BIOTECHNOLOGY : PRINCIPLES AND PROCESSES
INTRODUCTION
- Biotechnology may be defined as the use of micro-organism, animals, or plant cells or their products to generate different products at industrial scale and services useful to human beings.
- Old biotechnology are based on the natural capabilities of micro organisms.
E.g., formation of citric acid, production of penicillin by Penicillium notatum.
- New biotechnology is based on recombinant DNA technology. E.g., Human gene producing insulin has been transferred and expressed in bacteria like E.coli.
- In modern biotechnology, different types of valuable products are produced with the help of microbiology, biochemistry, tissue culture, chemical engineering and genetic engineering, molecular biology and immunology.
GENETIC ENGINEERING
- Genetic engineering (also referred to as recombinant DNA technology or gene splicing) is one kind of biotechnology involving manipulation of DNA. It deals with the isolation of useful genes from a variety of sources and the formation of new combinations of DNA (recombinant DNA) for repair, improvement, perfection and matching of a genotype.
- Genetic engineering may be defined as a technique for artificial and deliberately modifying DNA (gene) to suit human needs.
- In genetic engineering, for manipulation, breakage of DNA molecule occurs at two desired places with the help of restriction endonuclease to isolate a specific DNA segment and then insert it in another DNA molecule at a desired position.
- The new DNA molecule is recombinant DNA and the technique is called genetic engineering. Genetic engineering aims at adding, removing or repairing a part of genetic material.
- Paul Bergh (Father of genetic engineering) transferred gene of SV 40 virus (simian virus) into E.coli with the help of λ - phage. (Nobel prize - 1980)
The concept of genetic engineering was the outcome of two very significant discoveries made in bacterial research. These were –
- Presence of extrachromosomal DNA fragments called plasmids in the bacterial cell, which replicate along with chromosomal DNA of the bacterium.
- Presence of enzyme restriction endonucleases which cut DNA at specific sites.
- These enzymes are, therefore, called 'molecular scissors'.
TOOLS AND TECHNIQUES OF GENETIC ENGINEERING
RESTRICTION ENZYMES
- A number of specific kinds of enzymes are employed in genetic engineering.
- Lysing enzymes : These enzymes are used for opening the cells to get DNA for genetic experiment. Bacterial cell wall is commonly dissolved with the help of lysozyme.
- Cleaving enzymes : These enzymes are of three types-exonucleases, endonucleases and restriction endonucleases.
- Exonucleases cut off nucleotides from 5' or 3' ends of DNA molecule.
- Endonucleases break DNA duplex at any point except the end.
- Restriction endonucleases cleave DNA duplex at specific points in such a way that they come to possess short single stranded free ends.
- Restriction enzyme (EcoRI) was discovered by Arber, Smith & Nathans (1978 Nobel prize). These enzymes exist in many bacteria, beside cleavage, some restriction endonuclease also have capability of modification.
Restriction enzymes are used in recombinant DNA technology because they can be used in vitro to recognize and cleave within specific DNA sequence typically consisting of 4 to 8 nucleotides. These specific 4 to 8 nucleotide sequence is called restriction site and is usually palindromic, this means that the DNA sequence is the same when read in a 5'-3' direction on both DNA strand.
As a result, the DNA fragments produced by cleavage with these enzymes have short single stranded overhang at each end, these kinds of ends are called sticky or cohesive ends because base pairing between them can stick the DNA molecule back together again.
Therefore, by cutting two different DNA samples with the same restriction enzyme and mixing the fragments together a recombinant DNA molecule can be generated.
Exceptionally, some enzymes cleave both strands of DNA at exactly the same nucleotide position, typically in the center of the recognition sequence resulting in a blunt end or flush end.
Fig. : Steps in formation of recombinant DNA by action of restriction endonuclease enzyme - EcoRI
- These fragments can be separated by a technique known as gel electrophoresis, a method that exploits the fact that these molecules carry charged groups that cause them to migrate under an electric field through a matrix.
- The most commonly used matrix is agarose, a natural polymer extracted from sea weeds.
- The separated DNA fragments can be visualized only after staining the DNA with a compound known as ethidium bromide followed by exposure to UV radiations.
- The separated bands of DNA are cut out from agarose gel and extracted from the gel piece. This step is called elution.
- Synthesizing enzymes : These enzymes are used to synthesize new strands of DNA, complementary to existing DNA or RNA template. They are of two types: reverse transcriptases and DNA polymerases.
- Reverse transcriptases help in the synthesis of complementary DNA strands on RNA templates;
- DNA polymerases help in the synthesis of complementary DNA strands on DNA templates.
- Joining enzymes : These enzymes help in joining the DNA fragments. For example, DNA ligase from Escherichia coli is used to join DNA fragments. Joining enzymes are, therefore, called molecular glues.
- Alkaline phosphatases : These enzymes cut off a phosphate group from the 5' end of linearised circular DNA and prevent its recircularization.
