Class 12 Biology : Chapter 9 : Biotechnology : Principles and Processes

 

Chapter 9

Biotechnology - Principles and Processes

Syllabus: Genetic Engineering (Recombinant DNA Technology).

 

Important terms:

A.    Vector: A vector is a DNA molecule used as a vehicle to deliver genetic material into a cell. The cell used in the process is called host cell. The purpose of using vectors is to replicate or express the inserted DNA within the host cell.

i).     In a chromosome there is a specific DNA sequence called the origin of replication, which is responsible for initiating replication.

ii).   So, the vector should have origin of replication so that it could replicate in the host body along with the desired gene.

iii).            Commonly used vectors include plasmids ( a circular DNA very small in size as compared to normal DNA and has a origin of replication), viruses (such as retroviruses and adenoviruses), and artificial chromosomes .

Why It's Still Called "Artificial" Individual parts (like centromeres, origins of replication, telomeres) may come from natural organisms like bacteria or yeast. But, in nature, these parts do not exist together in the way scientists assemble them in the lab. Scientists combine them in a new way, designing a custom DNA molecule that has never existed before in any living organism.

 

iv).            These vectors are modified to carry and deliver the desired DNA sequences into the host organism's cells, where the introduced genetic material can be expressed, replicated, or manipulated for various purposes, such as producing therapeutic proteins, studying gene function, or modifying organisms for specific traits.

 

B.     Restriction endonucleases:

i).     It is a enzyme used to cut the desired gene.

 

C.     DNA ligase: It is a enzyme used to join the fragments of DNA.

D.    DNA Polymerase enzymes: It synthesize new DNA strands from 5’ to 3’ direction.

 

E.     Gene cloning: When the host cell multiplies the foreign gene and makes multiple copies of that gene, it is called gene cloning.

Introduction

1.     Biotechnology deals with techniques of using live organisms or enzymes from organisms to produce products and processes useful to humans.

2.     In this sense, making curd, bread or wine, which are all microbe-mediated processes, could also be thought as a form of biotechnology. We can call this as a traditional biotechnology.

3.     However, it is used in a restricted sense today, to refer to such of those processes which use genetically modified organisms to achieve the same on a larger scale. We can call this modern biotechnology.

4.     Further, many other processes/techniques are also included under biotechnology.

i).     For example, in vitro fertilisation leading to a ‘test-tube’ baby, synthesising a gene and using it, developing a DNA vaccine or correcting a defective gene, are all part of biotechnology.

 

5.     The European Federation of Biotechnology (EFB) has given a definition of biotechnology that encompasses both traditional view and modern molecular biotechnology.

The definition given by EFB is as follows:

i).     ‘The integration of natural science and organisms, cells, parts thereof, and molecular analogues (DNA) for products and services’.

 

9.1 PRINCIPLES OF BIOTECHNOLOGY

1.     Among many, the two core techniques that enabled birth of modern biotechnology are :

A.                Genetic engineering : Techniques to alter the chemistry of genetic material (DNA and RNA), to introduce these into host organisms and thus change the phenotype of the host organism.

 

B.                 Bioprocess engineering: Maintenance of sterile (microbial contamination-free) ambience (atmosphere) in chemical engineering processes to enable growth of only the desired microbe/eukaryotic cell in large quantities for the manufacture of biotechnological products like antibiotics, vaccines, enzymes, etc.

 

2.     Conceptual development of the principles of genetic engineering.

i).     You probably appreciate the advantages of sexual reproduction over asexual reproduction.

ii).   The former provides opportunities for variations and formulation of unique combinations of genetic setup, some of which may be beneficial to the organism as well as the population.

iii). Asexual reproduction preserves the genetic information, while sexual reproduction permits variation.

iv). Traditional hybridisation procedures used in plant and animal breeding, very often lead to inclusion and multiplication of undesirable genes along with the desired genes.

v).   The techniques of genetic engineering which include creation of recombinant DNA, use of gene cloning and gene transfer, overcome this limitation and allows us to isolate and introduce only one or a set of desirable genes without introducing undesirable genes into the target organism.

