Chapter 12: Biotechnology
In the early 1970s, two American scientists met at a conference and began a conversation that would quietly reshape biology. Stanley Cohen worked on plasmids — the small, circular pieces of DNA that bacteria carry apart from their main chromosome. Herbert Boyer worked on an enzyme from the bacterium Escherichia coli that could cut DNA at one exact sequence, leaving the cut ends slightly “sticky.”
Put those two ideas together and something remarkable follows. If an enzyme can cut a plasmid open at a chosen point, and cut a piece of foreign DNA in the same way, then the two cut ends should stick to each other. Seal the join, slip the plasmid back into a bacterium, and the bacterium will copy that foreign DNA every time it divides — millions of times, overnight, for free.
In 1973 Cohen and Boyer showed exactly this. They built the first recombinant DNA molecule and got bacteria to carry it. For the first time, a gene could be taken out of one organism and made to work inside another. A bacterial cell had become a living factory.
Everything in this chapter — human insulin brewed in bacteria, pest-resistant cotton, golden rice, gene therapy, and the arguments about who owns a gene — grows out of that single idea.
Diagram
Fig. 12.0 – Opening illustration: a bacterial cell with its chromosome and a plasmid; the plasmid is cut open, a foreign gene fragment is slotted in, and the cell divides to give many bacteria each carrying the new gene.
Keep these questions in mind as you read. This chapter will answer every one of them.
12.1 Biotechnology
Biotechnology is what happens when biological science and technology meet. Biology supplies the living material — cells, microbes, enzymes — and technology supplies the scale, the machinery and the control needed to turn that material into something useful. For this reason biotechnology is best described as an applied branch of biology: its purpose is not only to understand life, but to put life to work.
The word itself is only about a century old. The term biotechnology was first used by Karl Ereky in 1919, to describe a process for the large-scale production of pigs. Even in that early, rather agricultural sense, the essential idea was already present — using living organisms in an organised, industrial way to obtain a product.
How biotechnology is defined
Different organisations define biotechnology in slightly different ways, because each looks at it from its own angle. Broadly, it is understood as the development and utilisation of biological forms, products or processes to obtain maximum benefit to human beings and to other forms of life.
A more formal, industry-oriented definition was given by the OECD (Organization for Economic Cooperation and Development) in 1981. It stresses that biotechnology is not merely biology — engineering principles are applied to biological agents so that materials are processed into goods and services.
Notice how many subjects such a definition pulls together. Biotechnology uses the scientific principles of microbiology, genetics, biochemistry, chemical engineering, mathematics, statistics, computers and industrial processes. A biotechnologist must know how a microbe grows, how a gene is expressed, how a reaction is scaled up in a tank, and how data from thousands of samples is analysed.
The phrase “biological agent” in these definitions carries a precise meaning, and it is worth fixing it in your mind early, because the entire chapter depends on it.
Two phases of growth
The history of biotechnology is as old as human civilisation itself. Its growth, however, took place in two clearly different phases — traditional biotechnology and modern biotechnology. The difference between them is not only a difference of time but of understanding and of power over the genetic material. The overall path of this growth is summarised in FC 12.1.
Flowchart
FC 12.1 – Growth of biotechnology: traditional biotechnology (fermentation — curd, cheese, wine) → work of chemists, biochemists and microbiologists explains the mechanism and the microbes responsible → 1970: recombinant DNA technology developed → 1973: Cohen and Boyer establish the technique → modern biotechnology (genetic engineering-based)
Traditional biotechnology
Traditional biotechnology, also called old biotechnology, was based primarily on fermentation technology using microorganisms. Fermentation is the chemical breakdown of a substance by microbes — and long before anyone knew what a microbe was, human beings were already using it. The preparation of curd, ghee, soma, vinegar, yogurt (yoghurt), cheese-making and wine-making all belong here.
In Indian homes, this became an art of the kitchen, handed down from one generation to the next. And that is exactly the point: it was more an art than a science. Till that time people did not know how exactly the process occurred, nor which organisms caused it. A woman setting curd knew that a spoon of old curd added to warm milk would work; she did not know that she was adding Lactobacillus.
Over time, the contributions made by several chemists, biochemists and microbiologists could explain both the mechanism of the process and the nature of the microorganisms causing it. Once you know which organism does the job and how, you can select it, purify it, feed it better and control the conditions — and an art begins to turn into a science.
Modern biotechnology
The decisive break came in the 1970s. During 1970 a new technique, recombinant DNA technology, was developed, and it was then established by Stanley Cohen and Herbert Boyer in 1973. This technique changed the overall outlook of the subject, because for the first time it permitted scientists to change or modify the genetic (heritable) material itself in order to obtain new, specific products.
Think about what that means. In traditional biotechnology, you could only use the abilities an organism already had. In modern biotechnology, you can give an organism an ability it never possessed — because you can rewrite its instructions.
This combination of biology and production technology, built on genetic engineering, evolved into what we call modern biotechnology (or new biotechnology).
What sets modern biotechnology apart
There are two major features of technology that differentiate modern biotechnology from classical or old biotechnology.
The capability of science to change the genetic material for obtaining new specific products. This is done through rDNA technology, polymerase chain reaction (PCR), microarrays, cell culture and fusion, and bioprocessing. Each of these is a tool you will meet in the sections ahead.
The ownership of the technology and its socio-political impact. This second feature is not a laboratory technique at all — it is a social one. When a gene, a seed or a microbe can be owned, questions of patents, profit, and fairness between nations enter the picture. This is why a chapter on biotechnology must end with bioethics, biopatents and biopiracy.
Today the reach of this subject extends well beyond the laboratory. Conventional industries, pharmaceutical industries, agro industries and food industries are all now focusing their attention on producing biotechnology-based products — because a product made by a living cell is often cheaper, purer and more specific than one made by chemistry alone.
For NEET and board examinations, this section is usually tested through direct recall — who coined the term and in which year, the OECD definition, and above all the two features that separate modern from classical biotechnology. Assertion–reason questions frequently pair the statement “curd formation is biotechnology” with the reason “the genetic material of the bacteria is modified,” where the assertion is true but the reason is false.
12.2 Principles and Processes of Biotechnology
Modern biotechnology rests on two core techniques: genetic engineering and chemical engineering. The two are partners. One decides what the cell will make; the other makes sure the cell can be grown, safely and on a large scale, so that the product can actually be collected.
Genetic engineering deals with the alteration of the genetic material — DNA and RNA. Chemical engineering deals with maintaining a sterile environment for manufacturing a variety of useful products, including vaccines, antibodies, enzymes, organic acids, vitamins and therapeutics. The word sterile is the key one here. A culture in which even a single unwanted microbe grows will compete with the engineered cells, spoil the product, and waste an entire batch.
Genetic engineering
Genetic engineering is defined as the manipulation of genetic material towards a desired end, in a directed and predetermined way, using an in vitro process. Each phrase in this definition is doing work. Desired end means the outcome is chosen in advance. Directed and predetermined separates it from natural mutation, which is random. In vitro means the actual cutting and joining of DNA is done outside the living body, in glassware.
Manipulation of genes covers a wide range of operations:
- repairing defective genes, or replacing defective genes by healthy (normal) genes;
- artificially synthesising a totally new gene;
- transferring genes into a new location, or into a new organism;
- introducing an altogether new gene;
- manipulating genes for the improvement of living organisms;
- combining genes from two organisms, thereby altering the genotype;
- gene cloning.
Because the central act in all of these is the joining of DNA from different sources and its multiplication, genetic engineering is alternatively called recombinant DNA technology or gene cloning. The three names describe the same body of work from three angles: what is being changed (the genes), what is being made (recombinant DNA), and what is being obtained (many identical copies — clones).
What gene cloning means
In gene cloning, a gene of known function is transferred from its normal location into a cell that does not contain it, with the help of a suitable vector. Once inside, the transferred gene is replicated normally along with the host’s own DNA, and is handed over to the next progeny.
This last point is what makes the technique so powerful. You transfer the gene once; the host cell then copies it for you, generation after generation, at no further cost. A colony of bacteria carrying a human gene is a self-multiplying supply of that gene.
Tools and techniques for gene cloning / rDNA technology
Before we look at the procedure itself, we must know the basic requirements of the technique. These fall into two groups: the instruments (devices) used in the laboratory, and the biological tools taken from living organisms. The overall arrangement of these tools is shown in FC 12.2.
Flowchart
FC 12.2 – Tools for rDNA technology: Tools → (I) Instruments/devices → electrophoresis, chromatography, spectroscopy, mass spectrometry, PCR (thermal cycler); and (II) Biological tools → enzymes (restriction enzymes, ligases, polymerases and others) → cloning vectors (plasmids, bacteriophages and others) → competent hosts (mainly E. coli)
I. Instruments (devices)
Macromolecules such as DNA, RNA and proteins are synthesised inside living cells, and they differ from one another in molecular weight, solubility, the presence of charges, and the absorbance of light. Every one of these differences can be used to pull them apart from a mixture — which is exactly what a purification technique does.
Several techniques are used to isolate and characterise macromolecules. Those that work on the basis of molecular weight include gel permeation, ion exchange chromatography, spectroscopy, mass spectrometry and electrophoresis.
Electrophoresis
Electrophoresis is the separation of charged molecules by applying an electric field. It is applied for the separation of DNA, RNA and proteins.
Why does it work for DNA so neatly? Because DNA carries a phosphate group on every nucleotide, it is negatively charged along its whole length. Placed in an electric field, DNA therefore migrates towards the anode (the positive electrode). The gel through which it moves acts as a molecular sieve: small fragments of DNA slip through the pores easily and move faster, so they separate faster and travel further, while large fragments lag behind. The arrangement is shown in Fig. 12.2.
Diagram
Fig. 12.2 – Agarose gel electrophoresis: gel slab with wells at the cathode end, DNA samples loaded, bands separating along the gel towards the anode, with large fragments near the wells and small fragments furthest away; label cathode, anode, wells and bands.
The different methods of electrophoresis in common use are agarose gel electrophoresis, PAGE and SDS PAGE.
Polymerase chain reaction (PCR)
The polymerase chain reaction is another device used for gene cloning, or more precisely for gene multiplication in vitro. It is the amplification of the gene of interest.
In 1985, Kary Mullis made an important contribution in the form of an extremely powerful technique called the polymerase chain reaction (PCR). PCR can generate a billion copies of a desired segment of DNA or RNA, with high accuracy and specificity, in a matter of a few hours. The process of PCR is completely automated and involves automatic thermal cycles for the denaturation and renaturation of double-stranded DNA. The device required for PCR is called a thermal cycler.
PCR is the in vitro amplification of a desired DNA segment, and it requires:
- DNA containing the desired segment to be amplified (the template);
- several molecules of the four deoxyribonucleoside triphosphates (dNTPs) — the raw nucleotides from which new strands are built;
- an excess of two primer molecules — short DNA sequences that mark where copying must begin on each strand;
- heat-stable DNA polymerase — the enzyme that adds nucleotides;
- appropriate quantities of \(Mg^{++}\) ions — needed for the polymerase to work.
Mechanism of PCR
At the start of PCR, the DNA segment, an excess of two primer molecules, four deoxyribonucleoside triphosphates and the thermostable DNA polymerase are mixed together in an eppendorf tube. The following operations are then performed sequentially, as shown in Fig. 12.1 and summarised as a cycle in FC 12.3.
