Chapter 4 PRINCIPLES AND PROCESSES IN BIOTECHNOLOGY

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1 Chapter 4 PRINCIPLES AND PROCESSES IN BIOTECHNOLOGY Principles and processes in Biotechnology Biotechnology has been defined in various ways e.g., An integrated application of knowledge and techniques to draw benefits from the properties and capacities of microorganisms and animal and plant cell cultures and offers the possibilities of producing substances and compounds essential to life and the greater well being of man. Biotechnology makes it possible, through an integrated application of knowledge and techniques of biochemistry, microbiology, genetics and chemical engineering, to draw benefit at the technological level from the properties and capacities of microorganisms and cell cultures. OR The integration of natural science and organisms, cells, parts there of and molecular analogies for products and services is called biotechnology. Principles of biotechnology Origin of modern biotechnology can be traced under following headings : (i) Genetic engineering. The ability to isolate a gene coding for desired product and transfer it to another organism has opened the way either to the more effective production of useful proteins or to the introduction of novel characteristics in the host organism. Thus, this refers to methods to change the chemical structure of genetic material i.e., DNA and RNA and then to introduce in to host and alter its phenotype. (ii) To maintain the microbial conterminal free (sterile) ambience in chemical engineering. Due to such type maintenance only desired microbe / cells will be formed in huge number for manufacture of biotechnological products like hormones, vaccines, blood clotting factors, antibiotics, enzymes etc. It is necessary to have complete aseptic conditions. Recombinant DNA Technology Recombinant DNA technology includes techniques of cutting DNA into specific fragments using enzymes restriction endonucleases and joining the fragments with the help of enzyme, ligase. Using this technology, the fragment of foreign DNA can be inserted into a vector which may be plasmids or viruses. This technology bypasses the restriction in the gene transfer mechanisms between unrelated organisms. Recombinant DNA technology or genetic engineering involves the following steps : (i) DNA fragments coding for proteins of interest are synthesized chemically or isolated from an organism. (ii) These DNA fragments are inserted in a restriction endonuclease cleavage site of the vector that does not inactivate any gene required for vector s maintenance. (iii) The recombinant DNA molecules are now introduced into a host to replicate. (iv) Recipient host cells that have acquired the recombinant DNA, are selected. Selection pressure is applied to enrich bacteria with a selectable marker. (v) Desired clones are then characterized to ensure that they maintain true copies of the DNA segment that was originally cloned. Tools of Recombinant DNA Technology Key tools like restriction enzymes, polymerase enzymes, ligases, vectors and host organism are required for carrying on the recombinant DNA technology. Tools for this technique can be studied as under : 1. Restriction enzymes Streward Linn and Werner Arber (1963) isolated two enzymes which restricted the growth of bacteriophage in bacterium E. coli. One the these enzymes added methyl groups to DNA and second one cut DNA. The second enzyme was named as restriction endonuclease. (i) Exonucleases. They remove nucleotides from the ends of DNA.

