Biotechnology: Principles and Processes | CBSE Class 12 Biology Notes

This note covers the meaning of biotechnology and its two core techniques, the first recombinant DNA, restriction enzymes and sticky ends, gel electrophoresis, cloning vectors and pBR322, selection of recombinants, competent hosts and other ways of introducing DNA, the steps of recombinant DNA technology, isolation of DNA, the polymerase chain reaction (PCR), bioreactors and downstream processing.

What is biotechnology, and what are its two core techniques?

Biotechnology deals with techniques of using live organisms or enzymes from organisms to produce products and processes useful to humans. It is a twentieth century off-shoot of modern biology, and its products brought qualitative improvement in health and food production.

In this broad sense, making curd, bread or wine could also be thought of as biotechnology, because all three are microbe-mediated processes.

Today the word is used in a more restricted sense. It refers to those processes that use genetically modified organisms to achieve the same results on a larger scale.

Many other processes and techniques are also included under biotechnology. Examples are in vitro fertilisation leading to a 'test-tube' baby, synthesising a gene and using it, developing a DNA vaccine, and correcting a defective gene.

Definition: The European Federation of Biotechnology (EFB) defines biotechnology as 'the integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services'. This definition covers both the traditional view and modern molecular biotechnology.

Which two techniques gave birth to modern biotechnology?

Among many techniques, two core techniques enabled the birth of modern biotechnology.

Why is genetic engineering better than traditional hybridisation, and how does foreign DNA multiply in a host?

Asexual reproduction preserves the genetic information, while sexual reproduction permits variation and unique combinations of genetic set-up, some of which may benefit the organism and the population.

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

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

Why does foreign DNA need an origin of replication?

A piece of DNA that is somehow transferred into an alien organism would most likely not be able to multiply itself in the progeny cells of that organism.

If it gets integrated into the genome of the recipient, however, it may multiply and be inherited along with the host DNA. This happens because the alien DNA has become part of a chromosome, and a chromosome has the ability to replicate.

A chromosome contains a specific DNA sequence called the origin of replication, which is responsible for initiating replication. So an alien piece of DNA is linked with an origin of replication, and it can then replicate and multiply itself in the host organism.

This is called cloning: making multiple identical copies of any template DNA.

How was the first recombinant DNA molecule made?

The construction of the first recombinant DNA came from linking a gene encoding antibiotic resistance with a native plasmid of Salmonella typhimurium. A plasmid is an autonomously replicating, circular, extra-chromosomal DNA.

Stanley Cohen and Herbert Boyer accomplished this in 1972. The steps were as follows.

1. They isolated the antibiotic resistance gene by cutting out a piece of DNA from a plasmid that was responsible for conferring antibiotic resistance. Cutting at specific locations was possible with restriction enzymes, the so-called 'molecular scissors'.
2. The cut piece of DNA was linked with plasmid DNA, which acts as a vector to transfer the piece of DNA attached to it.
3. The linking was done by the enzyme DNA ligase, which acts on cut DNA molecules and joins their ends.
4. This new combination of circular, autonomously replicating DNA, created in vitro, is recombinant DNA.
5. The recombinant DNA was transferred into Escherichia coli, a bacterium closely related to Salmonella. There it replicated using the new host's DNA polymerase enzyme and made multiple copies.

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

A plasmid works like a mosquito, which acts as an insect vector to carry the malarial parasite into the human body. In the same way, a plasmid can be used as a vector to deliver an alien piece of DNA into a host organism.

What are the three basic steps in genetically modifying an organism?

1. Identification of DNA with desirable genes.
2. Introduction of the identified DNA into the host.
3. Maintenance of the introduced DNA in the host and transfer of the DNA to its progeny.

What are restriction enzymes, and how are they named?

Recombinant DNA technology can be carried out only with the key tools: restriction enzymes, polymerase enzymes, ligases, vectors and the host organism.

In 1963, two enzymes responsible for restricting the growth of bacteriophage in Escherichia coli were isolated. One of them added methyl groups to DNA, while the other cut DNA. The one that cut DNA was called restriction endonuclease.

Five years later, the first restriction endonuclease whose functioning depended on a specific DNA nucleotide sequence, Hind II, was isolated and characterised. Hind II always cut DNA molecules at a particular point by recognising a specific sequence of six base pairs.