vi). Do you know the likely fate of a piece of DNA, which is somehow transferred into an alien organism?

vii).                       Most likely, this piece of DNA would not be able to multiply itself in the progeny cells of the organism.

viii).                    But, when it gets integrated into the genome of the recipient, it may multiply and be inherited along with the host DNA.

ix). This is because the alien piece of DNA has become part of a chromosome, which has the ability to replicate.

x).   In a chromosome there is a specific DNA sequence called the origin of replication, which is responsible for initiating replication.

xi). Therefore, for the multiplication of any alien piece of DNA in an organism it needs to be a part of a chromosome(s) which has a specific sequence known as ‘origin of replication’.

xii).                       Thus, an 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.

xiii).                     This can also be called as cloning or making multiple identical copies of any template DNA.

 

3.     Process of cloning

i).     The construction of the first recombinant DNA emerged from the possibility of linking a gene encoding antibiotic resistance with a native plasmid (autonomously replicating circular extra-chromosomal DNA) of Salmonella typhimurium.

ii).   Stanley Cohen and Herbert Boyer accomplished this in 1972 by isolating the antibiotic resistance gene by cutting out a piece of DNA from a plasmid which was responsible for conferring antibiotic resistance.

iii). The cutting of DNA at specific locations became possible with the discovery of the so-called ‘molecular scissors’– restriction enzymes.

iv). The cut piece of DNA was then linked with the plasmid DNA.

v).   These plasmid DNA act as vectors to transfer the piece of DNA attached to it. You probably know that mosquito acts as an insect vector to transfer the malarial parasite into human body.

vi). In the same way, a plasmid can be used as vector to deliver an alien piece of DNA into the host organism.

vii).           The linking of antibiotic resistance gene with the plasmid vector became possible with the enzyme DNA ligase, which acts on cut DNA molecules and joins their ends.

viii).        This makes a new combination of circular autonomously replicating DNA created in vitro (In labs) and is known as recombinant DNA.

ix). When this DNA is transferred into Escherichia coli, a bacterium closely related to Salmonella, it could replicate using the new host’s DNA polymerase enzyme and make multiple copies.

x).   The ability to multiply copies of antibiotic resistance gene in E. coli was called cloning of antibiotic resistance gene in E. coli.

xi). You can hence infer that there are three basic steps in genetically modifying an organism —

a)     identification of DNA with desirable genes

b)     introduction of the identified DNA into the host by joining it with vector.

c)     maintenance of introduced DNA in the host and transfer of the DNA to its progeny (next generations).

 

9.2 TOOLS OF RECOMBINANT DNA TECHNOLOGY

1.     Now we know from the foregoing discussion that genetic engineering or recombinant DNA technology can be accomplished only if we have the key tools, i.e., restriction enzymes, DNA polymerase enzymes, ligases, vectors and the host organism. Let us try to understand some of these in detail.

 

9.2.1 Restriction Enzymes

1.     In the year 1963, the two enzymes responsible for restricting the growth of bacteriophage in Escherichia coli were isolated.

·        A bacteriophage, often referred to as a phage, is a type of virus that infects and replicates within bacteria. ·         These viruses are highly specific to bacterial hosts and can infect a broad range of bacterial species. Bacteriophages are composed of genetic material—either DNA or RNA—enclosed in a protein coat.

 

2.     One of these add methyl group to DNA (also known as methylase), while the other cut DNA. The later was called restriction endonuclease. In this way the E. coli. Protects itself but cutting the genome of bacteriophage entered in its body. The methylase enzyme adds the methyl group to its own genome in order to prevent it from the restriction endonuclease .

Restriction endonucleases (restriction enzymes) also recognize specific DNA sequences, but their function is to cleave DNA at these sequences. If the bacterial DNA is methylated at these recognition sites (modified by methylase enzymes), the restriction enzymes are unable to bind to and cleave the DNA.

 

3.     The first restriction endonuclease–Hind II, whose functioning depended on a specific DNA nucleotide sequence was isolated and characterised five years later.

i).     It was found that Hind II (Haemophilus influenza) always cut DNA molecules at a particular point by recognizing a specific sequence of six base pairs.

ii).   This specific base sequence is known as the recognition sequence for Hind II.