2 (ii) Endonucleases. They make cuts at specific positions within DNA. Thus, a restriction enzyme (or restriction endonuclease) recognizes a specific base pair sequene in DNA called a restriction site and cleaves the DNA (Hydrolyzes the phosphodiester back bones) with in the sequence. Restriction enzymes are widely found in prokaryotes and provide protection to host cell by destroying foreign DNA that makes entry into it. Here they act as a part of defence mechanism called Restriction Modification System. It bears two components. (a) First component is a restriction enzyme that selectively identifies a specific DNA sequence and degrades any DNA bearing that sequence. (b) In second component is a modification enzyme. It adds a methyl group to one or two bases within the sequence identified by restriction enzyme. If a base in DNA is modified due to addition of methyl group, restriction enzyme cannot identify and cleave that DNA. By this method bacteria are able to protect their chromosomal DNA from cleavage by restriction enzymes. Thus, bacteria bear sets of restriction endonucleases and corresponding methylases. Endonucleases are enzymes that produce internal cuts called cleavage in DNA molecules. Endonucleases cleave DNA molecules at random sites. A class of endonucleases cleaves DNA only within or near those sites with specific base sequences called restriction endonucleases. Sites recognized by them are called recognitation sites or recognition sequences. These sites differ for different restriction enzymes. Restriction endonuclease serves as the tools for cutting DNA molecules at predetermined sites, which is basic requirement for gene cloning or recombinant DNA technology. Types of Restriction endonucleases Three main types of restriction endonucleases i.e., Type I, Type II and Type III are known with slightly different mode of action. Type II restriction enzymes are used in rdna technology because they can be used in vitro to identify and cleave within specific DNA sequences usually having 4-8 nucleotides. More than 350 different type II endonucleases with 100 different recognition sequences are known. They need Mg 2+ ions for cleavage. The first type II enzyme isolated was Hind II in The recognition sequences for Type II restriction enzymes form palindromes with rotational symmetry. In a palindrome, base sequence of second half in DNA strand represents the mirror image of the base sequence of first half. Due to this in DNA double helix, complementary strand also represents the same mirror image. 5' GAA AAG3' Single strand 5' GAA AAG3' 3' CTT TTC 5' Double stranded DNA Palindromes are groups of letters that form the same words when read both forward and backward e.g., MALAYALAM. As against a palindrome when same work is read in both the directions, palindrome in DNA is a sequence of base pairs that reads same on the two strands when orientation of reading is kept the same. 5' G AA TTC 3' 3' CTT AA G 5' However in palindromes with rotational symmetry, second half of complementary strand in DNA double helix is the mirror image of base sequence in the first half of another strand. In such cases, base sequences in both the strands of DNA helix represents the same when read from same and i.e., either 5 or 3 of both strands in DNA duplex.. Some of the restriction endonuclease enzymes are given below alongwith their sources. Eco RV (Escherichia coli), AluI (Arthrobacter luteus), BamHI (Bacillus amyloliquefacines), EcoRI (Escherichia coli), EcoRII (Escherichia coli), PstI (Providencia stuartii), SalI (Streptomyces albus), Bam HI( Bacillus amylolique faciens)

3 Nomenclature Nomenclature of restriction enzymes is usually done by following technique : Principles and Processes in Biotechnology (i) The first letter of the genus is taken in which said enzyme was discovered. This letter is written in capital. (ii) Then, first two letters of species of that organism are written. (iii) All the above three letters should be written in italics. Examples. Eco from Escherichia coli, Hin from Haemophilus inflenzae and Hpa from Haemophilus parain influenzae. (iv) This followed by strain or type identification e.g., Eco. K. (v) When the enzyme is encoded by plasmid, the name of plasmid is written e.g., Eco RI i.e., Eco RI comes from Escherichia Coli RY13. Here R is derived from the name of strain. Roman numbers following the names indicate the order in which enzymes were isolated from the strain of bacteria. (vi) If an organism forms many enzymes, they are identified by sequential Roman numerals. Example. Enzymes formed by H. influenzae strain RD have been named as Hin II, Hin III etc. Discovery of Enzyme Eco RI led to award of Nobel Prizes to W. Arber, H. Smith and D. Nathans in Types of Cleavage produced by Restriction enzymes Many restriction enzymes like Smai isolated from Serratia marcescens cleave both the strands of DNA at exactly same nucleotide position almost in centre of recognition site resulting in blunt or flush end. Smai recognizes the 6 nucleotide palindromic sequence and cleave at both the ends. 5' 5' 3' 5' CCC GGG 3' CCC GGG 3' 3' 5' 3' 5' 3' 5' GGG CCC GGG CCC Still some other restriction enzymes cleave the recognition sequence asymmetrically. Thus due to cleavage, they produce short, single stranded hanging structures. Such ends are called sticky or cohesive ends because base pairing between them can stick the DNA molecule again. A 6 nucleotide palindromic nucleotide sequence recognized by Eco RI cleave both strands at different points. Cloning Vehicles (Vectors) 5' G AA TT C 3' 5' G 3' 5' AA TT C 3' ' 3' C TT AA 5' 3' G 5' 3' C TT AA G 5 A vector is a DNA molecule which has the ability to replicate in an host cell and into which the DNA fragment to be cloned known as DNA insert is integrated for cloning. Characteristics of vector : (i) Origin of replication (Ori). It represents the sequence from where replication initiates and any fragment of DNA when integrated to sequence can be made to replicate with in host cells. This sequence also controls the copy number of linked DNA. For getting several copies of target DNA, it is desirable that cloning should be carried out in a vector where origin facilitates high copy number. It should bear origin of replication (ori) due to which it is able to multiply with in the host cell i.e., it should be able to replicate autonomously. Due to this any foreign DNA introduced into vector with also replicate during this process.