This specific base sequence is known as the recognition sequence for Hind II. Today more than 900 restriction enzymes are known, isolated from over 230 strains of bacteria, and each recognises a different recognition sequence.

How is a restriction enzyme named?

The first letter of the name comes from the genus, and the next two letters come from the species of the prokaryotic cell from which the enzyme was isolated. EcoRI comes from Escherichia coli RY 13, and its letter R comes from the name of the strain. Roman numbers after the name give the order in which the enzymes were isolated from that strain.

How do exonucleases and endonucleases differ?

Restriction enzymes belong to a larger class of enzymes called nucleases, which are of two kinds.

• Exonucleases remove nucleotides from the ends of the DNA.
• Endonucleases make cuts at specific positions within the DNA. Restriction endonucleases belong here.

How do restriction enzymes cut DNA and produce sticky ends?

Each restriction endonuclease works by 'inspecting' the length of a DNA sequence. Once it finds its specific recognition sequence, it binds to the DNA and cuts each of the two strands of the double helix at specific points in their sugar-phosphate backbones.

What is a palindrome in DNA?

Each restriction endonuclease recognises a specific palindromic nucleotide sequence in the DNA. In language, a palindrome is a group of letters that forms the same word when read forward and backward, for example "MALAYALAM".

In DNA, a palindrome is a sequence of base pairs that reads the same on the two strands when the orientation of reading is kept the same. The sequence below reads the same on both strands in the 5′ to 3′ direction, and also in the 3′ to 5′ direction.

Upper strand: 5′ GAATTC 3′. Lower strand: 3′ CTTAAG 5′.

What are sticky ends?

Restriction enzymes cut the strand of DNA a little away from the centre of the palindrome site, but between the same two bases on the opposite strands. This leaves single-stranded portions at the ends.

These overhanging stretches on each strand are called sticky ends. They are named so because they form hydrogen bonds with their complementary cut counterparts. This stickiness of the ends makes the action of the enzyme DNA ligase easier.

A red helix labelled vector DNA and a black helix labelled foreign DNA each carry GAATTC on one strand paired with CTTAAG on the other. Labels say the enzyme cuts both strands at the same site, and that EcoRI cuts between bases G and A only when GAATTC is present.

After the EcoRI cut, the vector end shows G on one strand and CTTAA on the other, and the foreign DNA end shows single-stranded bases AATT; both overhangs are labelled sticky end. A curved arrow notes that DNA fragments join at sticky ends, and the bottom row shows one recombinant DNA, part red and part black.

See Fig. 9.1 in your NCERT textbook

Why must the vector and the source DNA be cut with the same enzyme?

Restriction endonucleases are used in genetic engineering to form 'recombinant' molecules of DNA, made of DNA from different sources or genomes.

When DNA is cut by the same restriction enzyme, the fragments have the same kind of sticky ends, and these can be joined end to end using DNA ligases. Normally, unless the vector and the source DNA are cut with the same restriction enzyme, the recombinant vector molecule cannot be created.

A strand of foreign DNA and a circular vector DNA (plasmid) are both cut by the same restriction enzyme at a specific point. The plasmid ring is opened, and ligases join the foreign DNA to it, giving a recombinant DNA molecule.

An arrow labelled transformation leads to E. coli (cloning host), a rod-shaped cell carrying the plasmid. The cells divide, and both daughter cells carry the plasmid.

See Fig. 9.2 in your NCERT textbook

How are DNA fragments separated by gel electrophoresis?

Cutting DNA with restriction endonucleases produces fragments of DNA. These fragments can be separated by a technique called gel electrophoresis.

DNA fragments are negatively charged molecules, so they can be separated by forcing them to move towards the anode under an electric field through a medium or matrix. Nowadays the most commonly used matrix is agarose, a natural polymer extracted from sea weeds.

1. The DNA sample is loaded into wells in the gel, and an electric field makes the negatively charged fragments move through the agarose towards the anode.
2. The fragments separate (resolve) according to their size through the sieving effect of the agarose gel. The smaller the fragment, the farther it moves.
3. The separated fragments are stained with a compound called ethidium bromide and then exposed to UV radiation. Pure DNA fragments cannot be seen in visible light without staining.
4. Bright orange coloured bands of DNA can then be seen in the stained gel under UV light.
5. The separated bands are cut out of the agarose gel and the DNA is extracted from the gel piece. This step is called elution.

The DNA fragments purified in this way are used to construct recombinant DNA by joining them with cloning vectors.