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

 

4.     Naming of restriction enzymes

i).     The convention for naming these enzymes is the first letter of the name comes from the genus and the second two letters come from the species of the prokaryotic cell from which they were isolated, e.g., EcoRI (here I =1) comes from Escherichia coli RY 13.

ii).   In EcoRI, the letter ‘R’ is derived from the name of strain. ‘R’ means rough colony without mucilaginous covering.

In microbiology and biology, the term "strain" refers to a specific genetic variant or subtype within a species. Like rough colony or smooth colony.

 

iii). Roman numbers following the names indicate the order in which the enzymes were isolated from that strain of bacteria.

 

5.     Restriction enzymes belong to a larger class of enzymes called nucleases.

6.     These are of two kinds;

a)     exonucleases

b)     endonucleases.

i).     Exonucleases remove nucleotides from the ends of the DNA whereas,

ii).   endonucleases make cuts at specific positions within the DNA.

 

7.     Working of restriction endonuclease

i).     Each restriction endonuclease functions by ‘inspecting’ the length of a DNA sequence.

ii).   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 (Figure 9.1).

 

iii). Each restriction endonuclease recognises a specific palindromic nucleotide sequences in the DNA.

iv). Do you know what palindromes are? These are groups of letters that form the same words when read both forward and backward, e.g., “MALAYALAM”.

v).   As against a word-palindrome where the same word is read in both directions, the palindrome in DNA is a sequence of base pairs that reads same on the two strands when orientation of reading is kept the same.

vi). For example, the following sequences reads the same on the two strands in 5' ------ 3' direction. This is also true if read in the 3' ------- 5' direction.

5' —— GAATTC —— 3'

3' —— CTTAAG —— 5'.

vii).                       Restriction enzymes cut the strand of DNA a little away from the centre of the palindrome sites, but between the same two bases on the opposite strands.

viii).                    This leaves single stranded portions at the ends. There are overhanging stretches called sticky ends on each strand .

ix). These are named so because they form hydrogen bonds with their complementary cut counterparts.

x).   This stickiness of the ends facilitates the action of the enzyme DNA ligase.

xi). Restriction endonucleases are used in genetic engineering to form ‘recombinant’ molecules of DNA, which are composed of DNA from different sources/genomes. When cut by the same restriction enzyme, the resultant DNA fragments have the same kind of ‘sticky-ends’ and, these can be joined together (end-to-end) using DNA ligases .

 

8.     Separation and isolation of DNA fragments :

i).     The cutting of DNA by restriction endonucleases results in the fragments of DNA.

ii).   These fragments can be separated by a technique known as gel electrophoresis.

iii).  Nowadays the most commonly used matrix is agarose which is a natural polymer extracted from sea weeds. It is available in the form of powder by which a gel like substance is formed.

iv). Material required for this process are as follows:-

a)     Electrophoresis tank with cathode and anode.

 

 

b)     Well comb to create wells for loading samples.

c)     Casting Dams are fixed in the tank so that the agarose solution does not flow out during setting.

d)     Ethidium bromide is added to the agarose solution formed earlier. It is intercalating agent.

An intercalating agent is a chemical compound or molecule that can insert itself between the stacked base pairs of DNA or RNA, causing the DNA or RNA helix to unwind or distort. When it is exposed to UV rays, it will fluoresce (glow) and DNA bands become visible. More DNA present, brighter the band.

e)     After the addition of ethidium bromide in the agarose solution, it is poured into the electrophoresis tank.

f)       As the gel solidifies, well comb and casting dams will be removed from the tank.

g)     Now a electrolyte is used to provide ions that carry a current.

h)     Now some chemicals and dyes (for colors) are added in the DNA samples to make their density high so that they can sink deep into gel.

i)       Now DNA samples are loaded in the wells formed.

 

j)       Now a lid is placed on the electrophoresis tank. Then electric current is applied to pull the samples through the gel.

k)     Based on their charge and size, the DNA molecules will travel through the gel at different speeds.

