Biotechnology - Principles and processes | ISC Class 12 Biology Notes

This note covers the principles of biotechnology, isolation and cutting of genetic material, separation of fragments, cloning vectors, transfer into host cells, selection of recombinants, gene amplification, production in bioreactors and recovery of the finished product.

What are the principles of biotechnology?

Definition: Biotechnology uses living organisms or their enzymes to produce products and processes useful to humans. Enzymes are biological catalysts, substances that speed up reactions.

Making curd, bread and wine involves microorganisms and can be understood as biotechnology in its broad sense. Modern biotechnology also includes processes using genetically modified organisms, whose genetic material has been altered, to obtain useful products on a larger scale.

Which two techniques support modern biotechnology?

Genetic engineering alters genetic material and introduces it into a recipient organism, called the host, to change its phenotype, meaning its expressed characteristics. The genetic material involved is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

Bioprocess engineering maintains sterile conditions, meaning freedom from microbial contamination, so that the desired microorganisms or cells can grow in large quantities. These cultures, populations grown in a suitable nutrient medium, can manufacture products such as antibiotics, vaccines and enzymes.

A gene is a functional unit of inheritance. Traditional hybridisation, the crossing used in plant and animal breeding, very often introduces undesirable genes along with desirable ones. Genetic engineering allows a selected gene or set of genes to be isolated and introduced into the target organism.

What must happen after gene transfer?

Recombinant DNA, abbreviated rDNA, combines DNA from different sources. Cloning means producing multiple identical copies of a DNA template, the DNA serving as the pattern for copying. Simply transferring a DNA fragment does not establish that it will multiply.

Most likely, an alien DNA fragment would not multiply in the recipient's progeny cells. If integrated into the recipient's genome, its complete genetic material, it may multiply and be inherited with host DNA. Replication means copying DNA; an origin of replication is the sequence where this begins.

The basic requirements are identification of desirable DNA, introduction into the host, and maintenance of that DNA with transfer to the host's progeny. Genetic engineering supplies the chosen genetic information; bioprocess engineering provides conditions for producing the desired material in quantity.

How is genetic material isolated from cells?

DNA must be purified before it is cut using specific enzymes. In the majority of organisms, DNA is the genetic material. Genomic DNA is the organism's chromosomal DNA, organised in chromosomes, structures that carry genes. Cell contents include other substances that must be separated from the DNA preparation.

What does cell lysis release?

Cell lysis means breaking open cells to release their contents. DNA is enclosed within membranes, so these must be disrupted. Cells with a wall also require treatment that deals with that barrier. Animal cells lack a cell wall, so no wall-digesting enzyme is needed for them.

Cell lysis releases DNA together with RNA, proteins, polysaccharides and lipids. Polysaccharides are long carbohydrate molecules, while lipids include fats and related substances. DNA is associated with proteins such as histones, proteins around which DNA can be wrapped.

1. Open the cells: use appropriate treatments to release the genetic material. Lysozyme is used for bacterial cells, cellulase for plant cells and chitinase for fungal cells; these enzymes act on their respective cell walls.
2. Remove RNA: treat the preparation with ribonuclease, an enzyme that breaks down RNA, to reduce this contaminating nucleic acid.
3. Remove proteins: use protease, an enzyme that breaks down proteins, and appropriate treatments to remove other unwanted molecules from the preparation.
4. Recover DNA: add chilled ethanol, which causes purified DNA to precipitate, meaning separate out of solution. The DNA appears as fine threads that can be collected by spooling, or winding them out.

The treatments perform different tasks. Lysozyme helps release material from bacterial cells; ribonuclease removes RNA; protease removes protein. Chilled ethanol is used for DNA precipitation. None of these steps by itself identifies the particular gene required for cloning.

Isolation therefore gives a purified DNA preparation. Subsequent cutting and separation yield DNA fragments from which the required fragment can be recovered. Keep the release of total DNA distinct from the later isolation of a selected fragment.