A slab of gel with four lanes numbered 1 to 4 and a row of wells at one end. Lane 1 holds undigested DNA and shows a single large band close to its well. Lanes 2 to 4 hold digested sets of DNA fragments and show several bands spread along the lane. The DNA bands are labelled from 'Largest', near the wells, to 'Smallest', farthest from the wells.

See Fig. 9.3 in your NCERT textbook

Note: The sample is loaded in the wells. Because smaller fragments move farther, the smallest bands lie farthest from the wells, towards the anode.

What features make a good cloning vector?

Plasmids and bacteriophages can replicate within bacterial cells independent of the control of chromosomal DNA. Because of their high number per cell, bacteriophages have very high copy numbers of their genome within bacterial cells.

Some plasmids may have only one or two copies per cell, whereas others may have 15 to 100 copies per cell, and the numbers can go even higher. If an alien piece of DNA is linked with bacteriophage or plasmid DNA, its number can be multiplied to equal the copy number of the plasmid or bacteriophage.

Vectors used at present are engineered so that they allow easy linking of foreign DNA and selection of recombinants from non-recombinants. The features below are required to make cloning into a vector easier.

Transformation is a procedure through which a piece of DNA is introduced into a host bacterium. Normal E. coli cells do not carry resistance against any of these antibiotics.

A circle labelled pBR322. Two long arcs mark the antibiotic resistance genes: ampᴿ on the upper left and tetᴿ on the upper right. The ori segment sits on the lower left and rop at the bottom.

Restriction sites are labelled around the circle: Pst I and Pvu I in the ampᴿ arc; EcoR I, Cla I and Hind III at the top, between ampᴿ and tetᴿ; BamH I and Sal I in the tetᴿ arc; and Pvu II at the bottom near rop. The rop region codes for the proteins involved in the replication of the plasmid.

See Fig. 9.4 in your NCERT textbook

How are recombinants told apart from non-recombinants?

Vectors are engineered so that recombinants can be selected from non-recombinants. There are two main ways of doing this.

How does selection by antibiotic resistance work in pBR322?

In pBR322, foreign DNA can be ligated at the BamH I site of the tetracycline resistance gene. The steps are as follows.

1. Foreign DNA is ligated at the BamH I site in the tetᴿ gene. The recombinant plasmids lose tetracycline resistance because of the insertion.
2. The transformants are plated on a medium containing ampicillin. Both recombinants and non-recombinants still carry ampᴿ, so both grow, while non-transformants do not.
3. The transformants growing on ampicillin are then transferred to a medium containing tetracycline.
4. Recombinants grow on the ampicillin medium but not on the tetracycline medium. Non-recombinants grow on the media containing both antibiotics.

Here one antibiotic resistance gene helps select the transformants, while the other gene is 'inactivated due to insertion' of alien DNA and helps select the recombinants.

How does a chromogenic substrate identify recombinants?

Selecting recombinants through inactivation of antibiotic resistance is a cumbersome procedure, because it needs simultaneous plating on two plates with different antibiotics.

So alternative selectable markers have been developed. They separate recombinants from non-recombinants by their ability to produce colour in the presence of a chromogenic substrate.

Here the recombinant DNA is inserted within the coding sequence of the enzyme β-galactosidase. This inactivates the gene for synthesis of the enzyme, which is called insertional inactivation.

If the plasmid in the bacteria does not have an insert, the chromogenic substrate gives blue coloured colonies. If an insert is present, the β-galactosidase gene is inactivated and the colonies do not produce any colour. These colourless colonies are identified as recombinant colonies.

How are genes delivered into plants and animals, and how is a host made competent?

We learnt how to transfer genes into plants and animals from bacteria and viruses, which have long known how to deliver genes that transform eukaryotic cells.

Which pathogens have been turned into vectors?

• Agrobacterium tumifaciens, a pathogen of several dicot plants, delivers a piece of DNA known as T-DNA. This transforms normal plant cells into a tumour and directs the tumour cells to produce the chemicals the pathogen needs.
• Its tumour inducing (Ti) plasmid has been modified into a cloning vector that is no longer pathogenic to plants, but can still deliver genes of our interest into a variety of plants.
• Retroviruses in animals can transform normal cells into cancerous cells. They have been disarmed and are now used to deliver desirable genes into animal cells.

Once a gene or DNA fragment has been ligated into a suitable vector, it is transferred into a bacterial, plant or animal host, where it multiplies.