 

l)       Since DNA fragments are negatively charged molecules (due to phosphate groups ), they can be separated by forcing them to move towards the anode under an electric field through a medium/matrix.

m)  The DNA fragments separate (resolve) according to their size through sieving effect provided by the agarose gel.

n)     Hence, the smaller the fragment size, the farther it moves.

o)    Look at the Figure 9.3 and guess at which end of the gel the sample was loaded.

 

p)     The separated DNA fragments can be visualised only after staining the DNA with a compound known as ethidium bromide followed by exposure to UV radiation (you cannot see pure DNA fragments in the visible light and without staining).

q)     You can see bright orange coloured bands of DNA in a ethidium bromide stained gel exposed to UV light (Figure 9.3).

r)      The separated bands of DNA are cut out from the agarose gel and extracted from the gel piece. This step is known as elution.

s)      The DNA fragments purified in this way are used in constructing recombinant DNA by joining them with cloning vectors.

 

9.2.2 Cloning Vectors

1.     You know that plasmids and bacteriophages (a type of virus) have the ability to replicate within bacterial cells independent of the control of chromosomal DNA.

It means they can replicate autonomously using their own genetic machinery and processes, separate from the bacterial chromosome.

 

2.     Bacteriophages because of their high number per cell, have very high copy numbers of their genome within the bacterial cells.

3.     Some plasmids may have only one or two copies per cell whereas others may have 15-100 copies per cell. Their numbers can go even higher.

4.     If we are able to link an alien piece of DNA with bacteriophage or plasmid DNA, we can multiply its numbers equal to the copy number of the plasmid or bacteriophage.

5.     Vectors used at present, are engineered in such a way that they help easy linking of foreign DNA and selection of recombinants from non-recombinants.

Essential features of a vector:

a)     Origin of replication (ori) :

i).     This is a sequence from where replication starts and any piece of DNA when linked to this sequence can be made to replicate within the host cells.

ii).   This sequence is also responsible for controlling the copy number of the linked DNA.

iii). So, if one wants to recover many copies of the target DNA it should be cloned in a vector whose origin support high copy number.

 

b)     Selectable marker :

i).     In addition to ‘ori’, the vector requires a selectable marker, which helps in identifying and eliminating non transformants and selectively permitting the growth of the transformants.

the term "transformants" refers to the host cells that have taken up and expressed a foreign DNA fragment or gene that has been introduced into them using a vector.

 

ii).   Transformation is a procedure through which a piece of DNA is introduced in a host bacterium .

iii). Normally, the genes encoding resistance to antibiotics such as ampicillin (ampR gene), chloramphenicol, tetracycline (tetR gene) or kanamycin, etc., are considered useful selectable markers for E. coli.

iv). The normal E. coli cells do not carry resistance against any of these antibiotics.

 

c)     Cloning sites:

i).     In order to link the alien DNA, the vector needs to have very few, preferably single, recognition sites for the commonly used restriction enzymes (They cut DNA at specific sequences called recognition sites).

ii).   Presence of more than one recognition sites within the vector will generate several fragments, which will complicate the gene cloning (Figure 9.4).

pBR322: Type: Plasmid vector. Origin: pBR322 was one of the first widely used plasmids for cloning, developed by Bolivar and Rodriguez (hence the "BR" in pBR322).

 

i).     This plasmid is formed by scientists in labs. ii).   Here ‘ori’ is site which is important for the replication. iii). ampR and tetR will work as selectable marker. iv). Here every single enzyme has single recognition site like one site for BamH I and Sal I etc.

iii). The ligation (joining) of alien DNA is carried out at a restriction site present in one of the two antibiotic resistance genes.

A restriction site is a specific sequence of DNA (usually 4–8 base pairs long) where a restriction enzyme (also called a restriction endonuclease) cuts the DNA.

 

·        For example, you can ligate a foreign DNA at the BamH I site of tetracycline resistance gene in the vector pBR322.

‘P’ stands for plasmid and "BR" in the name "pBR322" stands for "Bolivar and Rodriguez," which refers to the scientists who played a pivotal role in the creation and development of this particular plasmid vector.