How do restriction enzymes recognise and cut DNA?

Nucleases are enzymes that cut nucleic acids. Exonucleases remove nucleotides from DNA ends, whereas endonucleases cut within DNA. A nucleotide is a unit containing a sugar, a phosphate group and a nitrogenous base. Restriction endonucleases recognise particular DNA sequences before cutting.

How were restriction enzymes discovered and named?

In 1963, two enzymes associated with restriction of bacteriophage growth in Escherichia coli were isolated. A bacteriophage is a virus that infects bacteria. One enzyme added methyl groups to DNA; the other cut DNA and was called a restriction endonuclease.

A methyl group is a chemical group containing one carbon atom and three hydrogen atoms; such groups are added to DNA in this modification process. Five years later, Hind II was isolated and characterised. It recognises a particular sequence of six base pairs and always cuts at a particular point. A base pair consists of paired bases on opposite DNA strands.

In the name EcoRI, E comes from the genus Escherichia, co from the species coli, and R from the bacterial strain RY 13. The Roman numeral I indicates its order of isolation from that strain. Hind II is another restriction enzyme name.

What is a palindromic recognition sequence?

A recognition sequence is the specific sequence recognised by an enzyme. A DNA palindrome reads identically on the two strands when both are read in the same direction. Here A means adenine, T thymine, G guanine and C cytosine, the four DNA bases.

The symbols 5′ and 3′ identify DNA strand ends using the numbered carbon positions in its sugar. The two strands run in opposite directions. In the following pair, read the upper strand left to right and the lower strand right to left:

5′ GAATTC 3′
3′ CTTAAG 5′

Both readings in the 5′ to 3′ direction give GAATTC. EcoRI cuts between G and A in this sequence on each strand. The resulting sticky ends are single-stranded overhangs that can form hydrogen bonds, weak attractions between complementary bases, with matching ends.

Vector DNA and foreign DNA are drawn in different colours. EcoRI cuts at the marked recognition sequences, leaving sticky ends. Matching ends come together, and the final drawing shows a DNA molecule containing both differently coloured portions.

See Fig. 9.1 in your NCERT textbook

Complementary means having matching bases: A pairs with T, and G with C. DNA ligase joins cut DNA ends. Sticky-end pairing brings compatible fragments together and facilitates the action of ligase; recognising a sequence, cutting DNA and joining DNA are distinct operations.

How are DNA fragments separated and recombinant DNA formed?

Gel electrophoresis separates DNA fragments by their movement through a gel under an electric field. DNA is negatively charged and therefore moves towards the anode, the positive electrode. Agarose, a natural polymer obtained from seaweeds, is the most commonly used gel matrix.

How does separation reveal the required fragment?

The gel acts as a sieve. Smaller fragments move farther through it than larger fragments under the same separation conditions. A band is a visible group of separated DNA fragments. Movement through the gel separates fragments by size; it does not manufacture a new gene.

1. Digest the DNA: incubate purified source DNA with the required restriction enzyme under conditions optimal for that enzyme. Digestion here means enzymatic cutting, which produces DNA fragments.
2. Separate the fragments: subject them to agarose gel electrophoresis, allowing negatively charged DNA to move towards the anode and separate according to size.
3. Visualise the bands: stain DNA with ethidium bromide, a DNA-staining compound, and expose the gel to ultraviolet, abbreviated UV, radiation. Bright orange DNA bands become visible.
4. Recover the fragment: cut out the selected band and extract its DNA from the gel piece. This recovery step is called elution.

Pure DNA fragments cannot be seen in visible light without staining. Elution recovers the separated DNA for later use. The extracted fragment can then be joined to a cloning vector, a DNA carrier that transfers the attached DNA into a host for multiplication.

How does ligation create the new combination?