How is a bacterial cell made competent?

DNA is a hydrophilic molecule, so it cannot pass through cell membranes. To force bacteria to take up a plasmid, the cells must first be made competent to take up DNA.

1. The bacterial cells are treated with a specific concentration of a divalent cation, such as calcium. This increases the efficiency with which DNA enters the bacterium through pores in its cell wall.
2. The cells are incubated with recombinant DNA on ice.
3. They are placed briefly at 42°C. This is the heat shock.
4. They are put back on ice. This enables the bacteria to take up the recombinant DNA.

What other methods introduce alien DNA into host cells?

What are the steps of recombinant DNA technology, and how is pure DNA isolated?

Recombinant DNA technology involves several steps in a specific sequence.

1. Isolation of DNA.
2. Fragmentation of DNA by restriction endonucleases.
3. Isolation of a desired DNA fragment.
4. Ligation of the DNA fragment into a vector.
5. Transferring the recombinant DNA into the host.
6. Culturing the host cells in a medium at large scale.
7. Extraction of the desired product.

How is DNA isolated from cells?

Nucleic acid is the genetic material of all organisms without exception, and in the majority of organisms it is deoxyribonucleic acid, or DNA. To be cut with restriction enzymes, DNA needs to be in pure form, free from other macromolecules.

DNA is enclosed within membranes, so the cell has to be broken open. This releases DNA along with other macromolecules such as RNA, proteins, polysaccharides and lipids. Genes lie on long molecules of DNA intertwined with proteins such as histones.

Other molecules are removed by appropriate treatments. Purified DNA finally precipitates out after chilled ethanol is added, and it can be seen as a collection of fine threads in the suspension.

Two photographs of glass tubes. The upper one shows a pale mass of fine DNA threads suspended in the liquid. The lower one shows a rod lifting the stringy DNA out of a tube, which is what spooling means.

See Fig. 9.5 in your NCERT textbook

How is DNA cut and joined, and how does PCR amplify a gene?

How is the DNA cut at specific locations?

Restriction enzyme digestion is done by incubating purified DNA with the restriction enzyme at the optimal conditions for that specific enzyme.

Agarose gel electrophoresis is used to check the progression of the digestion. DNA is negatively charged, so it moves towards the positive electrode, the anode. The process is repeated with the vector DNA.

How is the recombinant DNA put together?

After the source DNA and the vector DNA have been cut with a specific restriction enzyme, the cut-out 'gene of interest' and the cut vector with space are mixed, and ligase is added. This produces the recombinant DNA.

What is PCR, and what happens in each cycle?

PCR stands for Polymerase Chain Reaction. In PCR, multiple copies of the gene (or DNA) of interest are synthesised in vitro using two sets of primers and the enzyme DNA polymerase.

Primers are small, chemically synthesised oligonucleotides complementary to regions of the DNA. The enzyme extends them using the nucleotides provided, with the genomic DNA as template.

1. Denaturation: heat separates the two strands of the double-stranded DNA.
2. Primer annealing: the primers bind to the single strands, one on each strand, at the two ends of the region to be amplified.
3. Extension: DNA polymerase extends the primers using the deoxynucleotides provided, giving two copies of the region.
4. The cycle is repeated many times, until about 1 billion copies of the segment are made.

Such repeated amplification is possible because of a thermostable DNA polymerase, isolated from the bacterium Thermus aquaticus. It remains active during the high temperature used to denature the double-stranded DNA. The amplified fragment can then be ligated with a vector for further cloning.

At the top is a double-stranded DNA with its 5′ and 3′ ends marked and the region to be amplified shaded in red. A downward arrow labelled heat leads to denaturation. Two primers then bind, one to each strand, at opposite ends of the red region; this step is labelled annealing.

An arrow labelled DNA polymerase (Taq polymerase) plus deoxynucleotides leads to extension, giving two double-stranded copies. After 30 cycles the region is amplified about 1 billion times.

See Fig. 9.6 in your NCERT textbook

How is recombinant DNA put into the host, and how is the product made on a large scale?

Competent recipient cells take up DNA present in their surroundings. If a recombinant DNA carrying an ampicillin resistance gene is transferred into E. coli, the cells become ampicillin-resistant. On agar plates containing ampicillin only the transformants grow, so the gene acts as the selectable marker.

What is a recombinant protein?