 

🧬 Goal: Insert a foreign gene into pBR322 and identify which E. coli cells received the recombinant plasmid (with foreign DNA) and which received the non-recombinant plasmid (without foreign DNA). 🔢 Step-by-Step Process 🔹 Step 1: Selection of Restriction Site ·         Choose a restriction enzyme like BamHI, which cuts within the tetʳ (tetracycline resistance) gene of pBR322. 🔹 Step 2: Cutting & Insertion ·         Use BamHI to cut: o    the pBR322 plasmid o    and the foreign DNA (same enzyme ensures compatible ends). ·         Mix and use ligase enzyme to insert the foreign DNA into the BamHI site. ·         This disrupts the tetʳ gene. ✅ Result: o    Recombinant plasmid → tetʳ gene is broken (non-functional). o    Non-recombinant plasmid → tetʳ gene intact. 🔹 Step 3: Transformation ·         Introduce the ligated plasmids into E. coli cells using a method like heat shock. ·         Not all bacteria take up plasmid. 🔹 Step 4: Selection using Ampicillin ·         Spread the transformed cells on agar plates containing ampicillin. ✅ Only E. coli cells with any plasmid (recombinant or not) will survive (because ampʳ gene is intact in both). 🔹 Step 5: Replica Plating on Tetracycline ·         Transfer colonies from ampicillin plate to another plate with tetracycline. ✅ Interpretation: o    Non-recombinant colonies will grow → tetʳ gene is intact. o    Recombinant colonies will NOT grow → tetʳ gene is disrupted by foreign DNA.

 

Example how can we use these cloning vector in medical field 🔹 1. Insertion of Human or Foreign Genes ·         Scientists use vectors like pBR322 to insert useful genes (like the insulin gene, pest-resistance gene, etc.) into bacteria or plants. 🔹 2. Selectable Markers Help Identify Recombinants ·         Selectable marker genes like ampʳ and tetʳ in pBR322 are used to: o    Detect whether the plasmid was taken up by the host. o    Find out if the plasmid became recombinant (i.e., if foreign DNA was successfully inserted).

 

·        The recombinant plasmids will lose tetracycline resistance due to insertion of foreign DNA, now it will become tet^S (sensitive) but can still be selected out from non-recombinant ones by plating the transformants on tetracycline containing medium. In this case non recombinant will grow in tetracycline containing medium but recombinant plasmid will not grow as it loosen it resistivity against tetracycline, in this way we can select out recombinant among non recombinant.

·        The transformants 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 because it loosen the tetracycline resistivity. But, non- recombinants will grow on the medium containing both the antibiotics.

·        In this case, 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 (complex or uneasy method) because it requires simultaneous plating on two plates having different antibiotics.

Final Summary of the Full Process: Main Goal: To find out recombinants. 1.     Start with E. coli (no plasmid). 2.     Insert foreign gene into vector (like pBR322). 3.     Transform the bacteria. 4.     Use ampicillin to select transformed cells. 5.     Use tetracycline to identify recombinant plasmids. 6.     Pick recombinant colonies. 7.     Grow, express, and purify the gene product or plasmid.

 

 

Limitations of the above process are as follows:-

i).     Cumbersome procedure as it is a two step process.

 

 

Alternative method for the selection of recombinants:

i).     As we know that the two step process is lengthy and cumbersome, 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.

ii).   In this case we use puc8 (p-plasmid, uc-‘University of California).

iii). When we provide a chromogenic substrate X-gal to B-galactosidase, it will give blue color.

iv). But when a recombinant DNA is inserted within the coding sequence of an enzyme, β-galactosidase. This results into inactivation of the gene (Lac Z gne) for synthesis of this enzyme, which is referred to as insertional inactivation.

v).   Case before insertion of a recombinant DNA: The presence of a chromogenic substrate with the B galactosidase enzyme gives blue coloured colonies if the plasmid in the bacteria does not have an insert.

vi). After the insertion of a recombinant DNA: Presence of insert results into insertional inactivation of the β-galactosidase gene and the colonies do not produce any colour, these are identified as recombinant colonies.