Cut the source DNA and the vector with the same restriction enzyme in the described sticky-end method. Mix the selected fragment with the cut vector and add DNA ligase. Joining these pieces, called ligation, creates the recombinant molecule.

Note: Normally, the source DNA and vector are cut with the same restriction enzyme so that their sticky ends are compatible. The restriction enzyme cuts DNA; DNA ligase joins its ends.

The sequence matters: purification allows controlled digestion, digestion supplies fragments, electrophoresis separates them, elution recovers a selected fragment, and ligation links it to the carrier. Gene transfer into a host follows construction of the recombinant DNA.

What makes a cloning vector useful?

A plasmid is an autonomously replicating, circular DNA molecule outside the bacterial chromosome. Plasmids and bacteriophages can replicate within bacterial cells independently of the control of chromosomal DNA. Linking a foreign DNA fragment to such a carrier allows the fragment to multiply with it.

Which features allow cloning and identification?

The copy number is the number of copies of the DNA molecule per cell. Some plasmids may have only one or two copies per cell, whereas others may have 15 to 100; their numbers can be even higher. The choice of origin affects recovery of cloned DNA.

A selectable marker identifies cells that have received vector DNA and helps eliminate those that have not. Transformation is introduction of DNA into a host bacterium. Cells that receive it are transformants; cells that do not are non-transformants.

A cloning site is a site at which foreign DNA can be inserted. A vector should have very few, preferably single, recognition sites for commonly used restriction enzymes. Multiple sites for the chosen enzyme would cut the vector into several fragments and complicate cloning.

pBR322 is a cloning plasmid used in Escherichia coli, abbreviated E. coli. Its map includes ori, ampᴿ, tetᴿ and rop. Ampicillin and tetracycline are antibiotics, substances that inhibit or kill susceptible microorganisms.

The circular map labels ori and rop, with ampᴿ and tetᴿ marking the resistance genes. Restriction sites labelled around it include EcoRI, Hind III, BamH I, Sal I, Pvu II, Pst I and Cla I; these names identify restriction enzymes.

See Fig. 9.4 in your NCERT textbook

The map relates physical insertion sites to vector functions. Inserting foreign DNA at the BamH I site within tetᴿ can interrupt tetracycline resistance while leaving ampicillin resistance available for selecting transformed bacteria.

Which vectors carry genes into different hosts?

Vectors must deliver DNA into a suitable host and permit its maintenance or multiplication there. Bacterial plasmids are useful carriers, but gene transfer into plants and animals also uses modified biological delivery systems. A pathogen is an organism or agent that causes disease.

How are natural delivery systems modified?

Agrobacterium tumefaciens is a pathogen of several dicot plants, flowering plants whose seeds have two cotyledons or seed leaves. It transfers a DNA segment called T-DNA, meaning transferred DNA, into plant cells, transforming normal cells into tumour cells.

A tumour is a mass produced by abnormal cell growth. The transformed plant cells produce chemicals required by the bacterium. The Ti plasmid, or tumour-inducing plasmid, is modified into a cloning vector that is no longer pathogenic to plants but retains the ability to deliver desired genes.

Retroviruses are RNA viruses that form a DNA copy during their life cycle. Retroviruses in animals can transform normal cells into cancerous cells. Disarmed retroviral vectors have their disease-causing ability removed and are used to deliver desirable genes into animal cells.

BAC and YAC expand the range of cloning vectors beyond ordinary plasmid examples. In genome sequencing work, fragments cloned in bacterial and yeast hosts are amplified before sequencing. Yeast is a fungus; amplification means increasing the number of copies of a DNA segment.

A disarmed vector retains the useful delivery mechanism of a natural biological system. Its use depends on the host and the purpose of the transfer. Distinguish the original pathogen from the modified vector prepared for gene delivery.

How is recombinant DNA introduced into a competent host?

A competent host is a recipient prepared to take up DNA. DNA is hydrophilic, meaning it interacts readily with water, and cannot pass unaided through the cell membrane in the described transformation procedure. Bacterial cells are therefore treated to increase DNA uptake.