In almost all recombinant technologies the ultimate aim is to produce a desirable protein, so the foreign gene has to be expressed, under appropriate conditions. A protein-encoding gene expressed in a heterologous host gives a recombinant protein.

Cells carrying the cloned gene may be grown on a small scale in the laboratory, and the desired protein extracted and purified by different separation techniques.

What is a continuous culture?

In a continuous culture system, used medium is drained out from one side while fresh medium is added from the other. This keeps the cells in their physiologically most active log, or exponential, phase. It produces a larger biomass and so higher yields of the desired protein.

What does a bioreactor do?

Small volume cultures cannot give appreciable quantities of product. Large-scale production needed bioreactors, in which large volumes (100 to 1000 litres) of culture can be processed.

Bioreactors are vessels in which raw materials are biologically converted into specific products, individual enzymes and so on, using microbial, plant, animal or human cells. They provide the optimum growth conditions: temperature, pH, substrate, salts, vitamins and oxygen.

The most commonly used bioreactors are of the stirring type. A stirred-tank reactor is usually cylindrical, or has a curved base to help mix the contents. The stirrer gives even mixing and oxygen availability throughout. Alternatively, air can be bubbled through the reactor.

The bioreactor has an agitator system, an oxygen delivery system, a foam control system, a temperature control system, a pH control system and sampling ports, through which small volumes of the culture can be withdrawn periodically.

Panel (a), the simple stirred-tank bioreactor, is a closed vessel filled with culture broth. A motor at the top turns a shaft carrying flat bladed impellers. Labels also mark the foam braker, acid/base for pH control, steam for sterilisation and sterile air entering at the bottom.

Panel (b), the sparged stirred-tank bioreactor, has sterile air bubbles sparged through the liquid. Labels note that bubbles dramatically increase the oxygen transfer area, point to an increased surface area for oxygen transfer at the liquid surface, and mark gas entrainment.

See Fig. 9.7 in your NCERT textbook

What is downstream processing, and how do all the steps fit together?

After the biosynthetic stage is complete, the product must pass through a series of processes before it is ready for marketing as a finished product. These processes include separation and purification, which together are called downstream processing.

• The product has to be formulated with suitable preservatives.
• Such a formulation has to undergo thorough clinical trials, as in the case of drugs.
• Strict quality control testing is required for each product.
• Downstream processing and quality control testing vary from product to product.

Biotechnology deals with large-scale production and marketing of products and processes using live organisms, cells or enzymes. Modern biotechnology using genetically modified organisms became possible only when we learnt to alter the chemistry of DNA and to construct recombinant DNA.

This key process is called recombinant DNA technology, or genetic engineering. The table links each stage to the tool it uses.

Glossary

• Biotechnology — Techniques of using live organisms or enzymes from organisms to produce products and processes useful to humans.
• Genetic engineering — Techniques to alter the chemistry of DNA and RNA, introduce it into a host organism and so change the phenotype of the host.
• Bioprocess engineering — Keeping a sterile ambience in chemical engineering processes so that only the desired microbe or eukaryotic cell grows in large quantities.
• Recombinant DNA — A new combination of DNA from different sources, made in vitro by joining a DNA fragment to a vector with DNA ligase.
• Restriction endonuclease — An enzyme that recognises a specific palindromic sequence in DNA and cuts both strands at specific points in the sugar-phosphate backbone.
• Recognition sequence — The specific base sequence that a restriction enzyme recognises; Hind II recognises a specific sequence of six base pairs.
• Palindromic sequence — A sequence of base pairs in DNA that reads the same on the two strands when the orientation of reading is kept the same.
• Sticky ends — Single-stranded overhanging stretches left by a restriction enzyme cut, which form hydrogen bonds with their complementary cut counterparts.
• DNA ligase — The enzyme that acts on cut DNA molecules and joins their ends, used to link a DNA fragment to a vector.
• Plasmid — An autonomously replicating, circular, extra-chromosomal DNA, used as a vector to carry alien DNA into a host.
• Origin of replication (ori) — The DNA sequence where replication starts; it also controls the copy number of the DNA linked to it.
• Selectable marker — A gene, often for antibiotic resistance, that helps identify and eliminate non-transformants and lets only transformants grow.
• Insertional inactivation — Loss of a gene's function because foreign DNA has been inserted within its coding sequence, used to identify recombinants.
• Elution — Cutting separated DNA bands out of an agarose gel and extracting the DNA from the gel piece.
• Downstream processing — Separation and purification of a product after the biosynthetic stage, before formulation and marketing.

