Pre-Medical NEET (UG) Coaching

Biotechnology is the process of using living organisms or enzymes from living organisms to create products and processes that are useful for humans. In this way, the production of curd, bread , or wine as well as other microbe-mediated, can be considered to be a type of biotechnology. However, it’s used in a limited sense for the purpose of referring to one processes that make use of genetically modified organisms to accomplish similar results on a bigger scale. Additionally, many other techniques and processes are also part of biotechnology. For instance in vitro fertilization leading to a baby in a test tube creating a genetic material and then using it to create DNA vaccines or correcting the defect in a gene are all elements of biotechnology. It is the European Federation of Biotechnology (EFB) has provided the definition of biotechnology which encompasses both the traditional view as well as the latest molecular biotechnology. The definition offered by EFB is as follows: ‘The fusion of biology and natural science including cells, components of them and molecular analogs for the production of products or services’.

 1. Principles of BIOTECHNOLOGY

In addition of the techniques which led to the birth of modern biotechnology are:

(i) Genetic engineering: techniques to alter the chemical properties of the genetic materials (DNA or RNA)  BIOLOGY to introduce them into host organisms to alter the phenotype for the living organism. 

(ii) bioprocess engineering Maintaining a the sterile (microbial contamination-free) atmosphere for chemical process engineering that permit the growth of just the required microbe/eukaryotic cells in large amounts to produce biotechnological substances like antibiotics enzymes, vaccines and so on. 

Let’s now look at the development of conceptual concepts that underlie genetic engineering. You may be aware of the advantages that sexual reproduction has over sexual reproduction. It is a way to allow modifications and the creation of unique genetic setups that could benefit the organism and the entire population. Sexual reproduction keeps the genetic information while sexual reproduction can allow for the possibility of variation. Traditional methods of hybridisation, employed in animal and plant breeding can result in the inclusion and multiplicity of undesirable genes, in addition to desired genes. Genetic engineering methods, that include the creating recombinant DNA, using gene cloning as well as gene transfer, eliminate this problem and allow us to create only one or a small set of desirable genes while avoiding the introduction of undesirable genes into the targeted organism. Are you aware of the probable outcome of a bit of DNA somehow transferred to an alien organism? The likelihood is that the particular piece of DNA wouldn’t be able to reproduce in the progeny cells that make up the living organism. However, once it is included in DNA of the person receiving it could multiply and become part of it. The host’s DNA. This is because the extra piece of DNA is now part of a genome, that is able to reproduce. Inside a chromosome is a particular DNA sequence, known as the origin of replication. It is responsible for triggering replication. This is why, in order to multiply of any other piece of DNA within an organism, it has to be part of the chromosome(s) that have the specific sequence called the ‘origin of replication’. So, an alien DNA is connected to the source of replication which means that the alien piece of DNA is able to replicate and multiply within the organism it is in. This is also referred to as cloning or the process of making several identical copies of any template DNA. Let’s now look at the first case of creation of an artificial recombinant DNA molecular. The first recombinant DNA came from the possibility of connecting the gene that encodes antibiotic resistance with the native DNA plasmid (autonomously replication of circular extra-chromosomal DNA) of Salmonella Typhimurium. Stanley Cohen and Herbert Boyer made this happen with the help of an antibiotic resistant gene cutting off a portion that contained DNA in a plasmid that is responsible for conferring resistance to antibiotics. Cutting DNA at specific sites was made possible by the discovery of the  “molecular scissors”which are restriction enzymes. The DNA fragment that was cut was then joined to the DNA from the plasmid. The plasmid DNA acts as vectors that transfer the DNA fragment connected to it. You are probably aware that the mosquitoes act like an insect in order to transmit malarial parasites into the the human body. Similar to that the plasmid could be utilized as a vector to introduce an element of DNA that is not native to the host. The linking of the antibiotic resistance genes to the plasmid vector was made possible thanks to the DNA ligase enzyme, that acts on DNA molecules and connects their ends. This creates a brand new mix of autonomously replicating circular DNA produced in vitro, and is called recombinantDNA. When the DNA is transferred to Escherichia Coli which is a bacterium that is closely related to Salmonella that could replicate with the hosts DNA polymerase enzyme to create numerous copies. The capacity for multiple copies to be made of the antibiotic resistance genes in E. Coli was referred to as the cloning process of the antimicrobial resistance genes in E. Coli. It is therefore possible to conclude the following three main steps to genetically altering an organism:

(i) the identification of DNA that contains desired genes

(ii) introduce the DNA that is identified into the host

(iii) maintaining the introduced DNA within your host, and then transfer to the progeny.