How does the heat-shock method work?

Bacteria are treated with a suitable concentration of a divalent cation, a positively charged ion carrying two positive charges, such as calcium. This increases the efficiency with which DNA enters through pores in the bacterial cell wall.

1. Prepare competent bacteria: treat the cells with a suitable concentration of calcium ions to increase their ability to take up DNA.
2. Incubate on ice: keep the treated cells together with recombinant DNA on ice as the first temperature stage.
3. Apply heat shock: briefly place the mixture at 42°C, where °C means degrees Celsius, to promote uptake of the recombinant DNA.
4. Return to ice: cool the cells again after the brief heat treatment, completing the described temperature-shock sequence.

What other transfer methods are available?

In microinjection, recombinant DNA is injected directly into the nucleus of an animal cell. The nucleus is the membrane-bound cell compartment containing chromosomes. This method introduces DNA physically into an individual recipient cell.

In biolistics, also called the gene gun method, plant cells are bombarded with high-velocity gold or tungsten microparticles coated with DNA. These tiny metal particles carry the DNA into cells. Disarmed pathogens can also transfer recombinant DNA when allowed to infect suitable hosts.

These methods differ in how DNA crosses the recipient's barriers: chemical treatment and temperature shock assist bacterial uptake, microinjection places DNA directly in a nucleus, and biolistics uses coated particles. Following transfer, selection is needed to identify the cells that received DNA.

How are transformants and recombinants selected?

A recombinant carries a vector containing inserted foreign DNA. A non-recombinant may carry vector DNA without the desired insert. Thus, a transformed bacterium is not necessarily a recombinant: it may have received an unchanged vector.

How does antibiotic selection work with pBR322?

Normally, genes giving resistance to antibiotics such as ampicillin, chloramphenicol, tetracycline or kanamycin are useful selectable markers for E. coli. In the described system, normal recipient cells lack the relevant resistance and cannot grow on medium containing that antibiotic.

If foreign DNA is inserted at the BamH I site within pBR322's tetᴿ gene, tetracycline resistance is lost. The ampᴿ gene remains functional. This is insertional inactivation, loss of gene function caused by insertion of DNA into that gene.

1. Select transformants: plate the treated bacteria on ampicillin-containing medium. Cells with the functioning ampicillin-resistance marker grow, while untransformed susceptible cells do not.
2. Transfer colonies: test the transformants from the ampicillin plate on a medium containing tetracycline. A colony is a visible population arising from bacterial growth.
3. Identify recombinants: transformants that grow with ampicillin but fail to grow with tetracycline have the resistance pattern expected after disruption of tetᴿ.
4. Identify non-recombinants: transformants with intact resistance genes grow on ampicillin medium and on tetracycline medium, distinguishing them from the insertional recombinants.

How does blue-white selection work?

An alternative marker uses beta-galactosidase, written β-galactosidase, where β is the Greek letter beta. Its activity produces a blue colour in the presence of a suitable chromogenic substrate, a substance that gives a coloured product through enzyme action.

Inserting foreign DNA into the enzyme's coding sequence inactivates its production. Non-recombinant colonies with an intact marker produce blue colour. Recombinant colonies with the disrupted marker do not produce colour and are identified as white or colourless colonies in blue-white selection.

Antibiotic selection tests survival under defined conditions; the colour method tests marker activity. In both methods, conclusions depend on the stated insertion site and marker. A missing colour or loss of resistance is meaningful because insertion has interrupted the corresponding function.

How does PCR amplify a gene outside a living cell?

Polymerase chain reaction, abbreviated PCR, produces multiple copies of a DNA segment in vitro, meaning outside a living organism in an experimental system. This differs from in vivo cloning, where a vector and its linked DNA multiply within living host cells.

What materials does PCR require?