Substrate	Recombinant (No B-galactosidase)	Non recombinant (B- galactosidase is present)
X gal substrate	No colour (white colour)	Blue colour colony
In this way we select recombinant and non recombinant in a single process.

 

 

Vectors for cloning genes in plants and animals :

1.     You may be surprised to know that we have learnt the lesson of transferring genes into plants and animals from bacteria and viruses which have known this for ages – how to deliver genes to transform eukaryotic cells and force them to do what the bacteria or viruses want.

i).     For example, Agrobacterium tumifaciens, a pathogen of several dicot plants is able to deliver a piece of DNA known as ‘T-DNA’ (‘t’- Transfer DNA) to transform normal plant cells into a tumor and direct these tumor cells to produce the chemicals required by the pathogen.

ii).   Similarly, retroviruses in animals have the ability to transform normal cells into cancerous cells.

·        Retroviruses are viruses that store their genetic information as RNA instead of DNA. When they infect a host cell, they convert their RNA into DNA using an enzyme called reverse transcriptase. ·        This new DNA is then inserted into the host cell’s own DNA, where it can be copied and used to make new viruses.

 

 

2.     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.

3.     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.

4.     Similarly, retroviruses have also been disarmed and are now used to deliver desirable genes into animal cells.

5.     So, once a gene or a DNA fragment has been ligated into a suitable vector it is transferred into a bacterial, plant or animal host (where it multiplies).

 

9.2.3 Competent Host (For Transformation with Recombinant DNA)

 

A.    1^st Method with the help of Ca ions, Heat shock followed by cooling in ice

1.     Since DNA is a hydrophilic molecule, it cannot pass through cell membranes. Why?

i).     DNA, as a molecule, is hydrophilic due to its structure and chemical properties. It contains phosphate groups and sugar molecules that form the backbone of the DNA strand, and these components are polar and hydrophilic, meaning they have an affinity for water. ii).   Cell membranes, on the other hand, are primarily composed of a lipid bilayer. Lipids are hydrophobic molecules, meaning they repel water and are nonpolar.

 

2.     In order to force bacteria to take up the plasmid, the bacterial cells must first be made ‘competent’ to take up DNA.

3.     This is done by treating them with a specific concentration of a divalent cation, such as calcium, which increases the efficiency with which DNA enters the bacterium through pores in its cell wall.

i).     Calcium ions can disrupt the stability of the bacterial cell membrane. ii).     By destabilizing the cell membrane, calcium ions can increase its permeability. This increased permeability allows for more efficient uptake of exogenous DNA molecules.

 

4.     Recombinant DNA can then be forced into such cells by incubating the cells with recombinant DNA on ice, followed by placing them briefly at 42⁰C (heat shock), and then putting them back on ice.

5.     This enables the bacteria to take up the recombinant DNA.

i).     Heat Shock: The cells and DNA mixture is briefly exposed to a higher temperature, often around 42 degrees Celsius (heat shock). This abrupt temperature change causes thermal stress to the bacterial cells, leading to the creation of temporary pores or disruptions in the cell membrane. ii).   DNA Uptake: The heat shock induces transient (brief) changes in the cell membrane, increasing its permeability. This temporary permeability allows the DNA, which was previously outside the bacterial cells, to be pulled into the cells. iii). Recovery: Finally, after the heat shock, the mixture is returned to ice or kept at a lower temperature. This helps the bacterial cells recover from the thermal stress and stabilizes the cell membrane, reducing further uptake of unnecessary molecules.

 

6.     This is not the only way to introduce alien DNA into host cells.

 

B.     By Micro-injection:

i).     In a method known as micro-injection, recombinant DNA is directly injected into the nucleus of an animal cell.

 

C.     Biolistics or Gene gun method:

i).     In another method, suitable for plants, cells are bombarded with high velocity micro-particles of gold or tungsten coated (because gold or tungsten are non reactive so cannot alter the chemical reactions within cell) with DNA in a method known as biolistics or gene gun.