4 (ii) Selectable marker. It should incorporate a selectable marker gene. This gene permits to select those host cells which bear the vector from amongst those which do not. Selectable marker helps in eliminating non-transformants and selectively permitting the growth of the transformants. In transformation DNA is introduced into host bacterium. Examples of few selectable markers are: (a) Genes which code for antibiotic resistance e.g., ampicillin, chloramphenicol, tetracycline or terramycin. (b) Genes which encode enzymes like -galactosidease (product of lac Z gene) which can be identified by colour reaction. (iii) It should be easy to isolate and purify. Cloning vector should be relatively smaller in size. Large molecules can breakdown during purification and difficult to manipulate. (iv) Vector should definitely and bear at least one restriction endonuclease recognition site. It will allow foreign DNA to be inserted into vector during the generation of recombinant DNA molecule. Plasmids and phages are the vectors that are used for cloning purposes in prokaryotes, particularly bacteria. (a) Plasmids Plasmids are the most widely used cloning vectors in the technique of gene-manipulation in bacteria. They are circular, double-stranded DNA molecules occurring in extra chromosomal state and selfreplicating. Some plasmids may have one or two copies per cell. Plasmids may be present in greater amounts, typically about per cell. However, some multi-copy plasmids are widely distributed throughout the prokaryotes, varying in size from less than daltons, to greater than daltons, and are generally dispensable. The plasmids posses a replication control system that maintains them in the bacterium at a characteristic level. There are two general types of plasmids: single-copy plasmids and multi-copy plasmids. Single-copy plasmids are maintained at one plasmid per host genome whereas the multi-copy plasmids are under relaxed replication control which means that they accumulate in very large amounts (about 1000) per cell when the bacteria stop growing. These plasmids are often used to provide cloning vectors. Three widely studied bacterial plasmids are: Plasmid pbr322 (i) F plasmids. They are responsible for conjugation. (ii) R plasmids. They bear genes for resistance to antibiotics. (iii) Col. Plasmids. Such plasmids code for colicin, the proteins that kill sensitive E. coli cells. They bear genes which provide immunity to colicin. (b) Phages as vectors Bacteriophages are viruses that infect bacterial cells by injecting their DNA into these cells Two phages which have been extensively modified for development of cloning vectors are lambda () phages and M13 phages. Lambda () phages provide another type of useful vector system for cloning in bacteria. Usually the DNA of phage,, in the form in which it is isolated from the phage particle, is a linear doublestandard molecule of about 48.5 kb paris. DNA of wild-type phage contains several target sites for most of the commonly used restriction enzymes and so is not itself suitable as vector.

5 (c) Cosmids Principles and Processes in Biotechnology Cosmids have been constructed by combining certain features of plasmid and cos sites of phage lambda. They have been constructed to add some of advantages of phage vectors to the plasmid vectors the cos sites endeavor in vitro packaging system to the plasmid vector. The cosmid vectors, however, provide an efficient means of cloning large fragments of foreign DNA (32-48 kb of foreign DNA) much more than a phage vector can accommodate. When injected into a bacterium, the recombinant-dna of a cosmid circularizes like phage DNA but replicates as a normal plasmid without the expression of any phage functions. Cosmid vectors are particularly attractive for constructing libraries of DNA fragments of eukaryotes because of their capacity to accommodate large fragments of DNA. (d) Phagmids Phagmids are also a type of plasmid vectors containing a fragment of phage DNA including its att site. Like cosmids, they have been constructed to exploit the advantages of both-plasmid vector and phage vector. The phagmid may insert into a phage DNA in the same way by which phage DNA inserts into the bacterial chromosome during lysogenic phage of life cycle. (e) Yac Vectors YACs or Yeast artificial chromosomes are being used as vectors to clone DNA fragments of more than 2500 Mb in size. They are being highly used in mapping large genomes like Human Genome Project. (f) BAC Vectors A typical yeast artificial chromosome (YAC). BACs or Bacterial artificial chromosomes are used as vectors which are based on natural extrachromosomal plasmid of E. coli the fertility or F-plasmid. This vector bears genes for replication and maintenance F-factor, a selectable marker and cloning sites. (g) Shuttle Vectors Genetic map of a bacterial artificial chromosome (BAC) vector. They are the plasmids capable of shuttling genes between two organisms. One of the organisms is prokaryote like E.coli and other is a eukaryote like yeast. Such vectors should bear unique origins of replication for every cell type. They should have separate markers for transformed host cells harbouring the vector.