The reaction uses template DNA, two primers, DNA polymerase and nucleotides. Primers are small, chemically synthesised DNA pieces, or oligonucleotides, complementary to regions of the template. They provide starting points for synthesis. DNA polymerase extends them using the supplied nucleotides.

Taq polymerase is the thermostable DNA polymerase obtained from the bacterium Thermus aquaticus. Thermostable means able to remain active through the high-temperature treatment used to separate DNA strands. This property allows the amplification cycle to be repeated.

What happens during each cycle?

Denaturation separates the two DNA strands by heating. Annealing allows primers to bind to complementary regions of the separated strands. During extension, DNA polymerase adds nucleotides to the primers using the template sequence.

The newly formed DNA provides material for further cycles. Repeated copying can amplify the target segment to approximately a billion copies. The amplified fragment may then be ligated into a vector for further cloning if required; amplification itself does not perform this ligation.

A coloured region marks the DNA to be amplified. Arrows lead through heat-induced denaturation, primer annealing and extension using Taq polymerase and nucleotides. The diagram labels 30 cycles and shows the amplified product as approximately one billion times the starting target.

See Fig. 9.6 in your NCERT textbook

When explaining PCR, keep each reagent tied to its function: the template supplies sequence information, primers identify starting regions, nucleotides supply building units, and Taq polymerase constructs new strands. The repeated temperature changes organise the three steps of amplification.

How does cloned DNA lead to a useful gene product?

Increasing DNA copy number and producing a protein are connected but different outcomes. In almost all recombinant technologies, the ultimate aim is a desirable protein. The inserted gene must therefore be expressed, meaning its information is used to produce the corresponding gene product.

Why are expression conditions important?

A foreign gene is expressed under appropriate conditions. After cloning the gene of interest, the conditions needed to produce its protein must be optimised. The presence of an inserted DNA fragment alone does not describe the conditions under which its product will be obtained.

A recombinant protein is a protein produced when its encoding gene is expressed in a heterologous host, a host different from the source of that gene. Cells carrying cloned genes can first be grown on a small scale, followed by extraction and purification of the desired protein.

How can a culture maintain productive growth?

In continuous culture, used medium is drained from one side while fresh medium enters from the other. This maintains cells in their physiologically most active log or exponential phase, the growth phase in which population increase is proportional to the number of cells already present.

This culturing method produces a larger biomass, the mass of living material, and higher yields of the desired protein. The nutrient supply and culture conditions support production after the genetic steps have established the required cells.

The overall relationship is therefore gene identification, cloning, transfer, selection, expression and production. A successful cloning step supplies copies of DNA; suitable expression and culture conditions make the desired product obtainable. Large-scale manufacture then requires equipment that can maintain those conditions in much larger cultures.

How do stirred-tank and sparged bioreactors support production?

A bioreactor is a vessel in which living cells or their biological systems convert raw materials into specific products. Small cultures cannot yield appreciable quantities for large-scale production. Bioreactors allow large culture volumes, such as 100 to 1000 litres, to be processed.

Which conditions does a bioreactor maintain?

The vessel provides optimum temperature, nutrients, salts, vitamins and oxygen for obtaining the desired product. It also controls pH, a measure of acidity or alkalinity. A substrate is material acted upon or used in the biological process.

A stirred-tank reactor is usually cylindrical or has a curved base to help mixing. An agitator, or stirring mechanism, mixes the contents and helps make oxygen available throughout the vessel. In a sparged stirred-tank reactor, sterile air is introduced as bubbles.

The simple reactor drawing labels a motor, foam breaker, flat-bladed impeller, culture broth, sterile air, steam for sterilisation and acid/base for pH control. The sparged reactor drawing shows bubbles and arrows indicating increased surface area for oxygen transfer.

See Fig. 9.7 in your NCERT textbook

An impeller is the rotating mixing part, and culture broth is the liquid medium containing the growing culture. Sparging means introducing gas as bubbles. The bubbles increase the area available for oxygen transfer, while stirring distributes the contents.