Common errors and misconceptions

• Misconception: Restriction enzymes cut DNA exactly at the centre of the palindrome. Correct: They cut a little away from the centre, between the same two bases on the opposite strands, which leaves single-stranded sticky ends.
• Misconception: In gel electrophoresis the largest DNA fragments travel farthest. Correct: Fragments separate by size through the sieving effect of agarose; the smaller the fragment, the farther it moves.
• Misconception: DNA moves towards the cathode in a gel. Correct: DNA is negatively charged, so it moves towards the positive electrode, the anode.
• Misconception: Blue colonies on a chromogenic substrate are the recombinants. Correct: Blue colonies have a plasmid without an insert. Recombinant colonies do not produce any colour, because the insert inactivates the β-galactosidase gene.
• Misconception: After foreign DNA is inserted at the BamH I site of pBR322, recombinants grow on tetracycline. Correct: Recombinants lose tetracycline resistance. They grow on ampicillin but not on tetracycline, while non-recombinants grow on both.
• Misconception: Any piece of DNA put into a cell will multiply in the progeny cells. Correct: Alien DNA usually multiplies only when it is linked with an origin of replication, for example by becoming part of a chromosome or a vector.
• Misconception: Vector DNA and source DNA can be cut with any two restriction enzymes. Correct: Normally both must be cut with the same restriction enzyme, so that they have the same kind of sticky ends that ligase can join.

Exam-style questions with model answers

Q1. Explain how the restriction enzyme EcoRI gets its name. [2 marks]

1. E comes from the genus Escherichia, and co comes from the species coli, of the bacterium from which the enzyme was isolated.
2. R comes from the strain, RY 13.
3. The Roman number I shows the order in which the enzyme was isolated from that strain: it was the first.

Q2. Why must bacterial cells be made competent before transformation? How is this done? [2 marks]

1. DNA is a hydrophilic molecule, so it cannot pass through cell membranes on its own.
2. The cells are treated with a specific concentration of a divalent cation such as calcium, then incubated with recombinant DNA on ice, given a brief heat shock at 42°C, and put back on ice. This lets them take up the recombinant DNA.

Q3. What are sticky ends? How are they formed, and why are they useful in genetic engineering? [3 marks]

1. A restriction enzyme recognises a palindromic sequence and cuts the DNA a little away from the centre of the palindrome, but between the same two bases on the opposite strands.
2. This leaves single-stranded overhanging stretches at the ends, called sticky ends.
3. They form hydrogen bonds with their complementary cut counterparts, which makes the action of DNA ligase easier.
4. Vector and source DNA cut with the same enzyme carry the same kind of sticky ends, so ligase can join them end to end to form recombinant DNA.

Q4. Describe how DNA fragments are separated, seen and recovered by agarose gel electrophoresis. [3 marks]

1. DNA fragments are negatively charged, so an electric field forces them through the agarose matrix towards the anode.
2. The fragments separate according to size through the sieving effect of the gel; the smaller the fragment, the farther it moves.
3. The DNA is stained with ethidium bromide and exposed to UV radiation, which shows bright orange bands.
4. The bands are cut out and the DNA is extracted from the gel piece; this is elution.

Q5. What is insertional inactivation? Explain how it is used with a chromogenic substrate to identify recombinant colonies. [3 marks]

1. Insertional inactivation is the inactivation of a gene because foreign DNA has been inserted within its coding sequence.
2. The recombinant DNA is inserted within the coding sequence of the enzyme β-galactosidase, so the gene for synthesis of this enzyme is inactivated.
3. In the presence of a chromogenic substrate, bacteria whose plasmid has no insert give blue coloured colonies.
4. Colonies with an insert do not produce any colour; these are identified as recombinant colonies.

Q6. Describe the features required in a cloning vector. Explain, using pBR322, how recombinants are selected by antibiotic resistance. [5 marks]

1. Origin of replication (ori): the sequence where replication starts. Linked DNA can replicate in the host, and ori controls its copy number.
2. Selectable marker: it helps eliminate non-transformants and lets only transformants grow, for example genes for resistance to ampicillin, chloramphenicol, tetracycline or kanamycin in E. coli.
3. Cloning sites: very few, preferably single, recognition sites for commonly used restriction enzymes, since several sites would give several fragments.
4. In pBR322, foreign DNA is ligated at the BamH I site in the tetracycline resistance gene, so recombinant plasmids lose tetracycline resistance.
5. Transformants are plated on ampicillin medium, where both kinds grow, then transferred to tetracycline medium, where only non-recombinants grow.