6 Competent host (for transformation with recombinant DNA) For propagation of DNA molecules host cells like E. coli, yeast and plant and animal cells are being used. Most popular and extensively used bacterium is E. coli due to following reasons : (i) E. coli a gram negative bacterium is easy to handle and grow. (ii) It can accept a variety of vectors. (iii) Under optimal conditions, bacteria double their numbers every 20 minutes. When bacteria reproduce, recombinant DNA also reproduces. Eukaryotic cells are also being used as host cells for expression of eukaryotic proteins. This leads to proper folding of polypeptide chain into exact 3- dimensional form. Simple eukaryotic organisms like yeast are being widely used being single celled, easy to grow and manipulate and genetically well characterized. A cell membrane does not permit DNA to pass through being hydrophilic in nature. Bacterial host cells are made competent to take up plasmid. For this bacteria are treated with divalent cation like calcium. This enhances the efficiency of entry of DNA into bacterium through pores on cell wall. Recombinant DNA is forced into such cells by: (a) Incubation of cells with recombinant DNA on ice. (b) It is followed by giving them a heart shock (42ºC) and again putting back on ice. Other methods to introduce alien DNA into host cells are microinjection, electroporation and gene gun methods. (a) Microinjection. DNA can also be stably introduced into tissue culture cells by its direct microinjection into the nuclei of the cells, using a glass micropipette that has been drawn out to an extremely thin diameter (from 0.1 to 0.5 microns). Such a procedure requires some fairly sophisticated equipment (a micropipette puller for making the needles, and a micromanipulator to position the needles correctly for injections); but given this equipment and enough practice, one can inject 500 to 1000 cells per hour with DNA, and have up to 50 per cent of the injected genes. It is found that injected DNA will integrate and express the injected genes. It is found that injected DNA will integrate at random into the nuclear DNA, and if an injected gene is attached to a suitable promoter it might be expressed. Microinjection of protoplasts or cells. The advantage of this procedure is that in principle any piece of DNA can be introduced into any cell; no selective pressure needs to be applied to maintain the transferred gene. This method has been used to transfer the gene for rat growth hormone into mice, in a few of which the gene was expressed, resulting in the production of gaint mice. The same procedure of introducing pieces of DNA into plant cells is also followed. The disadvantage of microinjection is the expensive equipment that is required, the extensive practice needed to master this tedious technique, and the relatively small number of cells that can be treated in one experiment. Also after microinjection, the egg must be re-implanted in a surrogate mother, and only after gestation can the progeny be screened for expression and correct regulation of the foreign DNA. This is therefore a slow, labour-intensive method of genetic manipulation, and the small size of population which can be produced for screening inevitably reduces the changes of success. (b) Electroporation This method is based on the use of short electrical impulses of high field strength. These impulses increase the permeability of protoplast membrane and facilitate entry of DNA molecules into the cells, if the DNA is in direct contact with the membrane. In view of this, for delivery of DNA to

7 protoplasts, electroporation is one of the several routine techniques for efficient transformation. However, since regeneration from protoplasts is not always possible, cultured cells or tissue explants are often used. Hence, it is important to test whether electroporation could transfer genes into walled cells. In most of these cases no proof of transformation was available. The electroporation pulse is generated by discharging a capacitor across the electrodes in a specially designed lectroporation chamber. Protoplasts in an ionic solution containing the vector DNA, are suspended between the electrodes, electroporated and then plated as usual. Transformed colonies are selected. Using electroporation method, successful transfer of genes was achieved with the protoplasts of Petunia, maize, rice wheat and sorghum. Transformation frequencies can be further improved by (i) using field strength of 1.25kV/ cm, (ii) adding PEG after adding DNA, (iii) heat shocking protoplasts at 45ºC for 5 minutes before adding DNA and (iv) by using linear instead of circular DNA. Shot-gun/gene gun method of DNA introduction In recent years, it has been shown that DNA delivery to plant cells is also possible, when heavy metallic