Both forms support controlled production. The sparged design emphasises gas delivery through bubbles in addition to stirring. Successful operation also requires sterile conditions so that the desired cells, rather than contaminating microorganisms, form the intended culture.

What happens during downstream processing?

Production inside a culture vessel does not by itself give a finished product. After the biosynthetic stage, the stage in which cells make the required substance, the material must be recovered and purified. These separation and purification steps are collectively called downstream processing.

How is the product prepared for use?

Separation removes the desired material from the production mixture. Purification reduces unwanted substances associated with it. The recovered product then needs a suitable formulation, the preparation in which it is supplied, including suitable preservatives that help maintain it.

For drugs, the formulation must undergo thorough clinical trials, studies that evaluate it in people. Each product also requires strict quality control, testing whether the finished material meets its required standards. Processing and quality-control methods vary from product to product.

How do all the stages fit together?

1. Obtain the genetic material: release DNA from cells, purify it, cut it with restriction enzymes and recover the desired fragment.
2. Construct recombinant DNA: prepare a suitable vector, join the selected DNA fragment to it and introduce the recombinant molecule into a competent host.
3. Select and grow the host: identify the required recombinants, multiply them and establish suitable conditions for expression of the foreign gene.
4. Produce and recover the product: grow the selected culture on an appropriate scale, then separate and purify the desired substance.
5. Prepare the finished material: formulate the product, perform the required testing and apply product-specific quality control before marketing.

Each stage solves a different problem. Genetic manipulation supplies the desired information, culture systems support its expression and production, and downstream processing yields a usable preparation. The final quality requirements depend on the particular product, so a single fixed recovery procedure cannot represent every biotechnology process.

Glossary

• Biotechnology — Use of organisms, cells or their enzymes to obtain products and processes useful to humans.
• Recombinant DNA — DNA combining genetic material from different sources into a new molecular arrangement.
• Restriction endonuclease — Enzyme that recognises a particular DNA sequence and cuts within the DNA molecule.
• DNA ligase — Enzyme that joins cut DNA ends during formation of a recombinant molecule.
• Sticky ends — Single-stranded DNA overhangs capable of pairing with complementary ends of another fragment.
• Elution — Recovery of separated DNA from a gel piece containing the selected band.
• Cloning vector — DNA carrier used to introduce attached foreign DNA into a host for multiplication.
• Origin of replication — DNA sequence where replication begins and which helps control the copy number of linked DNA.
• Selectable marker — Gene that helps identify transformants and eliminate cells that have not received the vector.
• Insertional inactivation — Loss of a gene's function caused by insertion of foreign DNA within it.
• Competent host — Recipient cell prepared so that it can take up DNA from its surroundings.
• PCR — Polymerase chain reaction, a method that repeatedly copies a selected DNA segment in vitro.
• Recombinant protein — Protein produced by expression of an introduced gene in a host different from its source.
• Bioreactor — Vessel providing controlled conditions for biological conversion of raw materials into desired products.
• Downstream processing — Separation and purification of a product after completion of its biological production stage.

Common errors and misconceptions

• Misconception: DNA ligase cuts out the desired gene. Correct: Restriction enzymes cut DNA at recognised sites; DNA ligase joins cut ends when the recombinant molecule is constructed.
• Misconception: A DNA palindrome must read identically in both directions along one strand. Correct: Compare the two strands in the same direction, such as 5′ to 3′.
• Misconception: Larger DNA fragments move farther through agarose. Correct: Smaller fragments move farther because the gel separates fragments through its sieving effect.
• Misconception: Every transformant contains the desired foreign insert. Correct: A cell can receive a non-recombinant vector. Recombinant selection must distinguish it from a cell carrying the inserted fragment.
• Misconception: Inserting DNA into pBR322's tetᴿ gene removes both antibiotic resistances. Correct: In the described insertion, tetracycline resistance is lost while ampicillin resistance remains useful for selection.
• Misconception: Blue colonies identify recombinants in the described colour test. Correct: Blue colonies have an active β-galactosidase marker; disruption by the insert gives colonies without that colour.
• Misconception: PCR joins the amplified gene to its vector. Correct: PCR copies DNA using primers and polymerase. Joining the product to a vector requires a separate ligation step.
• Misconception: A product is ready for use when culture growth ends. Correct: Recovery, purification, formulation and the appropriate quality testing are needed before the finished product is marketed.