D.    Disarm method:

i).     And the last method uses ‘disarmed pathogen’ vectors, which when allowed to infect the cell, transfer the recombinant DNA into the host.

a)     Infection and Entry: The disarmed pathogen vector is introduced into the target cells of the host organism. For instance, in the case of a virus-based vector, this could involve exposing the cells to the modified virus. b)     Cell Recognition and Attachment: The modified pathogen's surface proteins or other molecular components allow it to recognize and attach to specific receptors on the surface of the host cell. This binding facilitates the entry of the vector into the host cell.

 

9.3 PROCESSES OF RECOMBINANT DNA TECHNOLOGY

1.     Recombinant DNA technology involves several steps in specific sequence such as

a)     isolation of DNA,

b)     fragmentation of DNA by restriction endonucleases,

c)     isolation of a desired DNA fragment,

d)     ligation of the DNA fragment into a vector,

e)     transferring the recombinant DNA into the host,

f)       culturing the host cells in a medium at large scale and extraction of the desired product.

 

9.3.1 Isolation of the Genetic Material (DNA)

1.     Recall that nucleic acid is the genetic material of all organisms without exception.

2.     In majority of organisms this is deoxyribonucleic acid or DNA.

3.     In order to cut the DNA with restriction enzymes, it needs to be in pure form, free from other macro-molecules.

4.     Since the DNA is enclosed within the membranes, we have to break the cell open to release DNA along with other macromolecules such as RNA, proteins, polysaccharides and also lipids.

i).     For example RNA, lipids and proteins can be broken down by ribonuclease, lipase and protease respectively.

5.     This can be achieved by treating the bacterial cells/plant or animal tissue with enzymes such as lysozyme to break bacterial cell wall (bacteria cell wall made of peptidoglycan), cellulase to break plant cell wall (plant cells), chitinase to break fungus cell wall.(fungus).

6.     You know that genes are located on long molecules of DNA intertwined with proteins such as histones.

7.     Other molecules can be removed by appropriate treatments and purified DNA ultimately precipitates out after the addition of chilled ethanol.

8.     This can be seen as collection of fine threads in the suspension (Figure 9.5).

 

Spooling: A glass rod or a pipette tip is inserted into the solution where the DNA has precipitated. The DNA, being sticky and long, tends to adhere to the surface of the rod.

 

 

 

 

9.3.2 Cutting of DNA at Specific Locations

1.     Restriction enzyme (restriction endonuclease) digestions are performed by incubating purified DNA molecules with the restriction enzyme, at the optimal conditions for that specific enzyme.

2.     Agarose gel electrophoresis is employed to check the progression of a restriction enzyme digestion. Agarose is a natural polymer extracted by red algae. It is provided in the form of powder which is dissolved in the solvent for making a solution.

3.     DNA is a negatively charged molecule, hence it moves towards the positive electrode (anode) (Figure 9.3).

4.     The lighter the size of DNA, farther it will go as we can see in the diagram given below.

 

 

5.     The process is repeated with the vector DNA also.

6.     The joining of DNA involves several processes. Before joining amplification of the desired gene is required, so before joining desired gene with vector DNA we do amplification.

·         PCR amplification is a lab technique that produces many copies of a DNA fragment (like the desired gene), outside a living cell (in a test tube). ·         But to express the gene, make protein from it, or store and multiply it stably, we still need to insert it into a vector and then into a host.

 

7.     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.

8.     This results in the preparation of recombinant DNA.

 

8.3.3     Amplification of Gene of Interest using PCR

1.     PCR stands for Polymerase Chain Reaction.

Often, the amount of the desired gene available may be limited, especially if it's extracted from natural sources. Amplification ensures that there's enough of the gene to work with during the cloning process.

 

 

A.    Steps used in the amplification of gene are as follows:-

a)     Denaturation:

i).     In this step, the double-stranded DNA template is heated to a high temperature (usually around 94–98°C), causing the DNA strands to separate or denature into single strands.

 

b)     Annealing:

i).     Now we will add small segments of oligonucleotides primers (DNA primers) to initiate the process of replication.

ii).   The reaction temperature is lowered (around 55⁰C to 65⁰C) to allow short DNA primers (oligonucleotides) to bind specifically to complementary sequences on the single-stranded DNA template. This process is called annealing.