Step i — Denaturation. The reaction mixture is heated to a temperature of 90–98 °C to separate the two strands of the desired DNA. High heat breaks the hydrogen bonds between the base pairs, but not the strong phosphodiester backbone, so the two strands come apart intact.
Step ii — Annealing. The mixture is allowed to cool to 40–60 °C. This permits the pairing of the primer to the complementary sequences in the DNA. This step is called annealing. The temperature must be low enough for the primer to bind, but not so low that the two long strands simply re-join with each other.
Step iii — Primer extension. The temperature of 70–75 °C allows the thermostable Taq DNA polymerase to use the single-stranded DNA as a template and to add nucleotides, extending each primer into a full new strand. This is called primer extension, and it takes around two minutes.
Diagram
Fig. 12.1 – DNA replication through polymerase chain reaction: one copy of the desired gene → (1) denaturation at 90–98 °C, DNA strands separated → (2) annealing of primers at 40–60 °C → replication of DNA strands using Taq polymerase at 70–75 °C → (3) two copies of the desired gene (renaturation), first cycle complete → second cycle gives four copies → cycle continues to produce 2ⁿ copies.
Flowchart
FC 12.3 – One PCR cycle: heat to 90–98 °C (denaturation) → cool to 40–60 °C (annealing of two primers) → raise to 70–75 °C (primer extension by Taq polymerase) → 2 copies formed → back to denaturation for the next cycle; repeat 20–30 times
One cycle takes around 3 to 4 minutes. To begin the second cycle, the DNA is again heated to convert double-stranded DNA into single strands — and note that now there are two double-stranded molecules to work on instead of one.
In an automatic thermal cycler, the above three steps are automatically repeated 20–30 times. Thus, at the end of ‘n’ cycles, \(2^n\) copies of the DNA segment are produced. The machine performs the entire operation automatically and precisely.
\[\text{Number of copies after } n \text{ cycles} = 2^n\]
Once the desired number of cycles is completed, the amplified DNA segment is purified by gel electrophoresis. After its sequencing, the amplified DNA segment can be inserted into a cloning vector. A desired gene can also be obtained from a gene library.
II. Biological tools
There are three types of biological tools used for transformation with recombinant DNA: enzymes, cloning vectors (vehicle DNA), and competent hosts (cloning organisms). Together they answer three questions — what cuts and joins the DNA, what carries it, and what multiplies it.
A. Enzymes
Different enzymes are used at different stages of the work. They include lysozymes, nucleases (such as exonucleases and endonucleases), restriction endonucleases, DNA ligases, DNA polymerases, alkaline phosphatases and reverse transcriptases. Lysozyme opens the cell wall to release the DNA, nucleases cut it, ligases join it, polymerases copy it, and reverse transcriptase makes DNA from RNA.
i. Restriction enzymes
Enzymes that cut the phosphodiester bonds of polynucleotide chains are called nucleases. The phosphodiester bond is the linkage that holds one nucleotide to the next along the backbone of a DNA strand, so cutting it means cutting the strand itself.
Nucleases are of two types — exonucleases and endonucleases. Exonucleases cut nucleotides from the ends of DNA strands, whereas endonucleases cut DNA from within.
During the 1970s, it was found that bacteria contain nucleases that would recognise a short nucleotide sequence within duplex DNA and cut it. The phosphodiester backbone at highly specific sites on both strands of the duplex is cut by these enzymes, which are called restriction endonucleases (REN), or simply restriction enzymes (RE).
Why that curious name? They were given this name because they are used by the bacteria to destroy various viral DNAs that might enter the cell, thereby restricting the potential growth of the virus.
Thus, restriction enzymes serve as a defence mechanism. But this raises an obvious difficulty — if the enzyme cuts DNA wherever it finds its recognition sequence, why does it not cut the bacterium’s own DNA as well? The bacterium protects its own DNA from nucleolytic attack by methylating the bases at susceptible sites. This chemical modification blocks the action of the enzyme, so the enzyme cuts the invading viral DNA but leaves the host’s own, methylated DNA untouched.
The restriction enzymes are thus the molecular scissors used to recognise and cut DNA at specific sequences. The sites recognised by them are called recognition sequences or recognition sites. Different restriction enzymes found in different organisms recognise different nucleotide sequences, and therefore cut DNA at different sites. This is precisely what makes them so useful: by choosing the enzyme, the biotechnologist chooses where the cut will fall. Table 12.2 lists some restriction endonucleases and the sites at which they cleave DNA.
Table 12.2 : Source and recognition sequences of various restriction enzymes
| Restriction enzyme | Source (organism and strain) | Recognition sequence (arrow = site of cut) | Product | End products produced |
|---|---|---|---|---|
| Alu I | Arthobacter luteus | 5’ —A-G ↓ C-T— 3’ 3’ —T-C ↑ G-A— 5’ |
5’—A-G C-T—3’ 3’—T-C G-A—5’ |
Blunt ends |
| Bam HI | Bacillus amyloliquefaciens H | 5’ —G ↓ G-A-T-C-C— 3’ 3’ —C-C-T-A-G ↑ G— 5’ |
5’—G G-A-T-C-C—3’ 3’—C-C-T-A-G G—5’ |
Sticky ends |
| EcoRI | Escherichia coli Ry13 | 5’ —G ↓ A-A-T-T-C— 3’ 3’ —C-T-T-A-A ↑ G— 5’ |
5’—G A-A-T-T-C—3’ 3’—C-T-T-A-A G—5’ |
Sticky ends |
| Hind II | H. influenzae Rd | 5’ –G-T-C ↓ G-A-C– 3’ 3’ –C-A-G ↑ C-T-G– 5’ |
5’–G-T-C G-A-C–3’ 3’–C-A-G C-T-G–5’ |
Blunt ends |
ii. Recognition sequences
The sequences recognised by restriction enzymes are 4 to 8 nucleotides long, and are characterised by a particular type of internal symmetry. Consider the sequence recognised by the enzyme EcoRI:
3’ ——- C T T A A G —–5’ 5’ ——- G A A T T C —–3’
Read the top strand from right to left (3’ to 5’) and the bottom strand from left to right (5’ to 3’): the sequence is identical in both cases. A sequence with this type of symmetry is called a palindrome.
When the enzyme EcoRI attacks this palindrome, it breaks each strand at the same site in the sequence — between the A and the G residues, as indicated by the arrows. Notice in Fig. 12.3 that the two cuts are not opposite each other but staggered:
3’ —- C T T A A ↑ G ——-5’ 5’ —- G ↓ A A T T C ——-3’
Diagram
Fig. 12.3 – Mode of action of EcoRI to cleave DNA: DNA showing the palindromic recognition sequence GAATTC/CTTAAG, the EcoRI enzyme binding at the site, and the DNA cut into two fragments each carrying a single-stranded AATT projection (sticky end).
Restriction enzymes either cut straight across the DNA in the region of the palindrome, giving blunt ends, or make staggered cuts that produce short, single-stranded projections at each end of the DNA — these are called cohesive or sticky or staggered ends.
B. Cloning vectors (vehicle DNA)
Vectors are DNA molecules that carry a foreign DNA segment and replicate inside the host cell. Cutting a gene out is of no use unless something carries it in and keeps it multiplying — that “something” is the vector, which is why it is also called vehicle DNA.
Vectors may be plasmids, bacteriophages (M13, lambda virus), cosmids, phagemids, BAC (bacterial artificial chromosome), YAC (yeast artificial chromosome), transposons, baculoviruses and mammalian artificial chromosomes (MACs). The most commonly used vectors are plasmid vectors (pBR 322, pUC, Ti plasmid) and bacteriophages (lambda phage, M13 phage). Plasmids and bacteriophages are, in practice, the two workhorses of the field.
Features of a good vector
A good vector should have the ability of independent replication, so that as the vector replicates (through the ori gene, the origin of replication), a large number of copies of the DNA insert will be formed. Moreover, the vector should be able to get introduced into host cells easily.
Beyond this, a vector should have marker genes for antibiotic resistance; it must contain a unique cleavage site for a restriction enzyme in one of the marker genes; and it should have at least suitable control elements such as a promoter, an operator and ribosomal binding sites. The control elements matter because a gene that has been carried in but never switched on produces nothing — the promoter is what allows the host’s machinery to transcribe it.
The plasmids obtained naturally do not possess all of these characteristics. Hence they are constructed, by inserting a gene for antibiotic resistance into them. For example, pBR 322, pBR 320 and pACYC 177 are constructed plasmids. pBR 322 is mostly used in rDNA technology in plants.
i. Plasmid
The plasmids most commonly used in recombinant DNA technology are those that replicate in E. coli. Investigators have engineered these plasmids to optimise their use as vectors in DNA cloning. As seen in Fig. 12.4, such a plasmid carries a replication origin (ori), a drug-resistance gene (\(amp^r\), for ampicillin resistance) and a polylinker — a short region packed with unique cleavage sites into which foreign DNA can be inserted.
Diagram
Fig. 12.4 – Plasmid cloning vector showing a replication origin (ori), a drug resistance gene (ampʳ) and a polylinker region into which foreign DNA can be inserted, with the restriction sites HindIII, SphI, PstI, SalI, XbaI, BamHI, SmaI, KpnI, SacI and EcoRI marked.
ii. Plasmid vectors for plants
An important vector for carrying new DNA into many types of plants is a plasmid found in Agrobacterium tumefaciens. This bacterium lives in the soil and causes a plant disease called crown gall, which is characterised by the presence of overgrowths, or tumours, in the plant.
A. tumefaciens contains a plasmid called Ti (for tumor-inducing). The Ti plasmid contains a transposon, called T DNA, which inserts copies of itself into the chromosomes of infected plant cells. Here lies the opportunity: the transposon, loaded with new DNA, can still be inserted into the host cell’s chromosomes. A plant cell containing this DNA can then be grown in culture, or induced to form a new, transgenic plant.
C. Competent hosts (cloning organisms)
The competent hosts, or cloning organisms, used are usually bacteria like Bacillus haemophilus, Helicobacter pyroli and E. coli. Mostly E. coli is used for transformation with recombinant DNA.
“Competent” is a technical word, not a compliment. It means the cell has been treated so that its membrane will allow naked DNA to pass in — an ordinary bacterial cell will not take up DNA readily. E. coli is preferred because it grows very fast, is harmless in its laboratory strains, is genetically the best-understood organism on earth, and takes up plasmids readily once made competent.
For NEET and board examinations, this section is a favourite hunting ground. Expect questions on which enzyme cuts phosphodiester bonds, the exact temperature of each PCR step, the recognition sequence of EcoRI or Hind III, the meaning of a palindrome, and match-the-following items pairing a restriction enzyme with its source organism or with the type of end it produces.
12.3 Methodology for rDNA Technology
We now have the tools — scissors, glue, a vehicle and a host. This section puts them to work in order. The steps involved in gene cloning follow one another logically: get the gene out, put it into a vector, get the vector into a host, pick out the hosts that actually took it, grow them, and finally collect what they make. The whole sequence is outlined in Fig. 12.5 and summarised in FC 12.4.
Diagram
Fig. 12.5 – Outline of the process of recombinant DNA technology: (A) donor cell → isolation of desired DNA (target DNA); (B) enzymatic fragmentation by restriction enzyme, and a vector (plasmid or bacteriophage) cut with the same enzyme; (C) joining of target DNA and cloning vector by DNA ligase → rDNA; (D) introduction into a bacterial host cell (transformation / viral infection); (E) transformed host cell containing the rDNA molecule; (F) selection, identification and amplification; (G) cell multiplication → protein production from the cloned gene → protein harvesting.