2. Tools of Recombinant DNA TECHNOLOGY

As we have seen from the previous review that genetic engineering, also known as the recombinant DNA technology is done only with the essential instruments, i.e., restriction enzymes, polymerase enzymes vectors, ligases, along with the host. We will try to learn about certain of them in depth.

2.1 Restriction enzymes In the year 1963, two enzymes that control the growth rate of bacteriophage Escherichia coli were identified. One of them included methyl groups in DNA. The second cut DNA. The second was known as restriction endonuclease. The first restriction endonuclease, Hind II, whose activity was dependent on a particular DNA nucleotide sequence, was discovered and further characterized five years after. It was discovered it was the case Hind II always cut DNA molecules at a certain location by recognizing a particular pattern of 6 base pair. This specific sequence is also known as the recognition sequence of Hind II. Apart from Hind II, today we have about 900 restriction enzymes which were isolated from more than 230 bacteria strains each one of which recognizes distinct recognition sequences. The standard for Naming these enzymes is that the initial letter is derived from the genus, and the two other letters are directly from prokaryotic cells that they came from, e.g., EcoRI is derived from Escherichia Coli 13 RY. In EcoRI the letter ‘R’ taken from the name of the 2022-23 , 196 BIOLOGY strain. Roman numbers after the names denote the order in which enzymes came from the type of bacteria. Restriction enzymes fall under the larger category of nucleases or enzymes. There are two types which are endonucleases as well as exonucleases. Exonucleases eliminate nucleotides from the DNA’s ends and endonucleases create cuts in specific places within DNA. Each restriction endonuclease works by “inspecting” the length of the DNA sequence. Once it locates its specific recognition sequence it will bind the DNA and break both double helix strands at specific locations in their sugar-phosphate backbones. Each restriction endonuclease is able to recognize particular palindromic sequence of nucleotides in the DNA. The steps involved in the formation of Recombinant DNA due to the enzymes that regulate restriction – EcoRI Are you aware of palindromes? These are letters that make up the same words when read forward and reverse, e.g., “MALAYALAM”. Contrary to a word-palindrome, where the identical word is read in both directions, a palindrome within DNA is an series composed of bases that read the same on both strands when the direction of  Principles and Processes 197 reading remains the same. For instance the following sequence reads exactly the same on the two strands read in a 5′ 3 direction. The same is true for sequences you read it within the third 5 direction. Five’ —- GAATTC —- 3 3′ —- CTTAAG —- 5 The restriction enzymes cut the DNA strand just a bit away from the center of palindromes but they are located between the same two bases on opposite strands. The result is single stranded sections on the edges. Overhanging stretches are referred to as sticky ends that are present on each one of the strands. These are named for the reason that they create hydrogen bonds to their cut counterparts. The sticky nature of the ends aids in the actions of the DNA ligase enzyme. Endonucleases that are restricted for genetic engineering in order to create DNA molecules that are’recombinant comprised of DNA from multiple Genomes or sources. When cut with this same enzyme the resulting DNA fragments possess the same’sticky-ends which can be joined (end-to-end) by using DNA Ligases  Diagrammatic representation of the recombinant DNA technology Recombinant Molecule (Cloning Host) 2022-23 198 BIOLOGY You might have realized that, in normal circumstances, unless you cut the vector as well as the source DNA using similar restriction enzymes the recombinant vector molecule can’t be constructed. Separation and isolating DNA fragments : Cuts in DNA through restriction endonucleases leads to pieces of DNA. The fragments can be separated using a method called gel electrophoresis. Because DNA fragments are negatively charged , it is possible to separate them by making them move toward the anode in an electric field, which is accomplished by the use of a matrix or medium. Today, the most popular matrix is agarose, which is a naturally occurring polymer that is extracted from seaweeds. DNA fragments split (resolve) in accordance with their size due to the an effect of sieving that is provided by the gel of agarose. Therefore, the smaller the fragment’s size, the further it will move. Take a look at Figure 11.3 and try to guess where on that gel sample placed. The DNA fragments that are separated are visible only after staining the DNA using the chemical ethidium Bromide and then exposure to ultraviolet radiation (you are not able to see pure DNA fragments under visible light with no staining). There are the bright orange-coloured DNA bands on an staining with ethidium bromide and exposed to ultraviolet sunlight. The DNA bands that are separated are separated of the gel, and removed from the gel. This is referred to as elimination. The DNA fragments that are purified this manner are utilized in making recombinant DNA, by joining them together with Cloning Vector.