Exam-style questions with model answers

Q1. Distinguish the functions of a restriction endonuclease and DNA ligase during recombinant DNA formation. [2 marks]

1. A restriction endonuclease recognises a particular DNA sequence and cuts the DNA at specified positions.
2. DNA ligase joins cut DNA ends, linking the selected foreign fragment with the prepared vector.

Q2. Describe four stages used to isolate purified DNA from bacterial cells, from cell opening to DNA recovery. [4 marks]

1. Open the bacterial cells using an appropriate treatment involving lysozyme, releasing DNA together with other cellular substances.
2. Treat the preparation with ribonuclease to remove RNA, which otherwise remains mixed with the DNA.
3. Use protease to remove associated proteins and appropriate treatments to remove other unwanted cellular molecules.
4. Add chilled ethanol to precipitate purified DNA, which appears as fine threads and can be collected by spooling.

Q3. EcoRI recognises the paired sequence 5′ GAATTC 3′ / 3′ CTTAAG 5′ and cuts between G and A on each strand when read 5′ to 3′. Here A, T, G and C denote adenine, thymine, guanine and cytosine. Explain the palindrome, the resulting ends and how fragments from two sources can be joined. [3 marks]

1. The sequence is palindromic because reading each strand in the 5′ to 3′ direction gives GAATTC, despite the strands running in opposite directions.
2. The stated cuts leave single-stranded overhangs called sticky ends. Matching overhangs on fragments cut with EcoRI are complementary and can pair through hydrogen bonds.
3. Pairing brings the fragments together, and DNA ligase joins their cut ends to form recombinant DNA containing material from both sources.

Q4. A pBR322 vector has intact ampicillin- and tetracycline-resistance genes. Foreign DNA is inserted at the BamH I site within the tetracycline-resistance gene, inactivating it without affecting ampicillin resistance. Recipient E. coli cells initially lack both resistances. Explain, in five points, how growth on the two antibiotic media distinguishes non-transformants, recombinants and non-recombinant transformants. [5 marks]

1. First place the treated bacterial population on ampicillin-containing medium. Cells that received a vector with the intact ampicillin-resistance gene can grow as transformants.
2. Non-transformants lack the introduced resistance marker. Because the stated recipient cells are susceptible to ampicillin, these cells do not grow on the selection medium.
3. Transfer the colonies obtained on ampicillin medium to tetracycline-containing medium to test the second marker and distinguish the two kinds of transformant.
4. Recombinants grow with ampicillin but fail to grow with tetracycline, because the foreign insert has disrupted the tetracycline-resistance gene while leaving ampicillin resistance intact.
5. Non-recombinant transformants retain both functional resistance genes. They grow on each antibiotic medium, separating them from the transformants whose tetracycline marker was insertionally inactivated.

Q5. In a blue-white selection system, an intact β-galactosidase marker produces blue colonies with the supplied chromogenic substrate. A foreign insert inactivates this marker. What colour identifies each class of colony, and why? [2 marks]

1. Non-recombinant colonies are blue because the intact β-galactosidase marker produces enzyme activity with the supplied substrate.
2. Recombinant colonies are white or colourless because insertional inactivation prevents the marker from producing that colour.