CLONING VECTOR
- The DNA used as a carrier for transferring a fragment of foreign DNA into a suitable host is called vehicle or vector DNA. As plasmids and bacteriophages have the ability to replicate within bacterial cells independent of the control of chromosomal DNA.
The following are the features that are required to facilitate cloning into a vector.
- Origin of replication (ori) : This is a sequence from where replication starts. The vector requires an origin of replication (ori) so that it is able to multiply within the host cells. This sequence is also responsible for controlling the copy number of the linked DNA. This implies that any foreign DNA inserted into this vector will also be replicated in the process.
Fig.: E.coli cloning vector pBR322 showing restriction sites, ori and antibiotic resistane genes
- Selectable marker : Along with 'ori', the vector requires a selectable marker. Normally, the genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, tetracycline or kanamycin, etc., are considered useful selectable markers for E. coli.
- Cloning sites: In order to link the alien DNA, the vector needs recognition sites for the commonly used restriction enzymes. The ligation of alien DNA is carried out at a restriction site present in one of the two antibiotic resistance genes. For example, you can ligate a foreign DNA at the BamHI site of tetracycline resistance gene in the vector pBR322. The recombinant plasmids will lose tetracycline resistance due to insertion of foreign DNA (insertional inactivation). Now, it can be selected out from non-recombinant ones by plating the transformants on ampicillin containing medium. The transformants (plasmid transfer) 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.
Due to inactivation of antibiotics, selection of recombinants is a troublesome procedure because it requires simultaneous plating on two plates having different antibiotics. Therefore, alternative selectable markers have been 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, a recombinant DNA is inserted within the coding sequence of an enzyme, which is referred to as insertional inactivation. The presence of a chromogenic substrate X-gal (5-bromo-4chloro- β-D galactopyranoside) gives blue coloured colonies if the plasmid in the bacteria does not have an insert. Presence of insert results into insertional inactivation of the β-galactosidase (reporter enzyme) and the colonies do not produce any colour, these are identified as recombinant colonies.
- Vectors for cloning genes in plants and animals : Agrobacterium tumefaciens, deliver a piece of DNA (known as T-DNA) to transform normal plant cells into a tumour. Similarly, retroviruses in animals have the ability to transform normal cells into cancerous cells. A better understanding of the art of delivering genes by pathogens in their eukaryotic hosts has generated knowledge to transform these tools of pathogens into useful vectors for delivering genes of interest to humans.
- Plasmids : These are extra chromosomal DNA segments found in bacteria which can replicate independently. Plasmids can be taken out of bacteria and made to combine with desired DNA segments by means of restriction enzymes and DNA ligase. A plasmid carrying the DNA of another organism integrated with it, is known as recombinant plasmid or hybrid plasmid or chimeric plasmid.
For e.g., pBR vector plasmids (named after the discoverer Bolivar and Rodriguez, pUC vector plasmid university of california)
- Viruses : The DNA of certain viruses is also suitable for use as a vehicle DNA. Bacteriophage (bacterial virus) has been used to transfer genes for β galactosidase from Escherichia coli to human cells. Lambda phage (λ phage) has been used for transferring lac genes of E. coli into haploid callus of tomato.
- NOTE
- Vector type Insert size kb
- Plasmid 0.5-8
- Bacteriophage lamda 9-23
- Cosmid 30-45
- BAC 50-300
- YAC 1000-2500
- Passenger DNA : It is the DNA which is transferred from one organism into another by combining it with the vehicle DNA. The passenger DNA can be complementary, synthetic or random.
- Complementary DNA (cDNA) : It is synthesized on mRNA template with the help of reverse transcriptase and necessary nucleotides.
- Synthetic DNA (sDNA) : It is synthesized with the help of DNA polymerase on DNA template.
- Random DNA : It refers to small fragments formed by breaking a chromosome with the help of restriction endonucleases.
Fig. : Diagrammatic representation of recombinant DNA technology
PROCESS OF RECOMBINANT DNA TECHNOLOGY
- A recombinant DNA molecule is produced by joining together of two or more DNA segments usually originating from different organisms. More specifically, a recombinant DNA molecule is a vector (e.g., a plasmid, phage or virus) into which the desired DNA fragment has been inserted to enable its cloning in an appropriate host.
- Recombinant DNA molecules are produced with one of the following three objectives :
- To obtain a large number of copies of specific DNA fragments,
- To recover large quantities of the protein produced by the concerned gene.
- To integrate the gene in question into the chromosome of a target organism where it expresses itself.
- This technique developed by genetic engineering. In this technique, first of all isolation of desired gene from any organisms and its transfer and expression into any organism of choice. They are known as transgenic microorganisms. Transgenic microorganisms are produced with a view to obtain novel pharmaceutical proteins
For example - Human insulin is being produced commercially from transgenic E.coli strain.