Flowchart
FC 12.4 – Steps in rDNA technology: isolation of DNA (gene) from donor → cleavage by restriction enzyme → selection of the desired fragment (foreign/passenger DNA) → insertion into a cloning vector cut by the same enzyme, sealed by DNA ligase → recombinant (chimeric) DNA → transfer into a competent host (transformation, aided by Ca²⁺) → selection of transformed host cells using marker genes → multiplication of transformed cells in fresh culture medium → expression of the gene → downstream processing in a bioreactor → desired product
a. Isolation of DNA (gene) from the donor organism
- The desired gene to be cloned has to be obtained from the source organism, which is called the donor. Initially the cells of the donor organism are sheared with a blender and treated with a suitable detergent. There is a reason for this rough treatment: DNA lies protected inside the cell, wrapped in membranes, so the cell must be broken open. The detergent dissolves the lipid membranes and releases the contents.
The genetic material from the donor is then removed, isolated and purified by using several techniques. Purified DNA, when precipitated, appears as fine white threads and can be spooled on to a glass rod — the first moment in the whole procedure when a gene becomes something you can actually see.
- The isolated, purified DNA is then cleaved by using restriction enzymes, particularly restriction endonucleases (RE). These enzymes cleave DNA at specific sites, called restriction sites, and break the DNA into fragments. There are several types of restriction endonucleases, and the choice of enzyme decides where the breaks fall. The cleaved DNA fragments have cohesive, sticky, staggered ends or blunt ends.
From the cleaved DNA fragments, a fragment containing the desired gene is isolated and selected for cloning. This selected fragment is now called foreign DNA or passenger DNA. The name “passenger” is apt — from this point on, the gene is a traveller that will be carried by a vehicle.
A desired gene can also be obtained directly from a genomic library or a cDNA library, without going through the whole isolation procedure each time.
b. Insertion of the desired foreign gene into a cloning vector
The foreign DNA, or passenger DNA, is now inserted into a cloning vector, that is, into vehicle DNA. The most commonly used cloning vectors are plasmids of bacteria and bacteriophage viruses like lambda phage and M13. The most commonly used plasmid is pBR 322.
Plasmids are isolated from the vector organism, that is, from the bacterium. The plasmid — the vector DNA — is then cleaved by using the same restriction enzyme that was used to cut the desired gene out of the donor.
This detail is not a formality; it is the heart of the step. Because the same enzyme is used, the vector and the foreign DNA are left with identical sticky ends, whose single-stranded overhangs are complementary to one another. They therefore pair up spontaneously by hydrogen bonding.
The pairing of sticky ends only holds the two pieces together; the sugar–phosphate backbone is still broken. Now, by using the enzyme DNA ligase, the foreign DNA is inserted and integrated into the vector DNA. Ligase seals the nicks by forming phosphodiester bonds, making one continuous, closed molecule.
The combination of vector DNA and foreign DNA is now called Recombinant DNA or Chimeric DNA, and the technology is referred to as rDNA technology.
c. Transfer of rDNA into a suitable competent host or cloning organism
Finally the recombinant DNA is introduced — that is, transferred for expression — into a competent host cell of a suitable cloning organism, which is usually a bacterium.
The host cell takes up the naked rDNA by the process of transformation and incorporates it into its own chromosomal DNA, which finally expresses the trait controlled by the passenger DNA.
The transfer of rDNA into a bacterial cell is assisted by divalent \(Ca^{++}\) ions. Both the DNA and the bacterial cell surface carry negative charges and would otherwise repel each other; the divalent calcium ions neutralise this repulsion and help the DNA to pass through the membrane.
The cloning organisms used in plant biotechnology are E. coli and Agrobacterium tumifaciens. The host, or competent cell, which has taken up rDNA is now called a transformed cell.
Foreign DNA can also be transferred directly into the naked cell or protoplast of the competent host cell, without using a vector at all. This is done by using techniques like electroporation (brief electric pulses that open temporary pores in the membrane), microinjection (the DNA is injected directly with a fine needle), lipofection (the DNA is packed in lipid droplets that fuse with the membrane), the shot gun or biolistic method (DNA-coated metal particles are literally fired into cells), and ultrasonification. But in plant biotechnology, transformation is most often carried out through the Ti plasmids of A. tumifaciens.
d. Selection of the transformed host cell
Transformation is never complete. The process generates a mixed population of transformed (recombinant) and non-transformed (non-recombinant) host cells — and to the eye, they look exactly alike. So how do we find the few cells that actually received the gene, among the millions that did not?
For the isolation of recombinant cells from non-recombinant cells, the marker genes of the plasmid vector are employed. For example, the pBR 322 plasmid vector contains different marker genes — an ampicillin-resistant gene and a tetracycline-resistant gene.
When the Pst1 RE is used, it cuts within the ampicillin-resistant gene, and so knocks out the ampicillin-resistant gene from the plasmid. As a result, the recombinant cell becomes sensitive to ampicillin — while a cell carrying an unaltered, non-recombinant plasmid remains resistant. The loss of resistance is therefore a signal that the foreign DNA went in exactly where it was meant to.
e. Multiplication of the transformed host cell
Once transformed, the host cells are separated by the screening process just described. In this step, the transformed host cells are introduced into fresh culture media.
At this stage the host cells divide and redivide, and the recombinant DNA carried by them is replicated along with the cells. One transformed bacterium becomes a colony; a colony becomes a culture; and every single cell in that culture carries a faithful copy of the foreign gene. This is the moment at which “cloning” earns its name.
f. Expression of the gene to obtain the desired product
The next step involves the production of the desired products, such as alcohol, enzymes, antibiotics, and so on. Multiplying the gene is not the goal in itself — the goal is the protein or the metabolite the gene codes for, and this appears only when the gene is expressed.
Finally, the desired product is separated and purified through downstream processing, using a suitable bioreactor. A bioreactor is a large vessel in which the culture is grown under precisely controlled temperature, pH, oxygen supply and stirring — this is the chemical-engineering half of biotechnology doing its work. Downstream processing covers everything that happens after the reaction is over: separation of the cells, extraction of the product, purification, quality testing and formulation. A product that cannot be separated cleanly is, commercially speaking, no product at all.
For NEET and board examinations, this section is most often tested as a sequence-ordering question — “arrange the steps of rDNA technology in the correct order” — or through the two classic reasoning traps: why the same restriction enzyme must cut both donor DNA and vector, and why the recombinant cell loses resistance to one antibiotic. Diagram-based questions on Fig. 12.5, with steps A to G left unlabelled, are equally common.
12.4 Applications of Biotechnology
Biotechnology is an umbrella term. It covers a broad spectrum of scientific applications used in many sectors — health and agriculture, industry, environment and genomics. What holds this variety together is a single idea: once a gene can be moved and expressed at will, any organism can be asked to do a job it never did before.
The main branches of the subject are shown in Fig. 12.6. Plant biotechnology covers plant cell and tissue culture, GM crops and marker-assisted selection. Animal biotechnology covers tissue culture, regenerative medicine, immunotechnology, GM animals, pharmacogenomics and gene therapy. Industrial biotechnology covers enzyme biotechnology, protein engineering, metabolic engineering and metabolomics. Environmental biotechnology covers pollution control, biofuels, bioremediation and biodiversity. Microbial biotechnology covers microbial genomics, biofertilisers, biopesticides and IPM (integrated pest management). Genomics covers whole genome sequencing, gene function, reverse genetics and bioinformatics.
Diagram
Fig. 12.6 – Applications of Biotechnology: a central hub labelled “Biotechnology” with six arms radiating outward — plant biotechnology, animal biotechnology, industrial biotechnology, environmental biotechnology, microbial biotechnology and genomics — each arm listing its sub-fields.
a. Healthcare Biotechnology
Healthcare biotechnology refers to a medicinal or diagnostic product, or a vaccine. This branch has had a tremendous impact on meeting the needs of patients, because it can offer solutions that are unique, targeted and personalised — therapeutic and diagnostic solutions, including those needed for organ transplant.
Several familiar practices are outcomes of this branch: stem cell technology, genetic counselling, forensic medicine, gene probes, genetic fingerprinting and karyotyping. Each of these depends on the ability to read, copy or manipulate DNA.
Before rDNA technology, a human protein needed for treatment could be obtained only by extracting it from human or animal tissue — in tiny amounts, at great cost, and with a real risk of contamination. Today, the gene for that protein is simply cloned and expressed in a host cell. Table 12.7 lists the human proteins now produced by rDNA technology to treat human diseases.
Table 12.7 : Human proteins produced by rDNA technology to treat human diseases
| Disorder / Disease / Health condition | Recombinant protein(s) |
|---|---|
| 1. Anaemia | Erythropoeitin |
| 2. Asthma | Interleukin-1 receptor |
| 3. Atherosclerosis | Platelet derived growth factor |
| 4. Parturition | Relaxin |
| 5. Blood clots | Tissue Plasminogen Activator (TPA), Urokinase |
| 6. Cancer | Interferons, tumour necrosis factor, interleukins, macrophage activating factor |
| 7. Diabetes | Insulin |
| 8. Emphysema | \(\alpha_1\)-Antitrypsin |
| 9. Haemophilia A | Factor VIII |
| 10. Haemophilia B | Factor IX |
| 11. Hepatitis B | Hepatitis B vaccine |
Human insulin
Insulin is a peptide hormone produced by the β-cells of the islets of Langerhans of the pancreas. It was discovered by Sir Edward Sharpey Schafer (1916), while he was studying the islets of Langerhans.
Insulin is essential for the control of blood sugar levels. It signals the body’s cells to take up glucose from the blood after a meal, so that the blood glucose falls back to its normal range. Diabetes mellitus is a disease in which some people cannot make insulin themselves — so glucose remains in the blood instead of entering the cells, and over years this damages the eyes, kidneys, nerves and blood vessels.
How was it done? Hakura et al. (1977) chemically synthesised the DNA sequence of insulin for the two chains, A and B, and inserted them separately into two pBR 322 plasmid vectors. Insulin production by recombinant DNA technology was then designed by Gilbert and Villokomaroff in 1978.
The genes for chain A and chain B are inserted next to the B galactoside gene, separately, in separate plasmid vectors. The recombinant plasmids were then separately transformed into E. coli hosts. The host produced penicillinase + pre-pro insulin, and insulin is later separated by trypsin treatment. The sequence is set out in FC 12.5.
Flowchart
FC 12.5 – Production of human insulin by rDNA technology: DNA sequences for chain A and chain B synthesised chemically → each inserted separately next to the B galactoside gene in a separate pBR 322 plasmid → the two recombinant plasmids separately transformed into E. coli → host cells produce penicillinase + pre-pro insulin → insulin separated by trypsin treatment → purified human insulin
Vaccine production
A vaccine is a biological preparation that provides active acquired immunity against a certain disease. Usually a vaccine consists of a biological agent that represents the disease-causing microorganism. It is often made from a weakened or killed form of the microorganism, from its toxins, or from one of its surface protein antigens.
The word active in that definition matters. The vaccine does not hand the body ready-made protection; it presents a harmless sample of the enemy so that the body’s own immune system learns to recognise it and keeps a memory of it. When the real pathogen arrives, the response is immediate.
