating the copy number of the connected DNA.

(ii) Selectable marker: this assists in recognising and removing non-transformants while selectively allowing the development of transformants. Transformation is the process of inserting a fragment of DNA into a host bacteria. In general, genes encoding antibiotic resistance, such as ampicillin, chloramphenicol, tetracycline, or kanamycin, are thought to be helpful selection markers for E. coli.

(iii) Cloning sites: the vector must have a single recognition site for the generally used restriction enzymes for joining the foreign DNA, as multiple recognition sites inside the vector may yield several fragments, that may make the gene cloning process tricky. The foreign DNA is ligated at a restriction point found in one of the two antibiotic resistance genes.

e.g. E.coli cloning vector pBR322 shown in the diagram.

Insertional Inactivation:

the cloned DNA fragment disrupts the coding sequence of a gene, this is called insertional inactivation, the method is very important in screening recombinants. e.g. Blue white selection:

(i) The insertional inactivation of the lac Z gene contained on the vector is the basis for this approach. (ii) The lac Z gene encodes the beta-galactosidase enzyme, which may convert a chromogenic substrate into a blue product.

(iii) If this lac Z gene is inactivated by inserting a target DNA fragment into it, the formation of blue colonies is stopped, and white colonies result.

(iv) This allows us to distinguish between recombinant (white) and non-recombinant (blue) colonies.

• Host organisms:

these are the bacterial cells that take up the recombinant DNA, as DNA is hydrophilic, it cannot pass through the cell membrane of bacteria, thus the bacterial cells have to be made competent to take up the DNA. This is done as follows:

(a) A simple chemical treatment with divalent calcium ions boosts the efficacy of host cells to take up the rDNA plasmids (through cell wall pores).

(b) rDNA may also be turned into host cells by incubating both on ice, then temporarily placing them at 42 degree C (Heat Shock), and then returning to ice. This allows the bacteria to consume the recombinant DNA.

(d) Biolistics / Gene gun approach, which has been created to transfer rDNA into plant cells primarily by the use of a Gene / Particle gun. In this procedure, minute gold/tungsten particles are coated with the desired DNA and battered onto cells.

(e) The last technique employs "Disarmed Pathogen" Vectors (Agrobacterium tumefaciens), which, once infected, transport the recombinant DNA into the host.

✓ Transforming the recombinant DNA into the host

✓ Culturing the host cells in a medium at large scale

✓ Extraction of the desired product:

Processes involved in Recombinant DNA Technology:

Isolation of Genetic material: Enzymes such as lysozyme, cellulase, and chitinase are used to separate genetic material from other macromolecules (fungus). Spooling can be used to remove DNA that has separated. RNA can be removed by using ribonuclease, whereas proteins may be removed by using protease.

Cutting of DNA at specific location: To access the course of a restriction enzyme digestion, restriction enzyme and Agarose gel electrophoresis are used. After cutting the source and vector DNA with a particular restriction enzyme to remove the 'gene of interest' from the source DNA.

Amplification of gene of interest with PCR: here the gene of interest is amplified and multiple copies are made with help of primers, polymerases and a set of primers.

Insertion of Foreign DNA into the host cell: there are several methods of introducing the recombinant DNA into recipient cells, the recipient cells after making them competent to receive, take up DNA present in a surrounding, thus if a recombinant DNA bearing piece for resistance to an antibiotic (ampicillin) is transferred into E.coli cells, the host cells become transformed into ampicillin-resistant cells, if we spread the transformed cells on Agar plates containing ampicillin only transformants will grow and the untransformants will die.

Note:

A thermostable DNA polymerase (isolated from a bacterium, Thermus aquaticus), which remains active during the high temperature induced denaturation of double stranded DNA. 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. 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. Thus, bioreactors can be thought of as vessels in which raw materials are biologically converted into specific products, individual enzymes, etc., using microbial plant, animal or human cells. A bioreactor provides the optimal conditions for achieving the desired product by providing optimum growth conditions (temperature, pH, substrate, salts, vitamins, oxygen).

The most common types of bioreactors used are of stirring type.

A stirred-tank reactor is usually cylindrical or with a curved base to facilitate the mixing of the reactor contents. The stirrer facilitates even mixing and oxygen availability throughout the bioreactor.

Down Stream Processing

After completion of the biosynthetic stage, the product has to be subjected through a series of processes before it is ready for marketing as a finished product. The processes include separation and purification, which are collectively referred to as downstream processing. The product has to be formulated with suitable preservatives.

BIOTECHNOLOGY: PRINCIPLE AND PROCESSES