2.2 Cloning Vectors You are aware that bacteriophages and plasmids possess the capacity to replicate inside bacterial cells, without the control of the chromosomal DNA. Bacteriophages, due to their large numbers per cell, possess extremely high copies of their genome within bacteria’s cells. Certain plasmids might have just 2 or 3 copies in a cell, whereas others could have between 15 and 100 cells with copies. The numbers could be higher. If we can connect an alien DNA fragment with plasmid or bacteriophage DNA, we could multiply its numbers to equal its copy numbers of bacteriophage or the plasmid. The vectors we use today are designed in as to facilitate the easy joining of foreign DNA and the choice of Recombinants from non-recombinants. A typical electrophoresis of agarose showing the migration of undigested (lane 1) and digested sets of DNA fragments (lane 2 to 4) 2022-23 BIOTECHNOLOGY: PRINCIPLES and PROCESSES These are the elements that are needed for cloning to an underlying vector. 

(i) the origins of replication (ori)(ori): This is the sequence from which replication begins and every piece of DNA that is linked to this sequence may be allowed to replicate inside host cell. The sequence also is responsible in regulating the copy count of DNA linked to it. Therefore, if one wishes to recover multiple copies of the DNA target, it is recommended to clone it in an origin that has a large copy numbers. 

(ii) Selectable marker: In addition to the ‘ori’ marker, the vector also requires an able marker that aids to identify and eliminate non-transformants and allows for the selective growth of transformants. Transformation is the process by where a fragment of DNA is introduced into the host bacteria (you will examine the process in a subsequent section). The genes that encode resistance to antibiotics, such as chloramphenicol, ampicillin, Kanamycin or tetracycline. These are thought to be as markers that can be selected for E. coli. Normal E. bacteria cells don’t have resistance to one of these drugs. 

(iii) Cloning site: To connect the DNA from the other the vector must contain a minimum of one, recognized sites for the most commonly utilized restriction enzymes. The presence of multiple recognition sites within the vector can result in multiple fragments that will hinder the process of gene cloning. The ligation of DNA from an alien source occurs through a restriction region that is present in either of the two resistance genes. For instance, you could connect a foreign DNA to the BamH I site of the tetracycline resistance gene found in the vector called pBR322. Recombinant plasmids be unable to resist tetracycline due to the introduction of foreign DNA however they can be distinguished from non-recombinant ones, by placing the transformants onto media containing tetracycline. The transformants that are growing on ampicillin-containing media can then be transferred to the medium that is containing Tetracycline. Recombinants can thrive in ampicillin-containing medium however not in the medium that contains the tetracycline. Non-recombinants will develop on medium that has both antibiotics. In this instance one resistance gene to antibiotics aids in selecting transformants, while the other one is resistant to antibiotics. is used to select the transformants. E. coli cloning vector pBR322 displaying the restriction sites (Hind III EcoR I, BamH I Sal I Pst I, Pvu II and Cla I) ori, and antimicrobial resistance genes (ampR and TetR ). The rop gene codes for proteins that are involved in the replication process of the plasmid. 2022-23200 BIOLOGY gene becomes inactivated by insertion’ of foreign DNA. This assists in selecting the recombinant. The selection of recombinants based on the inactivation of antibiotics is difficult due to the requirement of simultaneously plating onto two plates with different antibiotics. So, alternative selectable markers have been created that distinguish recombinants from non-recombinants due to their capacity to create hues when they are in contact with the color-producing substrate. In this case, the recombinant DNA is introduced into the codon sequence in an enzyme called the b-galactosidase. This leads to the inactivation the gene responsible for the synthesis of this enzyme. This is known as an insertional inactivation. In the presence of a chromogenic substrate results in colonies that are blue when the plasmid of the bacteria is not inserted with an insert. Inserts result in an inactivation of the insert of the b-galactosidase gene . If colonies don’t produce any colour. They are referred to as recombinant colonies. 