Biotechnology has offered modern diagnostic test kits — rickettsial, bacterial and viral vaccines — along with radiolabelled biological therapeutics for imaging and analysis.
Vaccines have eliminated small pox, polio and other deadly diseases over the last several decades. Biotechnology has made further advancements in vaccination by making recombinant vaccines, which have the potential to eradicate even non-communicable diseases like cancer. Naked DNA vaccines, viral vector vaccines and plant-derived vaccines are found to be most effective against a number of bacterial and viral disorders.
Oral vaccines: a novel approach
The latest hot spot in the field of vaccine research is the development of a vaccine that can be taken orally. The immunogenic protein of certain pathogens is found to remain active when administered by mouth — it survives the passage far enough to be recognised by the immune tissue of the gut.
The gene corresponding to such a protein is isolated and a gene construct is produced. This is introduced and expressed in a plant genome, which results in the production of the immunogenic protein in the parts of the plant where the gene is expressed. When these plant parts are fed to animals, or mainly to humans, the person becomes vaccinated against that pathogen. Such vaccines are also known as edible vaccines.
Flowchart
FC 12.6 – Making an edible vaccine: identify the immunogenic protein of the pathogen → isolate its gene → prepare the gene construct → introduce and express it in the plant genome → the immunogenic protein accumulates in the edible part of the plant → the plant part is eaten → the immune system responds → the person is vaccinated
An exciting invention is the production of “melt in the mouth” vaccines, which can be administered by placing them under the tongue, from where they are delivered into the bloodstream. The most important example is the production of a flu vaccine by Bacillus subtilis, which melts in the mouth.
The tremendous benefit of such vaccines lies in the comfort of administration, the low cost and the ease of storage. Consider what those three words save: no needles, no trained staff to inject, no syringes to sterilise, and — most importantly for a tropical country — no unbroken cold chain of refrigeration from the factory to the remotest village.
b. Agriculture
Applications of biotechnology in agriculture involve the use of genetically modified crops, produced through gene manipulation. GM crops that are pest resistant, stress tolerant, and so on, are used to improve productivity.
Tissue culture is used in micropropagation — the large-scale propagation of plants in very short durations. From a small piece of tissue, thousands of identical plantlets can be raised on a nutrient medium, all year round, independent of season.
The tissue culture technique is also the best method for storing germplasm and for maintaining a specific genetic type (a clone). Because the plants are raised from tissue and not from seed, every one of them is genetically identical to the parent — nothing is lost or reshuffled, as it would be in sexual reproduction.
This technique is especially valuable for those plants which produce recalcitrant seeds, or which produce highly variable seeds.
Ordinary (orthodox) seeds can be dried and frozen and will still germinate years later; that is how seed banks work. Recalcitrant seeds cannot. For such species, tissue culture is not merely convenient — it is the only reliable way to conserve the germplasm at all.
c. Gene therapy
A gene is a stretch of DNA required to make a functional product, such as part of a protein or a whole protein. During gene therapy, DNA that codes for a specific gene is delivered to individual cells in the body.
Gene therapy is the treatment of a disease (or disorder) by replacing, altering or supplementing a gene that is absent or abnormal, and whose absence or abnormality is responsible for the disease.
Notice how different this is from every other treatment you know. A drug relieves the effects of a fault; gene therapy attempts to correct the fault itself, at its source.
Most, if not all, disorders have a genetic factor, and this factor can be wholly or partially responsible for the disorder. In disorders such as cystic fibrosis, haemophilia and muscular dystrophy, changes in a gene directly result in the condition. In other conditions, such as high cholesterol and high blood pressure, genetic and environmental factors interact to cause the disease.
There are more than 5000 different human genetic diseases known to be caused by single gene defects — for example sickle cell anaemia, thalassemia, Tay-Sach’s disease, cystic fibrosis, Huntington’s chorea, haemophilia, alkaptonuria and albinism. A single-gene disorder is, in principle, the easiest target for gene therapy, because there is only one fault to correct.
Gene therapy is being used in many ways. For example, to:
- replace missing or defective genes;
- deliver genes that speed up the destruction of cancer cells;
- supply genes that cause cancer cells to revert back to normal cells;
- deliver bacterial or viral genes as a form of vaccination;
- deliver DNA for antigen expression and generation of an immune response;
- supply a gene for impairing viral replication;
- provide genes that promote or impede the growth of new tissue; and
- deliver genes that stimulate the healing of damaged tissue.
Forms of gene therapy
a. Germ line gene therapy. In this method, healthy genes are introduced into germ cells — sperms, eggs, or early embryos. Because these are the cells from which the next generation arises, the modification is present in every cell of the resulting individual, and it allows transmission of the modified genetic information to the next generation.
Though germ line gene therapy is highly effective in counteracting genetic disorders — it would remove the fault from the family line permanently — it is not encouraged for application in human beings, due to a variety of technical and ethical reasons. The central ethical difficulty is plain: the individuals affected by the change are not yet born and cannot consent to it.
b. Somatic cell gene therapy. In this type, the gene is introduced only into somatic cells — bone marrow cells, hepatic cells, fibroblasts, endothelium and pulmonary epithelial cells, cells of the central nervous system, endocrine cells and the smooth muscle cells of blood vessel walls.
Modification of somatic cells affects only the person being treated, and the modified chromosomes cannot be passed on to future generations. Somatic cell gene therapy is therefore the only feasible option in practice, and clinical trials have already been employed for the treatment of acquired disorders such as cancer and rheumatoid arthritis, and of blood disorders including SCID, Gaucher’s disease, familial hypercholesterolemia, haemophilia, phenylketonuria, cystic fibrosis, sickle cell anaemia, Duchenne muscular dystrophy, emphysema and thalassemia.
d. Genetically Modified Organisms (GMOs)
Genetically modified organisms are living organisms whose genetic material has been artificially manipulated in a laboratory through genetic engineering. This creates combinations of plant, animal, bacterial and viral genes that do not occur in nature, or through traditional crossbreeding methods.
Most GMOs have been engineered either to withstand the direct application of a herbicide, or to produce an insecticide themselves — or both. However, new technologies are now being used to develop other traits artificially in plants, such as resistance to browning in apples, and to create new organisms using synthetic biology.
It is important to be aware that GMOs are also debated. Despite the promises of the biotech industry, the text notes that there is no evidence that any of the GMOs currently on the market offer increased yield, drought tolerance, enhanced nutrition, or any other direct consumer benefit. Sections 12.6 and 12.7 take up these concerns — for the environment and for human health — in detail. Hold the question open as you read what follows.
I. Transgenic plants
The human race is very dependent on agriculture, and as world populations continue to expand, there must be a continuous reassessment of agricultural practices to optimise their efficiency.
Since early times, human beings have sought to improve the quality and productivity of agriculturally important plants. This was done by selection and by traditional breeding procedures, which were painstakingly slow and difficult. Traditional breeding programmes involve sexual crosses, and they did result in the high quality of present-day food plants such as wheat, rice, corn and potato. But a sexual cross can only shuffle genes that already exist within closely related plants, and each generation takes a season.
More recently, biotechnological approaches have been applied to these plants to create genetic variations that are beneficial for mankind. A single, chosen gene — from any organism whatever — can be added in one step.
The first transgenic plant produced was tobacco. More than 60 transgenic dicot plants and several monocot plants like maize, oat, rice and wheat are now known. Tomato, soybean, potato, sugar beet, grapes, brinjal and cotton are other transgenic plants.
Transgenic plants are being looked upon as bioreactors for molecular farming — that is, for the production of novel drugs such as interferons, edible vaccines, antibodies, amino acids and immunotherapeutic drugs.
Advantages of GM food-plants
a. Insect pest resistance
Insect resistance can help farmers to reduce their use of chemical pesticides, which in turn can reduce the cost of producing food — and reduce the chemical load on soil, water and farm workers.
An alternative has, in fact, been available for more than 30 years: a biological insecticide obtained from the bacterium Bacillus thuringiensis (Bt). However, the use of B. thuringiensis sprays is limited because of the low stability of the protein in the field — sunlight and rain destroy it, so the spray must be repeated again and again.
Here the genetic solution is elegant. Instead of spraying the protein on the plant, put the gene into the plant, and let every cell of the plant make the protein continuously and freshly. Bt cotton is one of the best-known transgenic plants for its insect resistance property.
Insect-resistant plants contain either a gene from B. thuringiensis or the cowpea trypsin inhibitor gene. The gene called the cry gene, present in B. thuringiensis, produces a protein that forms crystalline inclusions in bacterial spores.
Now comes the crucial point — and the reason the toxin is safe for us. When ingested by a susceptible insect, a combination of the high pH and the enzyme proteinase of the insect’s midgut processes the crystals hydrolytically to release the core toxic fragments. In other words, the crystal is not itself a poison; it is a pro-toxin, and it is activated only in the alkaline, proteinase-rich midgut of the insect. The human stomach is strongly acidic, so the crystal is never activated there.
The effect of these fragments is seen within minutes of ingestion, beginning with midgut paralysis and ending with the disruption of the midgut cells of the insect. Bt toxin activity has been shown against many species of insects within the orders Lepidoptera, Diptera and Coleoptera. The sequence is summarised in FC 12.7.
Flowchart
FC 12.7 – Action of the Bt cry protein: cry gene in Bacillus thuringiensis (or in the transgenic plant) → inactive crystalline protein (pro-toxin) → ingested by the insect → high pH + proteinase of the insect midgut → hydrolysis releases the core toxic fragments → midgut paralysis within minutes → disruption of midgut cells → death of the insect. In the acidic human stomach → the crystal is not activated → no effect.
Similarly, the gene for the α-amylase inhibitor (αAI-Pv) has been isolated from the adzuki bean (Phaseolus vulgaris) and transferred to tobacco; this gene works against pests like Zabrotes subfasciatus and Callosobruchus chinensis. An amylase inhibitor blocks the insect’s starch-digesting enzyme, so the pest starves on a full stomach.
b. Improved nutritional qualities (biofortification)
Transgenic plants have also been produced to provide functional food and nutraceuticals — foods that do more than merely fill the stomach. Some of the substances introduced in this way, the benefit expected, the crop used and the transgene involved are listed in Table 12.8.
Table 12.8 : Some transgenic plants produced for functional food and nutraceuticals
| Substance | Potential benefit | Crop | Transgene |
|---|---|---|---|
| Provitamin A | Anti-oxidant | Rice | Phytoene synthase, lycopene cyclase |
| Vitamin E | Anti-oxidant | Canola | γ-tocopherol methyl transferase |
| Flavonoids | Anti-oxidants | Tomato | Chalone isomerase |
| Fructants | Low calories | Sugarbeet | 1-sucroese : sucrase fructosyl transferase |
| Iron | Iron fortification | Rice | Ferritin, metallothioein, phytase |
For millions of people in developing countries, rice is the main item in the diet. Because rice does not contain many essential nutrients, malnutrition is very common in these countries — a person may eat enough to feel full every day, and still be starved of a vitamin.
Especially terrible is the night blindness that results from a lack of vitamin A. This vitamin is abundant in milk, and in vegetables such as carrots, which most of the poor people of the world cannot easily afford.
To solve this problem, Swiss researchers created transgenic rice (golden rice) and transgenic mustard (golden mustard) varieties that are high in vitamin A. The golden colour is due to beta carotene, the precursor of vitamin A — a pigment the human body converts into the vitamin itself. Golden rice is thus a transgenic food crop used to reduce vitamin A deficiency disease. The researchers hoped that this rice, if grown and eaten in developing countries, would reduce the diseases associated with vitamin A deficiency (VAD).