pellets (tungsten or gold) coated with the DNA of interest are accelerated to a very high initial velocity (1,400 ft/sec.). these microprojectiles, normally 1-3 m in diameter, are carried by a macroprojectile other bullet and accelerated into living plant cells (target cells can be pollen, cultured cells, cells in differentiated tissues and meristerms) so that they can penetrate cells walls of intact tissue. The acceleration is achieved either by an explosive change (cordite explosion) or by using shock waves initiated by a high-voltage electric discharge. The design of two particle guns used for acceleration of microprojectiles are shown. Microprojectile acceleration devices The advantages of this method over microinjection include the following : (i) thousands of particles are accelerated at the same time, causing multiple hits resulting in transfer of genes into many cells simultaneously; (ii) since intact cells can be used, some of the difficulties encounter with the use of protoplasts are automatically circumvented (iii) the method is universal in its application, so that cell type, size and shape or the presence / absence of cell walls do not significantly alter its effectiveness. In view of this, particle bombardment method using microprojectiles has a great promise in a variety of plant species, particularly the cereals. DNA Ligase DNA ligase forms diester bonds between adjacent nucleotides. It is able to link two fragments of DNA by covalent bonds. The enzyme is utilized in cloning experiment is T 4 DNA ligase which is coded by phage T 4.

8 Process (Techniques) of recombinant DNA technology Methods for the cloning of specific DNA sequence involve insertion of these sequences into vector DNA which is capable of replicating within a host cell. The strategies of gene cloning in bacteria comprises four main stages viz; preparation of the gene, insertion into vector, transformation of host cell and detection of the cloned gene. 1. Isolation of the Genetic material (DNA) Following steps are required for isolation of genetic material DNA in pure form : (i) Bacterial cells or plant cells or animal cells are treated with enzymes like lysozyme for bacteria, cellulase for plant cells and chitinase for fungus. This will break the cell envelope open and macromolecules like DNA, RNA, proteins, polysaccharides and lipids will be released. (ii) As in eukaryotic cells DNA is inter wind with protein molecules like histones. Additional proteins can be removed by treating with enzyme protease. RNA can be removed by treated with enzyme ribonuclease. (iii) DNA sample can be further purified by using additional extraction techniques. (iv) By addition of chilled ethanol, DNA precipitates out and can be observed in the form of fine threads in suspension. Agarose gel electrophoresis is employed to check the progression of restriction enzyme digestion. DNA being negatively charged moves towards anode (positive electrode). Same technique is used for vector DNA. 2. Amplification of Gene of interest using PCR In PCR (Polymerase Chain Reaction) multiple copies of desired DNA (gene) can be formed in vitro. Polymerase Chain reaction (PCR) (Specific Sequences can be amplified) PCR technique was developed by Kary Mullis in If one knows the sequence of at least part of DNA segment of be cloned, a number of copies of that DNA segment can be hugely amplified using polymerase chain reaction. It is able to generate microgram (g) quantities of DNA copies (up to billion copes) of desired DNA segment, present even as a single copy with in short time. Polymerase chain reaction (PCR) The technique is based on principle that when a DNA molecule is subjected to high temperature due to denaturation the two DNA strands separate. As a result two single stranded DNA molecules

9 appear. DNA polymerase can copy these single stranded molecules. This leads to the formation of original DNA double stranded molecule. Due to repetition of this process, several copies of DNA sequences can be formed. 3. Preparation of the gene (How to cut DNA at specific locations) Gene cloning in bacteria is achieved by cleaving the purified DNA with enzyme restriction endonuclease which produces small fragments )approximately 4 kilo base pairs). Each fragment has a sticky complementary single-stranded end. Eukaryotic genes contain introns that are not processed in bacteria, therefore, DNA for cloning is usually obtained as a reverse transcriptase generated copy DNA (cdna) of the relevant mrna. In cases where nucleotide or amino acid sequences are known, synthetic DNA may be produced. The vector is an agent which is used to transfer DNA into a host cell e.g., plasmid, bacteriophage. The vector is cut with the same enzymes (restriction endonuclease) as that used to generate the chromosomal DNA fragments. The chromosomal fragments and linearised vector are incubated with DNA ligase which covalently joins the DNA molecules. Those plasmids which contain an inserted fragment are called recombinant plasmid. 