Many valuable recombinant proteins are also being produced using transgenic animal cells lines and transgenic plants.
At the same time, a number of these proteins of great medicinal value could not be produced on a commercial scale using the non-transgenic cells or organisms.
Proteins produced by transgenes are called recombinant proteins. Such type of recombinant genes are utilized for the formation of different products.
- Application of recombinant DNA technology
The technique of recombinant DNA can be employed in the following ways :
- It can be used to elucidate molecular events in the biological processes such as cellular differentiation and ageing. The same can be used for making gene maps with precision.
- In biochemical and pharmaceutical industry, by engineering genes, useful chemical compounds can be produced cheaply and efficiently.
- Production of transgenic plants.
- Production of genetically modified microorganisms
- Recombinant DNA technology involves several steps in specific sequence such as
- Isolation of a specific genetic material
- Cutting of DNA at specific locations
- Amplification of gene of interest using polymerase chain reaction.
- Insertion of recombinant DNA into the host cell/organisms.
- Obtaining the foreign gene product.
- Downstream processes.
ISOLATION OF A SPECIFIC GENETIC MATERIAL (DNA)
- The removal of plasmid or genomic DNA from cells is termed isolation.
- Isolation usually involves the breaking of the cell's membrane (and possibly nuclear membrane) and possibly also a cell wall (plant cells). DNA is obtained by treating the bacterial cells/plant or animal tissue with enzymes such as lysozyme (bacteria), cellulase (plant cells), chitinase (fungus). The RNA can be removed by treating with ribonuclease while proteins can be removed by treating with protease. Other molecules are removed by suitable treatments. The purified DNA ultimately precipitates out after the addition of chilled ethanol.
CUTTING OF DNA AT SPECIFIC LOCATIONS
- DNA purified from an organism can be prepared for cloning only after it has been cut into smaller molecules.
- Restriction endonucleases make possible the cleavage of DNA.
- Agarose gel electrophoresis is employed to check the progression of a restriction enzyme digestion. The process is repeated with the vector DNA also. After having cut the source DNA as well as the vector DNA with a specific restriction enzyme, the cut out 'gene of interest' from the source DNA and the cut vector with space are mixed and ligase is added. DNA ligase joins DNA to DNA. This results in the preparation of recombinant DNA.
AMPLIFICATION OF GENE OF INTEREST USING POLYMERASE CHAIN REACTION (PCR)
- The polymerase chain reaction is a repetitive bidirectional synthesis of DNA. In this reaction, multiple copies of the gene (or DNA) of interest is synthesized in vitro using two sets of primers and the enzyme DNA polymerase.
- The enzyme extends the primers using the nucleotides provided in the reaction and the genomic DNA as template.
- As amplification proceeds, the DNA sequence between the primers doubles after each cycle. The process of replication of DNA is repeated many times. The segment of DNA can be amplified to approximately a billion times. The amplified fragment can now be used to ligate with a vector for further cloning.
Fig. : Polymerase chain reaction (PCR) : Each cycle has three steps: (i) Denaturation; (ii) Primer annealing; and (iii) Extension of primers
INSERTION OF RECOMBINANT DNA INTO THE HOST CELL/ORGANISM
- The desired DNA sequence, once attached to a DNA vector, must be transferred to a suitable host. Transformation is defined as the introduction of foreign DNA into a recipient cell. Transformation of a cell with DNA from a virus is usually referred to as transfection. There are several methods of introducing the ligated DNA into recipient cells. Escherichia coli is usually the host, and transformation of E. coli is an essential step in these experiments. Several methods are available for the transfer of DNA into cells of higher eukaryotes.
OBTAINING THE FOREIGN GENE PRODUCT
- The ultimate aim of recombinant DNA technology is to produce a desirable protein. Therefore, there is a need for the recombinant DNA to be expressed. The foreign gene gets expressed under appropriate conditions. The cultures may be used for extracting the desired protein and then purifying it by using different separation techniques.
- 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 commonly used bioreactors are of stirring type.
Fig. : (a) Simple stirred-tank bioreactor; (b) Sparged stirred-tank bioreactor
- A stirred-tank reactor is usually cylindrical or with a curved base to facilitate the mixing of the reactor contents. The stirrer facilities even mixing and oxygen availability throughout the bioreactor. Alternatively, air can be bubbled through the reactor. The bioreactor has an agitator system, an oxygen delivery system and a foam control system, a temperature control system, pH control system and sampling parts so that small volumes of the culture can be withdrawn periodically.
Microorganisms can be grown in bioreactors in two ways :
- Support growth system : In this method, microorganisms are grown as a thin layer or film in the solid medium.
- Suspended growth system : By suspending cells or mycelia in the liquid medium is called suspended growth system.
- Manufacturing unit : During the designing of bioreactor for the process, often very large sized unit is used so that it accommodate huge amount of medium.
DOWNSTREAM 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.
Study Notes for NEET/AIIMS/JIPMER