Improvement in the oil content and oil quality of oil crops like soybean, oil palm, rapeseed and sunflower has been achieved by the transfer of ‘Arabidopsis genes’.
Iron deficiency is also a serious nutritional problem, affecting an estimated 30% of the world population. For the production of transgenic crops that will produce food rich in iron, an iron storage protein — ferritin — is targeted. Ferritin is found in many animals, plants and bacteria. The genes for the ferritin protein, isolated from soybean and from Phaseolus, have been transferred to rice.
c. Modification in post-harvest characteristics
A crop is not safe once it is harvested. Diseases and pests, bruising on soft fruits and vegetables, heat and cold storage, over-ripeness, and the loss of flavours and odours all lead to a great deal of loss during the storage and transport of crops. In a country with long distances and hot weather, a large part of the harvest may never reach the plate at all.
Most of these physiological changes are due to endogenous enzyme activity — the plant’s own enzymes, still working after the fruit is picked. Genetic engineering has made it possible to slow down these activities.
In the tomato, the enzyme polygalacturonase breaks down the cell wall constituent pectin, leading to the softening of the fruit during ripening. Thus, the fruits become easily bruised and damaged on shipment. By inhibiting the polygalacturonase through antisense genes, the genetically modified tomato — called the “Flavr savr” tomato — can remain on the vine until mature, has a long shelf life, and can be transported in a firm, solid state.
An antisense gene works by producing an RNA that is complementary to the mRNA of the target gene. The two RNAs pair with each other, the mRNA can no longer be translated, and the enzyme is simply never made.
d. Plants as factories
To produce novel biochemicals and vaccines — biopharmaceuticals — plants are potential factories, or bioreactors. They can be used for high-value biochemicals like starch, sugar, lipids and proteins, and for products like fine chemicals, perfumes and adhesive compounds, as well as industrial lubricants, biodegradable plastic, and even ‘renewable’ energy crops to replace fossil fuels.
A plant is an attractive factory for an obvious reason: it runs on sunlight, water, carbon dioxide and soil minerals, and it multiplies itself.
Some of the proteins that are being produced by transgenic crop plants are:
- Human growth hormone, with the gene inserted into the chloroplast DNA of tobacco plants.
- Humanised antibodies against infectious agents such as HIV, Respiratory syncytial virus (RSV) and Herpes simplex virus (HSV), the cause of “cold sores”.
- Protein antigens to be used in vaccines — for example, patient-specific antilymphoma (a cancer) vaccines. B-cell lymphomas are clones of malignant B cells, each expressing on its surface a unique antibody molecule; a vaccine can therefore be tailored against that one unique molecule, and so against that one patient’s tumour.
Currently many novel products have been commercially exploited, such as:
A ‘superglue’ produced by tobacco plants carrying genes that encode the powerful adhesive proteins which enable marine mussels to stick to rocks. It will be especially valuable as a biochemical glue for body repairs during surgery — an adhesive that works on wet, living tissue.
Transgenic plants containing an oil-encoding gene from marine algae, which produce oil having a nutritional value similar to cod-liver oil.
A plant that will produce the antimalarial drug Artemisinin.
The product of genetically engineered opium (poppy), used to produce more powerful painkillers, that is, analgesics and sedatives.
e. Transgenic plants producing edible vaccines
Genetically altered plants can provide protection against infectious diseases. Plant products acting as vaccines would be inexpensive to produce, and can thus easily be made available in developing countries.
GM potatoes, tomatoes, bananas, soybeans, alfalfa and cereals are the most common foods proposed for edible vaccine delivery. The fruits of GM bananas can be used as an oral edible vaccine against hepatitis B. A banana needs no cooking — and cooking would destroy the protein — which is one reason it is such a favoured candidate.
II. Transgenic animals
Many transgenic animals — mice, rats, rabbits, pigs, sheep, cows, fowls and fish — have been produced through rDNA technology.
The term transgenic animal refers to an animal in which there has been a deliberate modification of the genome — the material responsible for inherited characteristics — in contrast to spontaneous mutation. Foreign DNA is introduced into the animal using recombinant DNA technology, and it must then be transmitted through the germ line, so that every cell of the animal, including its germ cells, contains the same modified genetic material.
Diagram
Fig. 12.9 – Transgenic animals: transgenic fowl for meat and eggs; transgenic fish for protein-rich meat; transgenic mouse as an animal model for research; transgenic cow as a bioreactor; with a central image of DNA being microinjected into an egg cell.
A representative, though non-inclusive, list of the purposes for which transgenic animals have been used indicates the wide-ranging application of this biotechnology:
- in medical research, transgenic animals are used to identify the functions of specific factors in complex homeostatic systems, through the over-expression or under-expression of a modified gene (the inserted transgene);
- in toxicology, as responsive test animals for the detection of toxicants;
- in mammalian developmental genetics;
- in molecular biology, where the analysis of the regulation of gene expression makes use of the evaluation of a specific genetic change at the level of the whole animal;
- in the pharmaceutical industry, for the targeted production of pharmaceutical proteins, for drug production and for product efficacy testing;
- in biotechnology, as producers of specific proteins;
- as sources of genetically engineered hormones to increase milk yield and meat production; in the genetic engineering of livestock and in aquaculture, affecting the modification of animal physiology and/or anatomy; and in cloning procedures to reproduce specific blood lines;
- in developing animals specially created for use in xenografting.
a. Transgenic mice and cancer research
Through laboratory investigations with transgenic mice that have been modified using a particular oncogene (a cancer-causing gene), and which therefore develop a certain type of cancer, questions concerning the relationship between oncogenes and cancer development could be answered. Theoretically, such animals can also be used for research into cancer treatment and the prevention of malignancy.
The logic is worth pausing on. In an ordinary animal, cancer appears unpredictably, and you cannot tell which of thousands of possible causes was responsible. In a transgenic mouse, exactly one known gene has been added — so if the cancer appears, that gene is implicated. The animal turns an association into an experiment.
b. Transgenic farm animals
With the advent of the technology, the intention of researchers has turned to producing transgenic farm animals from which mankind can derive greater benefits. Many farm animals are improved for their meat production ability, while others are improved for milk yield and quality, and for disease-free status.
It is worth seeing how far conventional improvement has already carried us. At the beginning of the last century, a dairy cow provided 2,000 to 3,000 litres of milk a year. Today, a Holstein cow provides 6,000 litres on average, and up to 8,000–10,000 litres for the best animals. A century ago, a hen laid about 70 eggs a year, whereas today the best breeds lay up to 250 eggs per year. This has been possible through breeding and, increasingly, through the advent of biotechnology.
The main objectives of improved animal breeding programmes, coupled with the new technology of gene transfer, are:
- efficiency of meat production;
- improved quality of meat;
- milk quality and quantity;
- egg production;
- wool quality and quantity;
- disease resistance in animals;
- production of low-cost pharmaceuticals and biologicals.
c. Transgenic cattle for food production
Of the few research reports describing the use of transgenic technologies in cattle, only one is directed towards a food production application. Researchers introduced additional copies of the bovine beta or kappa casein genes into dairy cattle, and evaluated the effect on milk production and composition. The transgenic offspring had an 8 to 20% increase in beta casein and a two-fold increase in kappa casein.
Casein is the main protein of milk, and it is what makes milk set into curd and cheese — so more casein means a richer, more nutritious milk, and a higher yield of cheese from the same volume.
d. Transgenic cattle for human therapeutic production
A second application for genetically modified cattle is the production of human therapeutic proteins. Human proteins that have been expressed in milk include human lactoferrin, human alpha lactalbumin, human serum albumin and human bile salt stimulated lipase.
The mammary gland in dairy cows is an excellent protein production factory. Consider why: it is built to secrete large quantities of protein, day after day, into a fluid that can be collected without harming the animal. If a human gene is placed under the control of a milk-protein promoter, the human protein appears in the milk and nowhere else — and the milk is simply milked.
So productive is this system that one transgenic cow would be more than sufficient for the production of the annual world supply of factor IX (plasma thromboplastin component), which is used in the treatment of haemophilia.
In 1990, Tracy was born in Scotland — a transgenic animal able to produce a human protein in her milk, for human therapeutics.
Antibodies are currently used for many different human clinical applications, including the treatment of infectious disease and cancer, the prevention of transplanted organ rejection, autoimmune diseases, and use as antitoxins. To make a human antibody product, genetically modified cows are immunised with a vaccine containing the disease agent — the cow’s own immune system then does the work of producing the antibody.
e. Transgenic sheep
Gene transfer technology is applied to sheep to produce transgenic sheep, which are able to achieve better growth and meat production as well as to serve as bioreactors. The human growth hormone gene is introduced into sheep for promoting growth and meat production.
The bacterial genes cys E and cys M are concerned with the biosynthesis of the amino acid cysteine, which is involved in the formation of the keratin protein found in wool. Both these genes have been identified, cloned and introduced into sheep, to increase wool production and to improve the quality of wool.
Why cysteine in particular? Because keratin owes its strength to disulphide bridges, and those bridges are formed between the sulphur-containing side chains of cysteine residues. More cysteine available means more keratin, and better wool.
f. Transgenic pigs
The objective of gene transfer in pigs is to increase growth and meat production, and to make the animals act as bioreactors.
Pigs are also regarded as the most suitable animals to be bred for heart transplant, because a pig’s heart is about the same size as a human heart, and pig heart valves have already been used in human heart surgery for over a decade. The pig clone is the first step towards providing animal organs and tissues for human transplants — xenotransplantation.
g. Transgenic fish
Commercially important fish like Atlantic salmon, catfish, goldfish, Tilapia, zebra-fish, common carp and rainbow trout have been transfected with the growth hormone gene, the chicken crystalline protein gene and the E. coli hygromycin resistance gene.
Transgenic fish have shown increased cold tolerance and improved growth. Since the quantity and quality of fish protein, as well as its preservation, are the factors affecting the economic value of fish, both these traits translate directly into value: a fish that grows faster reaches market size sooner, and a fish that tolerates cold can be farmed in waters that were previously unusable.
h. Transgenic chicken
Transgenic chickens also carry and express foreign genes. They could be used to improve the genetic make-up of existing strains with respect to built-in (in vivo) resistance to viral and coccidial diseases, better feed efficiency, lower fat and cholesterol levels, eggs with a high protein content, and better meat quality.
For NEET and board examinations, this section is the richest source of application-based questions in the chapter. Expect match-the-following items pairing a recombinant protein with its disorder (Table 12.7), or a transgene with its crop (Table 12.8); assertion–reason questions on why the Bt toxin does not harm humans; and short-answer questions on the purpose of golden rice, the Flavr savr tomato, and the difference between germ line and somatic cell gene therapy.
12.5 Bioethics, Bio-piracy and Bio-patent
Everything so far in this chapter has answered the question “what can we do?” This section begins a different and harder question: “what should we do?” No experiment can settle it, and no enzyme can help.
Bioethics
Ethics usually deals with matters related to socially acceptable moral duty, conduct and judgement. In other words, it helps to regulate the behaviour of a community by some set of standards.
These standards, however, are not fixed. The concepts differ according to culture and traditions, and they also change with time, because our perception of values shifts — and that shift is itself affected by progress in science and technology. A practice that was unthinkable in one generation may be routine in the next, precisely because the technology made it possible.