Q6. Describe the three stages of a PCR cycle and explain the role of thermostable Taq polymerase. [4 marks]

1. During denaturation, heating separates the two strands of the template DNA so they can be copied.
2. During annealing, short DNA primers bind to complementary regions on the separated template strands, establishing starting points for synthesis.
3. During extension, DNA polymerase extends the primers by adding supplied nucleotides according to the template sequences.
4. Taq polymerase from Thermus aquaticus remains active during high-temperature denaturation, allowing repeated cycles that amplify the selected DNA segment.

Q7. Explain four features of a stirred-tank bioreactor that support controlled production, followed by two requirements for preparing its product after biological synthesis. [6 marks]

1. The agitator mixes the culture contents and helps maintain oxygen availability throughout the vessel, supporting the growing cells during production.
2. The oxygen delivery system supplies oxygen to the culture; in a sparged stirred-tank reactor, sterile air is introduced as bubbles.
3. The temperature control system maintains suitable thermal conditions so that growth and biological production proceed under the required conditions.
4. The pH control system maintains appropriate acidity or alkalinity, another environmental condition that must be controlled during culture and product formation.
5. After biological synthesis, downstream processing separates and purifies the desired product from the production mixture to obtain the recovered material.
6. The product needs suitable formulation and strict quality-control testing; drug formulations also require thorough clinical trials before being supplied as finished products.

Q8. Describe the heat-shock method for introducing recombinant DNA into bacteria, including competence treatment and the temperature sequence. [4 marks]

1. Treat the bacterial cells with a suitable concentration of a divalent cation such as calcium to increase their efficiency of DNA uptake.
2. Incubate the prepared competent cells with recombinant DNA on ice, bringing recipient cells and DNA together.
3. Place the mixture briefly at 42°C to apply the heat shock that assists entry of recombinant DNA.
4. Return the cells to ice after the brief heat treatment, completing the temperature sequence used in this transformation procedure.

Key takeaways

• Modern biotechnology combines genetic engineering with controlled, sterile production conditions that support the desired cells and their products.
• DNA isolation releases and purifies genetic material; restriction digestion, electrophoresis and elution subsequently provide the selected fragment.
• Restriction enzymes recognise specific DNA sequences, while DNA ligase joins compatible fragments during construction of recombinant DNA.
• A useful cloning vector requires an origin of replication, suitable cloning sites and markers that help identify transformed cells.
• Transformation and recombinant selection answer different questions: whether cells received DNA and whether their vector contains an insert.
• PCR repeatedly uses denaturation, primer annealing and extension to amplify a selected DNA segment outside living cells.
• Bioreactors provide controlled growth conditions, with stirring and oxygen delivery supporting production in large culture volumes.
• Downstream processing separates and purifies the product; formulation and appropriate testing prepare it for use.

Test yourself

What does ori do in a cloning vector?

It marks the sequence where replication starts and helps control the copy number of DNA linked to it.

Why is a single recognition site for the selected restriction enzyme useful?

Multiple recognition sites would cut the vector into several fragments, complicating insertion and cloning of the desired DNA.

Which electrode attracts DNA during gel electrophoresis, and why?

The positive electrode, or anode, attracts DNA because DNA fragments carry a negative charge.

What is the purpose of elution?

Elution extracts the desired DNA from a selected gel piece after electrophoretic separation of fragments.

How does microinjection differ from the gene gun method?

Microinjection places DNA directly into an animal-cell nucleus. The gene gun bombards plant cells with DNA-coated gold or tungsten microparticles.

What do BAC and YAC stand for?

BAC means bacterial artificial chromosome, and YAC means yeast artificial chromosome; both are specialised cloning vectors.

Why can Taq polymerase support repeated PCR cycles?

It is thermostable and remains active through the high-temperature treatment used to denature double-stranded DNA.

How does continuous culture support a high yield?

Replacing used medium with fresh medium maintains cells in their physiologically most active exponential phase, producing greater biomass and higher protein yields.