(iv) Vectors for cloning genes within animals and plants may be shocked to discover that we’ve learned the art of transmuting genes into animals and plants from viruses and bacteria. We have been around for years How to release genes that transform eukaryotic cells and force them into doing what the viruses or bacteria require. For instance, Agrobacterium tumifaciens, a pathogen found in several dicot plants can deliver the fragment of DNA known as ‘TDNA to turn ordinary plant cells to tumor , and then direct the tumor cells to make the chemical that the pathogen requires. Similar to retroviruses in mammals, retroviruses can transform healthy cells to cancerous ones. A greater understanding of of delivering genes through pathogens that live in eukaryotic host cells has resulted in the creation of knowledge to transform these pathogens’ tools into effective vectors to deliver the genes that are of interest to humans. The tumor-inducing (Ti) the plasmid from Agrobacterium tumifaciens has been modified to become an cloning vector that is no longer pathogenic to plants however it is still able to utilize the mechanisms to transfer genes that we are interested in to various plants. In addition, retroviruses have been weakened and can be employed to introduce beneficial genes to animal cells. Thus, once a particular DNA fragment or gene is ligated to an appropriate vector, it can be transferred into a bacterial animal or plant host (where it expands). 11.2.3 A Competent Host (For transformation using recombinant DNA) Because DNA is a hydrophilic molecule it is not able to cross the cell membranes. Why? To force bacteria to absorb the plasmid, bacterial cells have to first be made “competent” to accept DNA. This is achieved by treatment with a particular concentration of divalent cations that includes calcium, which improves the speed at the speed at which DNA is introduced into 2022-23 BIOTECHNOLOGY Processes and Principles the bacterium through the pores within the cell wall. Recombinant DNA may then be transferred into cells by incubating them with DNA recombinant on ice, then exposing them for a short time in a temperature of 420C (heat shock) after which returning them to frozen. This allows bacteria to absorb the DNA recombinant. It isn’t the only method of introducing the DNA of an alien into cells of the host. In a technique called micro-injection where recombinant DNA is introduced into the nucleus of the animal cell. Another method that is suitable for plants cells are bombarded with high-speed micro-particles of gold or tungsten that are coated with DNA, in a process called biolistics, or a gene gun. The final method employs disarmed pathogen vectors which , when allowed to infect the cells and then transmit the recombinant DNA back to the host. After we’ve learned about the techniques used to construct the recombinant DNA examine the procedures that enable the development of recombinant DNA technology