Bioethics helps us to study the moral vision, the decisions and the policies of human behaviour in relation to biological phenomena or events. It is ethics applied to ‘Life’ itself — for example, to in vitro fertilisation, sperm banks, gene therapy, cloning, gene manipulations, euthanasia, death, the maintenance of those who are in a comatose state, and prenatal genetic selection.
Look again at that list of examples. Every item on it exists because a biological technique made it possible. Sperm banks were not an ethical question before sperm could be frozen; prenatal genetic selection was not a question before a foetus could be genetically tested. Each new power creates a new choice, and each new choice creates a new duty.
Why biotechnology raises ethical questions
The era of biotechnology has brought a wide spectrum of new topics — cloning, transgenics, gene therapy, eugenics and rDNA technology among them. The use of all of these has drawn a wide range of reactions from society, and those reactions are based on each individual’s own perception and morality.
It is honest to say that the ethical aspects pertaining to the use of biotechnology seem more controversial, and to many people more frightening, than the science itself. The main concerns can be broadly summarised, and they are set out in FC 12.8:
- the use of animals causes great suffering to them;
- the integrity of a species is violated by transgenosis — that is, by the transfer of genes across the species barrier;
- human genes are transferred into animals, and animal genes into human beings;
- the indiscriminate use of biotechnology poses risks to the environment, to health and to biodiversity.
Flowchart
FC 12.8 – Bioethical concerns arising from biotechnology: biotechnology → (i) concern for animals → suffering caused by experimentation; (ii) concern for species → violation of species integrity by transgenosis → transfer of human genes into animals and vice versa; (iii) concern for the world outside the laboratory → indiscriminate use → risk to environment, to human health, and to biodiversity
Regulation: the need and the machinery
All this shows that the vast advances in the life sciences, taking place within multicultural and pluralistic modern societies, create numerous bioethical problems that require some stringent regulation. Where individual moral opinions differ so widely, the decision cannot be left to the individual scientist or the individual company alone.
In terms of GMOs, the Indian Government has set up the Genetic Engineering Approval Committee (GEAC). This organisation makes decisions regarding the validity of research involving GMOs, and addresses the safety of GMOs introduced for public use.
The wider GMO debate
The introduction of Genetically Modified Organisms has led to a wider debate on bioethical concerns affecting the social, economic and environmental spheres.
These concerns include the effects on non-target organisms; the consequences of insect-resistant crops; gene flow — the escape of introduced genes into wild relatives through pollen — and the resulting loss of diversity; as well as the deeper objection of interfering with nature, in which the modification process itself is seen as disrupting the natural processes of biological entities.
Ethics in biotechnology also includes the general subject of what should and what should not be done in using recombinant DNA techniques. Notice that the objections listed above are of two quite different kinds. Some are consequence-based — they warn of harm that might follow, and can in principle be answered with evidence. Others are principle-based — they hold that crossing the species barrier is wrong in itself, whatever the consequences. Evidence cannot settle the second kind of objection, and it is important not to confuse the two while arguing.
The next two sections take up the consequence-based questions directly: what biotechnology may do to the environment (12.6), and what it may do to human health (12.7).
For board and NEET examinations, this section is usually tested through short-answer or long-answer questions: define bioethics, list the ethical concerns raised by biotechnology, and state the full form and function of the GEAC. The expansion “Genetic Engineering Approval Committee” and its role in approving GMOs for public use are frequently asked directly.
12.6 Effects of Biotechnology on the Environment
Effects on the environment are of particular concern with regard to GMO crops and food production. A transgenic plant is not grown in a sealed vessel like a bacterial culture — it stands in an open field, exchanging pollen, insects, water and soil with everything around it. Whatever gene we place in it therefore enters an ecosystem, not just a crop.
a. Herbicide use and resistance
One major area of development involves adding to a crop the ability to produce a pesticide, and resistance to specific herbicides. These traits are genuinely helpful in food production: they allow farmers to use fewer chemicals, and to grow crops in less than ideal conditions.
The reasoning behind a herbicide-tolerant crop is simple. A herbicide kills plants; it cannot normally tell a weed from a crop. If the crop is made resistant to one particular herbicide, the field can be sprayed with that herbicide and only the weeds will die.
But the benefit carries a cost. Because the crop can now survive the chemical, herbicide use could actually be increased, which will have a larger negative effect on the surrounding environment — the very chemicals that were meant to be reduced may be applied more freely and more often.
There is a second, subtler danger. Unintended hybrid strains of weeds and other plants can develop resistance to these herbicides through cross-pollination. Pollen from the resistant crop reaches a wild relative growing at the edge of the field, and the resistance gene passes into the weed population. The weed then survives the spray — and the whole benefit of the herbicide is negated. This escape of an introduced gene into wild populations is what is meant by gene flow, and its path is shown in FC 12.9.
Flowchart
FC 12.9 – Gene flow producing herbicide-resistant weeds: herbicide-tolerant transgenic crop → pollen carries the resistance gene → cross-pollination with a wild relative or weed at the field margin → hybrid weed carrying the resistance gene → the weed survives spraying → herbicide loses its effect → a “superweed” spreads
One such herbicide that has already been used in this way is RoundUp. Crops of RoundUp-ready soybeans have already been implemented into agricultural practices, possibly conferring RoundUp resistance to neighbouring plants.
b. Effects on untargeted species
Bt corn, which produces its own pesticide, is also in use today. But a toxin produced in every tissue of a plant does not stay neatly inside the plant. It is present in the pollen too, and pollen drifts.
Bt corn has been reported to have adverse effects on Monarch butterfly populations — and the Monarch is not the original target of the pesticide at all. It can likewise have unintentional effects on neutral, or even beneficial, species.
Two lessons run through this section, and both are worth carrying into the next one. First, an environmental effect is rarely confined to the organism that was engineered. Second, the harm and the benefit often come from the same property: the very stability that makes a Bt crop better than a Bt spray is what allows the toxin to persist and spread.
For board examinations, this section is commonly tested as “what are the harmful effects of GM crops on the environment?” or through assertion–reason questions such as: Assertion — herbicide-tolerant crops reduce herbicide use; Reason — the crop is unaffected by the herbicide. Read such statements carefully: the reason may be true while the assertion is not.
12.7 Effects of Biotechnology on Human Health
GMO crops could potentially have negative effects on human health as well as on the environment. The reason lies in a simple fact: when a gene is moved, a protein is moved with it — and a protein that was perfectly harmless in its original organism may behave very differently once it appears in a food that people eat every day.
a. Allergies
Consumers have developed unexpected allergic reactions to some genetically modified foods.
Follow the logic of what happened, because it explains why allergenicity is treated so seriously. A person allergic to Brazil nuts avoids Brazil nuts. He does not avoid soybean — he has no reason to. But the soybean now contains a Brazil-nut protein, and the immune system recognises that protein, not the plant it came in. The person is exposed to his allergen without ever knowing it. The path is set out in FC 12.10.
Flowchart
FC 12.10 – How a transgene can transfer an allergy: allergenic protein in the source organism (Brazil nut) → its gene is transferred to a new crop (soybean) → the protein is now expressed in the new food → a consumer allergic to the original source eats the new food, unaware → the immune system recognises the protein → allergic reaction
b. Long-term effects
Because GMO technology has been available for such a short amount of time, there is relatively little research on its long-term effects on health — effects which, by definition, we cannot anticipate at this point.
This is not a claim that harm exists; it is an admission that the evidence is young. A crop introduced two or three decades ago has simply not been eaten by anybody for a lifetime, and an effect that appears only after forty years of consumption would not yet have been seen. Honest science distinguishes between “shown to be harmless” and “not yet shown to be harmful”, and this section belongs firmly in the second category.
c. New proteins
Proteins that have never before been ingested by human beings are now part of the foods that people consume every day. Their potential effects on the human body are as yet unknown — whey proteins are an example.
Every transgene means a new protein in a familiar food. Most such proteins are digested to amino acids like any other, and pose no problem at all. But “most” is not “all”, and the only way to know is to test each one — which is precisely why regulatory bodies such as the GEAC exist.
d. Food additives
The same technology also carries clear promise. GMOs present us with the possibility of introducing additional nutrients into foods, as well as antibiotics and vaccines. This availability of technology can provide nutrition and disease resistance to the people of those countries which do not otherwise have the means to provide them — the edible vaccine and the biofortified crop are both examples you have already met.
However, there is a real danger in this convenience: the possibility of the creation of antibiotic-resistant and vaccine-resistant strains of pathogens.
Taken together, sections 12.6 and 12.7 do not say that biotechnology is dangerous, nor that it is safe. They say that its power to change a living organism permanently deserves careful, case-by-case scrutiny — of the environment it is released into, and of the body it enters.
For board and NEET examinations, this section is commonly tested by asking for the harmful effects of GMOs on human health, and through the Brazil nut–soybean example, which is a favourite illustration of unintended allergenicity.
12.8 Biopatent and Biopiracy
a. Biopatent
A patent is a special right granted to an inventor by the government. It is the personal property of the inventor, and like any other property, it can be sold.
The purpose behind a patent is worth understanding before its problems are discussed. Developing a new product may take years of work and enormous expense, while copying it may take a week. If anyone could copy freely, no one would invent. A patent gives the inventor a limited period of exclusive right, so that the effort is rewarded — and in exchange, the invention is fully disclosed to the public, so that others may learn from it.
A patent consists of three parts:
- the grant — the agreement with the inventor;
- the specification — the subject matter of the invention;
- the claims — the scope of the invention that is to be protected.
A biopatent is a biological patent. Biopatents are awarded for strains of microorganisms, cell lines, genetically modified strains, DNA sequences, biotechnological processes, product processes, products, and product applications.
Biopatents are awarded to recognise the real innovative contributions made by an inventor to the cause of human welfare. Such awards are given to encourage the development of a scientific culture and its values, and to emphasise the role of biology in shaping human society.
The Indian patent system allows a ‘process patent’ and not a ‘product patent’. This distinction is important. A process patent protects the particular method by which something is made; anyone who finds a different method to make the same substance is free to do so. A product patent would protect the substance itself, no matter how it was made — a far wider monopoly.
A biopatent allows the patent holder to exclude others from making, using, selling or importing the protected invention for a limited period of time. The duration of biopatents is five years from the date of the grant, or seven years from the date of filing the patent application — whichever is less.
The first biopatent, and a troubling one
The first biopatent was granted for a genetically engineered bacterium, Pseudomonas, used for clearing oil spills. It was a landmark, because for the first time a living organism was accepted as a patentable invention.
A very different case is the patent issued under the title ‘control of plant gene expression’, granted jointly to the Delta and Pineland company and the U.S. Department of Agriculture. The patent is based on a gene that produces a protein toxic to the plant, and which therefore does not allow the seeds to germinate.
Think about what that means for a farmer. The crop grows, and yields grain — but the grain saved from that harvest will not sprout the following season. The farmer must buy fresh seed every year, from the company, forever.
This patent was not granted by the Indian government. Such a patent is considered morally unacceptable and fundamentally unequitable. The reason is that financially powerful corporations would thereby acquire a monopoly over a biotechnological process, and this in turn would pose a threat to global food security.
b. Biopiracy
Pirates, in general terms, are those who steal and kill to enrich themselves. Biopirates do not kill — but they steal the patent, that is, they misuse the patent system.