Q7. Describe the polymerase chain reaction. Why is a thermostable DNA polymerase needed? [5 marks]

1. PCR makes multiple copies of a gene of interest in vitro, using two sets of primers (small, chemically synthesised oligonucleotides complementary to regions of the DNA) and DNA polymerase.
2. Denaturation: heat separates the two strands of the double-stranded DNA.
3. Primer annealing: the primers bind to the single strands at the two ends of the region to be amplified.
4. Extension: DNA polymerase extends the primers using the nucleotides provided, with the genomic DNA as template.
5. Repeating the cycle amplifies the segment about a billion times.
6. A thermostable DNA polymerase from Thermus aquaticus is needed because it stays active during the high temperature used to denature the DNA.

Q8. What is a bioreactor? Describe a stirred-tank bioreactor and state what happens during downstream processing. [5 marks]

1. A bioreactor is a vessel in which raw materials are biologically converted into specific products using microbial, plant, animal or human cells. It processes large volumes (100 to 1000 litres) and provides optimum temperature, pH, substrate, salts, vitamins and oxygen.
2. A stirred-tank reactor is usually cylindrical or has a curved base to help mixing. The stirrer gives even mixing and oxygen availability; alternatively, air can be bubbled through.
3. It has an agitator system, an oxygen delivery system, a foam control system, a temperature control system, a pH control system and sampling ports for withdrawing small volumes of culture.
4. Downstream processing is the separation and purification of the product after the biosynthetic stage. The product is formulated with suitable preservatives, goes through thorough clinical trials as for drugs, and needs strict quality control testing.

Key takeaways

• Biotechnology uses live organisms or their enzymes to make useful products and processes; in its modern, restricted sense it uses genetically modified organisms on a larger scale.
• Genetic engineering and bioprocess engineering are the two core techniques that enabled the birth of modern biotechnology.
• Alien DNA multiplies in a host only when linked to an origin of replication; Cohen and Boyer made the first recombinant DNA in 1972.
• Restriction endonucleases recognise palindromic sequences and cut both strands a little away from the centre, leaving sticky ends that DNA ligase can join.
• In agarose gel electrophoresis, negatively charged DNA moves towards the anode, smaller fragments move farther, and ethidium bromide with UV light shows orange bands.
• A cloning vector needs an origin of replication, a selectable marker and very few, preferably single, recognition sites for commonly used restriction enzymes.
• Recombinants are selected by insertional inactivation, either of an antibiotic resistance gene such as tetᴿ in pBR322 or of the β-galactosidase gene.
• DNA enters host cells through calcium-treated competent cells with heat shock, micro-injection, biolistics or disarmed pathogen vectors such as the Ti plasmid.
• PCR repeats denaturation, primer annealing and extension, using thermostable DNA polymerase from Thermus aquaticus, to amplify DNA about a billion times.
• Bioreactors of 100 to 1000 litres grow cells on a large scale, and downstream processing separates, purifies and formulates the product before marketing.

Test yourself

What is a recognition sequence?

It is the specific base sequence that a restriction enzyme recognises before it cuts DNA. Hind II, for example, recognises a specific sequence of six base pairs.

Why is DNA stained with ethidium bromide after electrophoresis?

Pure DNA fragments cannot be seen in visible light without staining. After ethidium bromide staining and exposure to UV light, the DNA shows as bright orange bands.

Which enzymes are used to break open bacterial, plant and fungal cells when isolating DNA?

Lysozyme is used for bacteria, cellulase for plant cells and chitinase for fungi.

What does rop code for in pBR322?

rop codes for the proteins involved in the replication of the plasmid.

What is a recombinant protein?

A protein made when a protein-encoding gene is expressed in a heterologous host is called a recombinant protein.

How does a continuous culture system lead to higher yields of the desired protein?

Used medium is drained from one side while fresh medium is added from the other. This keeps cells in their most active log or exponential phase, giving a larger biomass and higher yields of protein.

Name the two metal particles used in biolistics, and the kind of cell this method suits.

Biolistics, or the gene gun, uses high velocity micro-particles of gold or tungsten coated with DNA. It is suitable for plant cells.