3. PROCESSES of RECOMBINANT DNA Technology

Recombinant DNA technology involves a variety of steps that are specific to a particular sequence, like the isolation of DNA, the fragmentation of DNA using restriction endonucleases, obtaining the desired DNA fragment, the ligation of the DNA fragment to an RNA vector, then transferring the recombinant DNA into a host, growing your host’s cells within a culture medium on a high-volume and then extracting what you want to produce. Let’s look at each step in greater detail. 11.3.1 Separation of the genetic Material (DNA) It is important to remember it is that DNA, the nucleic material of all living organisms and is the same for all organisms. In the majority of organisms, this is called deoxyribonucleic acid, or DNA. To cut DNA with restriction enzymes it must be pure and free of other macromolecules. Since DNA is encased in membranes, it is necessary to cut the cell in order in order to release DNA, as well as other macromolecules, such as proteins, RNA, polysaccharides and fats. This can be accomplished by treating bacterial cells or plant or animal tissues by utilizing enzymes like Lysozyme (bacteria) cellulase (plant cells) and chitinase (fungus). It is known that genes are found on long DNA molecules that are interspersed with proteins, such as histones. The RNA is removed by treatment with ribonuclease . Similarly, proteins can be eliminated by treatments with protease. The other molecules may be eliminated with appropriate treatment and the that purified DNA will eventually precipitate out following the addition of chilled alcohol. It is a fine threads that are sucked up into the suspension (Figure 11.5). The figure 11.5 DNA that splits is removed by Spooling 2022-23 BIOLOGY 11.3.2 Cutting DNA at specific locations Restriction enzyme digestions happen by incubating DNA molecules purified in the presence of the restriction enzyme at the right conditions required for the enzyme. The electrophoresis of Agarose gel is used to observe the progress of digestion by restriction enzymes. DNA is an molecule that is negatively charged and therefore it is moved toward its positive electrode (anode) (Figure 11.3). This process repeats with the DNA vector as well. The process of joining DNA involves multiple steps. After cutting the DNA source and the vector DNA using the restriction enzyme of a particular type The cut-out “gene of curiosity” of the DNA source as well as the cut vector along with space are mixed before the ligase is then added. This is the result of Recombinant DNA. 11.3.3 Amplification of the Gene of interest using PCR stands as Polymerase Chain reaction. In this procedure, several duplicates of the genetic material (or DNA) that is of interest are synthesized in vitro, using two figures 11.6 Polymerase Chain Reaction (PCR) each cycle includes 3 steps: (i) Denaturation; (ii) Primer annealing and (iii) extension of primers Biotechnology 202 2022-23: Principles and PROCESSES 203 groups of primers (small chemically synthesized oligonucleotides which are compatible with the areas of DNA) and the DNA enzyme polymerase. The enzyme expands the primers by using the nucleotides that are present during the reaction, and also DNA that is the genomic template. When the procedure of replicating DNA is repeated several times DNA, the DNA fragment could be amplified up to around trillion times i.e. one billion copies can be produced. Repeated amplification of DNA is accomplished through the use of a thermostable DNA polymerase (isolated from a bacterium Thermus aquaticus) that is active throughout the high temperature-induced denature of double-stranded DNA. The amplified DNA fragment can be used to bind with an RNA vector to further clone (Figure11.6). 11.3.4 Introduction of Recombinant DNA in the Host Cell or Organ are many ways of introducing the DNA that has been ligated into cells of the recipient. Cells that are recipients after becoming “ready” to accept, absorb DNA from the environment around. Thus, if a DNA-containing gene that confers the resistance of an antibiotic (e.g. ampicillin) is introduced in E. coli cells, the host cells transform to ampicillin resistant cells. If we place the transformed cells onto plates containing ampicillin only transformants will develop and the cells that are not transformed will cease to exist. Due to the ampicillin’s resistance gene it is possible to identify cells that have been transformed with ampicillin. This ampicillin-resistant gene is known as a marker that can be selected. 11.3.5 How to obtain The Foreign Gene Products If you insert a fragment of foreign DNA in a vector for cloning, and insert it into a plant, bacterial or animal cells, that alien DNA is multiplied. In the majority of recombinant technology The ultimate goal is to create desired proteins. Therefore, it is necessary to allow the DNA recombinant be expressed. The gene that is foreign gets expression under the appropriate conditions. Expression of genes from foreign sources within host cells involves figuring out a lot of technical terms. After having created a clone of the gene in question and adjusting the conditions that trigger it to express the targeted protein, one must think about the possibility of producing it on a massive scale. Do you know of any reason for the requirement for production on a large scale? If a protein-encoding gene has been expressed by a heterologous environment, it is known as an recombinant protein. The cells that contain gene clones of interest could grow on a smaller scale in the lab. These cultures can be used to extract the desired protein , and later making it purified using various methods of separation. The cells may be further multiplied using continuous culture systems where the media used is drained out from one end as fresh culture medium gets introduced from the other side to keep the cells biologically best condition.

A stirred tank reactor is usually cylindrical or has a curving base to aid in mixing of the contents of the reactor. Stirrers facilitate an even mix and oxygen supply within the bioreactor. Additionally, air can be pumped throughout the reactor. If you study the diagram closely, you will notice that the bioreactor includes an agitator system as well as an oxygen delivery system , foam control systems that controls temperature as well as a pH control system. There are also sampling ports, so that tiny amounts of the culture may be taken out regularly. 11.3.6 Downstream Processing After the completion of the biosynthetic process it is necessary for the product to undergo a variety of procedures before it’s available for commercialization as an end-to-end active log or exponential phase. This method of culturing results in a higher biomass, leading to greater yields of the desired proteins. Small volumes of cultures do not yield substantial quantities of protein. To make massive quantities, invention of bioreactors that can handle huge volumes (100-1000 tonnes) of culture could be processed was needed. Bioreactors can also be described as vessels where raw materials are converted into specific products, enzymes or enzymes, etc. made through microbial plant, animal and human cell. Bioreactors provide the ideal conditions to achieve the desired result by providing the ideal conditions for growth (temperature as well as pH substrate, salts, oxygen, vitamins).