There is a striking imbalance in the world that makes biopiracy possible. The developed, industrialised and financially rich nations are poor in biodiversity and in traditional knowledge. The developing and underdeveloped nations have abundant biodiversity, and traditionally they know far better how to use their own bio-resources.
Traditional knowledge naturally includes a deep understanding of ecological processes, and the ability to sustainably extract useful products from the local habitat. It is handed down through the generations. For a company, such knowledge is immensely valuable, because it saves the time, money and effort of searching blindly — it points directly at which of a hundred thousand plants is worth testing.
The components of traditional knowledge that are especially relevant to our global survival include knowledge of:
- food, crop varieties and agricultural or farming practice;
- the sustainable management of natural resources and the conservation of biological diversity;
- biologically important medicines.
The conservation of species, of habitat and of biodiversity is essential to the continued survival of tribal people. And the relationship runs both ways: by conserving the customs and the habitat of tribal people, we simultaneously reduce emissions from deforestation and ecosystem degradation. Furthermore, the opportunity for cultural survival is a basic human right. It is precisely this traditional knowledge that is now facing the problem of biopiracy.
The act of piracy is the unauthorised publication or reproduction of another person’s work or material. When someone indulges in piracy, the accused is using someone else’s work illegally, or without taking any permission. Many innovations and discoveries in pharmaceutical and agricultural research are, in truth, not new enough to qualify as inventions at all — they are based on centuries of knowledge held by traditional societies.
Examples of biopiracy
Patenting of Neem (Azadirachta indica)
The people of India have used neem in a variety of ways since time immemorial, and Indians have shared their knowledge of the properties of neem with the entire world.
Pirating this knowledge, the USDA and an American multinational, W.R. Grace, in the early 90s sought a patent from the European Patent Office (EPO) on the “method for controlling on plants by the aid of hydrophobic extracted neem oil”. The patenting of the fungicidal properties of neem was a clear example of biopiracy — a property of the tree that Indian farmers had relied on for generations.
Patenting of Basmati
Basmati is a long-grained, aromatic variety of rice indigenous to the Indian subcontinent.
In 1997, the US Patent and Trademark Office (USPTO) granted a patent to a Texas-based American company, Rice Tec Inc, for “Basmati rice line and grains”, having the trade name Texmati. The patent application was based on 20 very broad claims of having “invented” the said rice.
Due to a people’s movement against Rice Tec, in March 2001 the USPTO rejected all the claims.
Haldi (Turmeric) biopiracy
Two American researchers of Indian origin, at the University of Mississippi Medical Center, put a claim before the US Patent and Trademark Office maintaining that they had discovered haldi’s healing properties. Surprisingly, they were granted a patent in March 1995 — for something that Indian households had known for years, and the ayurvedas for centuries.
The consequence was serious: it meant they held exclusive rights over any such haldi drug, and were in a position to make millions of dollars from it.
The Council of Scientific and Industrial Research (CSIR) applied to the US Patent Office for a re-examination. The Office realised the mistake and cancelled the patent. This came about only after Indian scientists protested loudly about how the country was losing its traditional knowledge to foreign companies that had begun poaching on ancient healing techniques.
Flowchart
FC 12.11 – How biopiracy happens, and how it is defeated: traditional community holds knowledge of a plant (neem, haldi, basmati) → a foreign company or institution learns of it → files a patent claiming it as an “invention” → the patent office finds no documented prior art → the patent is granted → exclusive commercial rights and profits, with no benefit to the source country → challenge by the host country (CSIR re-examination, or a people’s movement) → prior art is documented and presented → the patent is cancelled or the claims are rejected
The way ahead
It is the need of the hour to launch a genetic literacy movement in Indian schools and colleges, so that there is a better understanding of the opportunities and the risks related to biotechnology, and so as to promote the safe and meaningful use of the technologies of modern life sciences.
This closing appeal is aimed at you. A citizen who understands what a gene is, what a patent does, and what a GM crop can and cannot do, is able to argue about these questions on evidence rather than on fear — and that, ultimately, is the only reliable protection a country has, both against its own ignorance and against the misappropriation of what belongs to it.
For board examinations, this section is regularly examined through the long-answer question “Define and explain the terms biopiracy, biopatent and bioethics”, and through short notes on the neem, basmati and turmeric cases. Remember the exam-critical specifics: the three parts of a patent (grant, specification, claims), the duration of a biopatent (five years from the date of grant or seven years from the date of filing, whichever is less), and that India allows a process patent and not a product patent.
12.9 Summary
Biotechnology is the product of interaction between biological science and technology — an applied branch of biology. The term was first used by Karl Ereky in 1919, for a process of large-scale production of pigs.
Broadly, biotechnology is the development and utilisation of biological forms, products or processes for maximum benefit to man and other forms of life. The OECD (1981) defined it as the application of scientific and engineering principles to the processing of materials by biological agents to provide goods and services for human welfare. A biological agent means plant and animal cells, microorganisms, enzymes, or their products.
Biotechnology grew in two phases. Traditional (old) biotechnology was based on fermentation using microorganisms — curd, ghee, soma, vinegar, yogurt, cheese and wine — and was more an art than a science. Modern (new) biotechnology began when recombinant DNA technology, developed in 1970, was established by Stanley Cohen and Herbert Boyer in 1973.
Modern biotechnology differs from the classical form in two major features: (i) the capability of science to change genetic material for new specific products, through rDNA technology, PCR, microarrays, cell culture and fusion, and bioprocessing; and (ii) the ownership of the technology and its socio-political impact.
Modern biotechnology rests on two core techniques: genetic engineering, which alters the genetic material (DNA and RNA), and chemical engineering, which maintains the sterile environment needed to manufacture vaccines, antibodies, enzymes, organic acids, vitamins and therapeutics.
Genetic engineering is the manipulation of genetic material towards a desired end, in a directed and predetermined way, using an in vitro process. It is also called recombinant DNA technology or gene cloning.
The instruments used include gel permeation, ion exchange chromatography, spectroscopy, mass spectrometry and electrophoresis. In electrophoresis, charged molecules are separated in an electric field; DNA, being negatively charged, migrates to the anode, and smaller fragments move faster. Agarose gel electrophoresis, PAGE and SDS PAGE are its methods.
PCR, discovered by Kary Mullis in 1985, is the in vitro amplification of a desired DNA segment. It requires template DNA, four dNTPs, an excess of two primers, heat-stable DNA polymerase and \(Mg^{++}\) ions. Its three steps are denaturation (90–98 °C), annealing of primers (40–60 °C) and primer extension by Taq polymerase (70–75 °C). One cycle takes 3–4 minutes; after n cycles, \(2^n\) copies are produced. The device used is the thermal cycler.
The three biological tools of rDNA technology are enzymes, cloning vectors (vehicle DNA) and competent hosts (cloning organisms).
Nucleases cut the phosphodiester bonds of polynucleotide chains. Exonucleases cut from the ends; endonucleases cut from within. Restriction endonucleases recognise specific sequences and cut both strands — they are the molecular scissors. In bacteria they act as a defence against viral DNA; the bacterium protects its own DNA by methylation. Smith, Nathan and Arber received the Nobel Prize in 1978 for their discovery.
Recognition sequences are 4 to 8 nucleotides long and show internal symmetry; such a sequence is a palindrome, reading the same on both strands in the 5’→3’ direction. Restriction enzymes give either blunt ends (straight cut) or sticky (cohesive, staggered) ends. Type II enzymes, which cut within the palindrome, are the ones used in rDNA technology.
Cloning vectors carry foreign DNA into a host and replicate there. They include plasmids, bacteriophages, cosmids, phagemids, BAC, YAC, transposons, baculoviruses and MACs. A good vector must replicate independently (through the ori gene), enter host cells easily, carry marker genes for antibiotic resistance with a unique cleavage site in one of them, and possess control elements such as a promoter, an operator and ribosome binding sites. pBR 322, pBR 320 and pACYC 177 are constructed plasmids. The Ti plasmid of Agrobacterium tumefaciens, which causes crown gall, carries the transposon T DNA and is the chief vector for plants.
Competent hosts are usually bacteria such as Bacillus haemophilus, Helicobacter pyroli and E. coli; E. coli is used most often.
The steps of rDNA technology are summarised in FC S.1.
Flowchart
FC S.1 – Complete sequence of rDNA technology: isolation of DNA from the donor (cells sheared and treated with detergent; DNA purified and spooled) → cleavage by restriction enzymes at restriction sites → selection of the fragment carrying the desired gene (foreign or passenger DNA) → insertion into a cloning vector cut with the same restriction enzyme, and sealing by DNA ligase → recombinant (chimeric) DNA → transfer into a competent host by transformation, assisted by divalent \(Ca^{++}\) ions → transformed cell → selection of transformed cells using marker genes (e.g. Pst1 knocks out the ampicillin-resistance gene of pBR 322) → multiplication of transformed cells in fresh culture medium → expression of the gene → downstream processing in a bioreactor → desired product
Foreign DNA can also be transferred without a vector, by electroporation, microinjection, lipofection, the shot gun or biolistic method, and ultrasonification. In plants, transformation is usually through the Ti plasmid.
A gene library stores cloned DNA sequences. A genomic library represents the complete genome and is suitable for prokaryotes; a cDNA library is made from mRNA by reverse transcription and is meaningful for eukaryotes, whose genomes contain introns, regulatory genes and repetitive DNA.
In healthcare, rDNA technology produces human proteins such as erythropoietin, insulin, Factor VIII, Factor IX, TPA, interferons and α₁-antitrypsin. Insulin, a peptide hormone of the β-cells of the islets of Langerhans, was discovered by Sir Edward Sharpey Schafer (1916). Hakura et al. (1977) synthesised the DNA of chains A and B and inserted them into pBR 322 vectors; Gilbert and Villokomaroff designed recombinant insulin production in 1978, using E. coli hosts, with insulin later separated by trypsin treatment.
A vaccine gives active acquired immunity. Recombinant vaccines, naked DNA vaccines, viral vector vaccines and plant-derived vaccines are new advances. Edible (oral) vaccines are produced by expressing an immunogenic protein in a plant genome; “melt in the mouth” vaccines include the flu vaccine produced by Bacillus subtilis. Their benefits are comfort of administration, low cost and ease of storage.
In agriculture, GM crops that are pest resistant and stress tolerant raise productivity. Tissue culture is used for micropropagation, for storing germplasm and for maintaining a clone — essential for plants with recalcitrant seeds, which lose viability on drying and freezing.
Gene therapy treats a disorder by replacing, altering or supplementing an absent or abnormal gene. Genes are delivered ex vivo or in vivo, and also by virosomes and bionic chips. Germ line gene therapy treats germ cells and is heritable, but is not encouraged in humans for technical and ethical reasons; somatic cell gene therapy treats body cells only, is not heritable, and is the only feasible option. More than 5000 human genetic diseases are caused by single gene defects.
GMOs are organisms whose genetic material has been artificially manipulated in the laboratory. The first transgenic plant was tobacco. Transgenic plants act as bioreactors for molecular farming.
Insect resistance is obtained from the cry gene of Bacillus thuringiensis, whose crystalline protein is activated by the high pH and proteinase of the insect midgut, causing midgut paralysis and cell disruption; it acts on Lepidoptera, Diptera and Coleoptera. Bt cotton is the best-known example. The α-amylase inhibitor gene (αAI-Pv) from Phaseolus vulgaris protects against Zabrotes subfasciatus and Callosobruchus chinensis.