 

c)     Extension/Amplification:

1.     A DNA polymerase enzyme, such as Taq polymerase, extends the primers by adding nucleotides to the 3' end of each primer, synthesizing a new DNA strand complementary to the template.

2.     It is thermostable DNA polymerase (isolated from a bacterium, Thermus aquaticus), which remain active during the high temperature induced denaturation of double stranded DNA.

2.     In this reaction, multiple copies of the gene (or DNA) of interest is synthesised in vitro using two sets of primers and the enzyme DNA polymerase.

3.     If the process of replication of DNA is repeated many times, the segment of DNA can be amplified to approximately billion times, i.e., 1 billion copies are made.

4.     The amplified fragment if desired can now be used to ligate with a vector for further cloning (Figure 9.6).

 

9.3.4 Insertion of Recombinant DNA into the Host Cell/Organism

1.     There are several methods of introducing the ligated DNA into recipient cells.

2.     Recipient cells after making them ‘competent’ to receive, take up DNA present in its surrounding.

3.     So, if a recombinant DNA bearing gene for resistance to an antibiotic (e.g., ampicillin) is transferred into E. coli cells, the host cells become transformed into ampicillin-resistant cells.

4.     If we spread the transformed cells on agar plates containing ampicillin, only transformants will grow, untransformed recipient cells will die.

5.     Since, due to ampicillin resistance gene, one is able to select a transformed cell in the presence of ampicillin.

6.     The ampicillin resistance gene in this case is called a selectable marker.

 

9.3.5 Obtaining the Foreign Gene Product

1.     When you insert a piece of alien DNA into a cloning vector and transfer it into a bacterial, plant or animal cell, the alien DNA gets multiplied.

2.     In almost all recombinant technologies, the ultimate aim is to produce a desirable protein.

3.     Hence, there is a need for the recombinant DNA to be expressed.

4.     The foreign gene gets expressed under appropriate conditions.

5.     The expression of foreign genes in host cells involve understanding many technical details like Promoters and Regulatory Sequences, Vector Selection, Host Cell Type etc.

6.     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.

7.     If any protein encoding gene is expressed in a heterologous host, it is called a recombinant protein.

In the context of genetic engineering or biotechnology, a heterologous host refers to an organism or cell in which a gene from a different species or source is introduced and expressed. This host organism is different from the species in which the gene naturally occurs or is native to.

 

8.     The cells harbouring (nurture) cloned genes of interest may be grown on a small scale in the laboratory.

9.     The cultures (Groups of host cells (like bacteria, yeast, or other cells) that are grown in the lab) may be used for extracting the desired protein and then purifying it by using different separation techniques.

10.The cells can also be multiplied in a continuous culture system wherein the used medium is drained out from one side while fresh medium is added from the other to maintain the cells in their physiologically most active log/exponential phase.

11.This type of culturing method produces a larger biomass leading to higher yields of desired protein.

12.Small volume cultures cannot yield appreciable quantities of products.

13.To produce in large quantities, the development of bioreactors, where large volumes (100-1000 litres) of culture can be processed, was required.

i).     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.

ii).   A bioreactor provides the optimal conditions for achieving the desired product by providing optimum growth conditions (temperature, pH, substrate, salts, vitamins, oxygen).

iii). The most commonly used bioreactors are of stirring type, which are shown in Figure 9.7.

iv). A stirred-tank reactor is usually cylindrical or with a curved base to facilitate the mixing of the reactor contents.

v).   The stirrer facilitates even mixing and oxygen availability throughout the bioreactor.

vi). Alternatively air can be bubbled through the reactor.

vii).                       If you look at the figure closely you will see that the bioreactor has an agitator system, an oxygen delivery system and a foam control system, a temperature control system, pH control system and sampling ports so that small volumes of the culture can be withdrawn periodically.

 

9.3.6 Downstream Processing

1.     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.

2.     The processes include separation and purification, which are collectively referred to as downstream processing.

3.     The product has to be formulated with suitable preservatives.

4.     Such formulation has to undergo thorough clinical trials as in case of drugs.

5.     Strict quality control testing for each product is also required.

6.     The downstream processing and quality control testing vary from product to product.

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