4. Detection of the cloned gene (Recombinant) Sequential steps in formation of recombinant DNA Cells with recombinant DNA (rdna) are selected on the expression or non-expression of some traits like resistance to antibiotic chloramphenicol. Direct selection of recombinants is made due to encoding of these traits by vector or cloned DNA sequence. a. various methods for identification of recombinants are : Transformants (host cells with foreign DNA) can be selected by : (i) Host cells transformed with plasmid having amplicillin resistant gene are grown on medium having antibiotic ampicillin, only those cells bearing the above plasmid will be able to grow on it. (ii) But one is not able to know that which colony bear recombinant plasmid and which bear religated vector plasmid. b. Insertional inactivation method : It is based on basic principle that cloned DNA fragment disrupts the coding sequence of gene. To identify recombinants, one of the important approaches is to use DNA probe. In a DNA molecule, the two complementary standards are held together by hydrogen bonds. If two similar DNA pieces are mixed together and hydrogen bonds broken (by heating) the strands will separate. Upon lowering the temperature, the hydrogen bonds are formed again. Some of the resultant double-stranded DNA will be hybrids i.e., composed of one strand of one type and one strand of the other type. This concept of DNA hybridization has been exploited for utilizing the DNA molecules as probes. The transformed colonies are replica plated to a nitrocellulose filter and are lysed to release the DNA. This DNA is denatured (by raising the temperature and fixed to the nitrocellulose so as to produce a DNA print corresponding exactly to the position of the colonies on the original plate. The DNA print is then hybridized with the probe which has been previously radioactively

10 labeled. After washing off unhybridized DNA, the position of the radioactive spots on the filter in indicated by autoradiography in order to identify the presence of the required DNA. Obtaining the foreign gene product When alien DNA is inserted into cloning vector and then transferred to host cell, DNA gets multiplied. Due to expression of this foreign gene, proteins can be formed. Such target proteins (recombinant proteins) are to produced in large scale. The cells having cloned genes may be grown on small scale in laboratory. Such cultures are used to make proteins with required characters. For multiplication of cells continuous culture for system is used. Here medium is drained out and fresh medium is added. This helps in active growth of cells during log / exponential phase. To produce this product in large quantities bioreactors are needed. In such bioreactors litres of culture can be processed. Most commonly used in stirring type bioreactor, whose details are given below : Fermenter (bioreactor) Structure. The basic design of a Stirredtank fermenter. It consists of a large stainless steel vessel with a capacity of up to 500,000 ltr. around which there is a jacket of circulatory water used to control the temperature within the fermenter. There is also an agitator, comprising of a series of flat blades, which can be rotated with the help of a motor. This ensures the thoroughly mixing of the contents so that nutrients come in close with the micro-organisms. The agitator also prevents settling out of the cells at the bottom. Fermenter also has adequate arrangement for aeration, temperature and ph control. For proper aeration, air can be forced in at the bottom of the tank aeration, air can be forced in at the bottom of the tank through a porous ring, called sparger, by the process called sparging while there is an outlet to remove air and waste gases at the top of the tank. Basic design of a simple stirred-tank fermenter The top of the tank also a number of inlet tubes called ports, through which materials can be introduced or withdrawn e.g., Inoculation port for introducing initial inoculum; Nutrient port for introducing more nutrients; Antifoam port for introducing antifoaming agents; optimal ph. ph port for introducing acid or alkali to maintain optical ph. At the base of the tank, there is a harvest line to extract culture medium and microbial product. To regularly detect the ph and temperature changes, tank is fitted with certain probes. Downstream processing Products formed are separated and purified. Steps are collectively called as downstream processing. Suitable preservatives are used. For medicinal purposes, clinical trials are carried out. Quality control is also maintained.