Biofortification has produced golden rice and golden mustard, rich in beta carotene (the precursor of vitamin A), to combat vitamin A deficiency; ferritin genes from soybean and Phaseolus have been transferred to rice against iron deficiency, which affects an estimated 30% of the world population.
Post-harvest characteristics are improved in the “Flavr savr” tomato, where antisense genes inhibit polygalacturonase, so pectin is not broken down and the fruit stays firm with a long shelf life.
Plants act as factories for biopharmaceuticals — proteins, hormones, antibodies, vaccines and enzymes from transgenic plants — and for products such as a surgical superglue from tobacco, cod-liver-type oil, artemisinin, and improved analgesics. Edible vaccines are proposed in GM potatoes, tomatoes, bananas, soybeans, alfalfa and cereals; GM banana can serve as an oral vaccine against hepatitis B.
Transgenic animals carry deliberately introduced foreign DNA, transmitted through the germ line. Transgenic mice carrying the oncogenes myc and ras were developed in Philip Leder’s laboratory at Harvard for breast cancer research. Transgenic cattle give increased beta and kappa casein, and produce human proteins in milk; Tracy, born in Scotland in 1990, produced a human protein in her milk. Transgenic sheep carry the human growth hormone gene and the bacterial cys E and cys M genes for wool. Transgenic pigs serve growth, meat production and xenotransplantation. Transgenic fish show improved growth and cold tolerance; transgenic chickens give disease resistance, better feed efficiency and better eggs.
Bioethics studies the moral vision, decisions and policies of human behaviour in relation to life — IVF, sperm banks, gene therapy, cloning, gene manipulation, euthanasia and prenatal genetic selection. Concerns include animal suffering, violation of species integrity by transgenosis, the transfer of genes between humans and animals, and risks to environment, health and biodiversity. In India, the Genetic Engineering Approval Committee (GEAC) decides on the validity and safety of GMO research and public use.
Environmental effects of GMOs include increased herbicide use, herbicide-resistant weeds arising by cross-pollination (as with RoundUp-ready soybean), and harm to untargeted species — Bt corn has adverse effects on Monarch butterfly populations.
Health effects include allergies (a Brazil nut gene transferred into soybean caused allergic reactions), unknown long-term effects, new proteins in the diet, and the risk that antibiotics and vaccines in food may create resistant strains of pathogens.
A patent has three parts — grant, specification and claims. Biopatents are awarded on microbial strains, cell lines, GM strains, DNA sequences, biotechnological processes, products and applications. India allows a process patent, not a product patent. The duration of a biopatent is five years from the date of grant or seven years from the date of filing, whichever is less. The first biopatent was on a genetically engineered Pseudomonas used to clear oil spills. The patent on ‘control of plant gene expression’, which prevents seeds from germinating, was refused in India as morally unacceptable and a threat to food security.
Biopiracy is the theft of natural products and indigenous knowledge, patented and sold without benefit or compensation to the host country. Indian examples are the neem patent sought by the USDA and W.R. Grace, the basmati patent granted to Rice Tec Inc (Texmati, 1997; all claims rejected in March 2001), and the turmeric patent granted in March 1995 and later cancelled after the CSIR sought re-examination. A genetic literacy movement in Indian schools and colleges is needed for the safe and meaningful use of modern life sciences.
12.10 Points to Ponder
Restriction enzymes evolved in bacteria as a defence against invading viruses, and had nothing to do with human purposes. If the most powerful tool of genetic engineering was found rather than designed, how many other tools might still be waiting, unnoticed, inside ordinary organisms?
Taq polymerase came from a bacterium living in a hot spring — a habitat that seemed to have no practical value at all. What does this suggest about the worth of conserving organisms whose usefulness we cannot yet see?
In gene cloning, a bacterium copies a human gene faithfully, without any way of “knowing” whose gene it is. Why does the cell not distinguish between its own DNA and foreign DNA — and what does that tell you about how universal the genetic machinery of life must be?
The same restriction enzyme must be used to cut both the donor DNA and the vector. Think through what would happen at every later step of rDNA technology if this one condition were quietly ignored.
The Bt crystal protein is harmless until an insect’s alkaline midgut activates it. Would you call such a protein a poison at all? Where, exactly, does the toxicity reside — in the molecule, or in the encounter between the molecule and a particular gut?
Golden rice was created to prevent childhood blindness, and yet it has been resisted in some of the very countries where vitamin A deficiency is common. What arguments could a person make against a crop that is meant to save eyesight, and how would you weigh them?
Germ line gene therapy could remove a disease from a family line permanently, and is precisely for that reason not permitted in human beings. Whose interests are being protected by that restriction — and who is being denied a benefit by it?
A herbicide-tolerant crop was expected to reduce the use of chemicals, and may end up increasing it. Can you think of other technologies where the solution quietly enlarged the very problem it was meant to solve?
Traditional knowledge of neem and turmeric was held for centuries by communities that never wrote a patent claim. If the knowledge cannot be traced to a single inventor, who — if anyone — owns it, and who should receive the benefit when it becomes a commercial product?
India refused a patent on a gene that prevents seeds from germinating, calling it morally unacceptable. Should there be inventions that a country declines to protect even though they are scientifically valid — and who should decide where that line falls?
Biotechnology has made it possible to do many things. Sections 12.5 to 12.8 ask what should be done. Is it reasonable to expect the scientist who develops a technique to also judge whether it ought to be used — or is that a responsibility that belongs to society as a whole?
12.11 Objective Questions
- The bacterium which causes a plant disease called crown gall is …………….
- Helicobacter pylori
- Agrobacterium tumifaciens
- Thermophilus aquaticus
- Bacillus thuringienesis
- The enzyme nuclease hydrolyses …………… of the polynucleotide chain of DNA.
- hydrogen bonds
- phosphodiester bonds
- glycosidic bonds
- peptide bonds
- In vitro amplification of a DNA or RNA segment is known as ……………
- chromatography
- southern blotting
- polymerase chain reaction
- gel electrophoresis
- Which of the following is the correct recognition sequence of the restriction enzyme Hin d III?
- 5’ —A-A-G-C-T-T— 3’ / 3’ —T-T-C-G-A-A— 5’
- 5’ —G-A-A-T-T-C— 3’ / 3’ —C-T-T-A-A-G— 5’
- 5’ —C-G-A-T-T-C— 3’ / 3’ —G-C-T-A-A-G— 5’
- 5’ —G-G-C-C— 3’ / 3’ —C-C-G-G— 5’
- Recombinant protein ……………… is used to dissolve blood clots present in the body.
- insulin
- tissue plasminogen activator
- relaxin
- erythropoietin
- Recognition sequences of restriction enzymes are generally …………… nucleotides long.
- 2 to 4
- 4 to 8
- 8 to 10
- 14 to 18
- Fill in the blanks and complete the chart.
| GMO | Purpose |
|---|---|
| i. Bt cotton | ……………….. |
| ii. ……………….. | Delay the softening of tomato during ripening. |
| iii. Golden rice | ……………….. |
| iv. Holstein cow | ……………….. |
- Match and write the pairs.
| Recombinant protein | Its use in or for |
|---|---|
| a. Platelet derived growth factor | i. Anaemia |
| b. α-antitrypsin | ii. Cystic fibrosis |
| c. Relaxin | iii. Haemophilia A |
| d. Erythropoietin | iv. Diabetes |
| e. Factor VIII | v. Emphysema |
| f. DNA ase | vi. Parturition |
| vii. Atherosclerosis |
12.12 Check Your Concepts
Bacteria produce restriction enzymes that cut DNA at specific sequences. Why is the bacterium’s own DNA not destroyed by these enzymes? Explain the mechanism that protects it.
Assertion: The same restriction enzyme must be used to cut the donor DNA and the vector DNA. Reason: Sticky ends produced by the same enzyme are complementary to each other. State whether the assertion and the reason are true, and whether the reason correctly explains the assertion.
Why is a heat-stable DNA polymerase essential for PCR? What would happen if an ordinary DNA polymerase from a human cell were used instead?
Arrange the following steps of rDNA technology in the correct sequence: selection of transformed host cells; expression of the gene and downstream processing; isolation of DNA from the donor; transfer of rDNA into a competent host; cleavage by restriction enzyme; joining of foreign DNA to the vector by DNA ligase; multiplication of transformed host cells.
A student performs electrophoresis on a mixture of DNA fragments and finds one band very close to the well and another far from it. Which band contains the larger fragments, and why? Towards which electrode did the DNA migrate, and what property of DNA decides this?
What happens if a recombinant plasmid enters a host cell but the host cell is not “competent”? Explain what the term competent means and how competence is brought about.
In pBR 322, the foreign DNA is inserted within the ampicillin-resistance gene using Pst1. Explain how the loss of a useful property helps a scientist to identify the recombinant cells.
Why is a cDNA library, and not a genomic library, preferred when a eukaryotic gene is to be expressed in a bacterium? Explain with reference to the structure of the eukaryotic gene.
Assertion: The Bt toxin does not harm human beings who eat Bt cotton seed oil or Bt brinjal. Reason: The cry protein is ingested as an inactive crystal and requires an alkaline pH and midgut proteinases for activation. State whether the assertion and reason are true, and whether the reason explains the assertion.
Draw a labelled diagram of a plasmid cloning vector and label the following: ori, the antibiotic-resistance marker gene, and the region into which foreign DNA can be inserted. State the function of each of the three labelled parts.
Sketch the recognition sequence of EcoRI on a double-stranded DNA, mark the site of cleavage on both strands, and show the ends produced. Why is the sequence called a palindrome?
Distinguish between blunt ends and sticky ends. Which of the two is preferred in gene cloning, and why?
What happens to a tomato fruit if the polygalacturonase enzyme is not inhibited? Explain how the antisense gene technology used in the “Flavr savr” tomato solves this problem.
Golden rice is engineered to accumulate beta carotene rather than vitamin A itself. Give a reason for this choice.
Why is germ line gene therapy not permitted in human beings, even though it is more effective than somatic cell gene therapy? Give both a technical and an ethical reason.
Why is the mammary gland of a dairy cow described as an excellent “protein production factory”? Explain the structure–function reasoning behind using transgenic cattle as bioreactors.
A herbicide-tolerant crop is grown next to a field where a wild relative of the crop is also growing. Predict what may happen over several seasons, and explain why the benefit of the herbicide may eventually be lost.
Bt corn was engineered against crop pests, yet it has been reported to affect Monarch butterfly populations. Explain why a non-target insect can be harmed by a toxin that was never aimed at it.
A gene from the Brazil nut was transferred to soybean to increase methionine. Explain what went wrong, and why an allergen is dangerous even when the food itself is not toxic.
Match the following.
| Column A | Column B |
|---|---|
| a. Taq polymerase | i. Joins DNA fragments |
| b. DNA ligase | ii. Cuts DNA at a specific palindrome |
| c. EcoRI | iii. Extends the primer at 70–75 °C |
| d. Reverse transcriptase | iv. Vector for plants |
| e. Ti plasmid | v. Makes DNA from mRNA |
- Match the following.
| Column A | Column B |
|---|---|
| a. cry gene | i. Wool quality in sheep |
| b. Ferritin gene | ii. Insect resistance |
| c. cys E and cys M genes | iii. Vitamin A in rice |
| d. Phytoene synthase | iv. Iron fortification of rice |
Give scientific reasons: (a) E. coli is the most commonly used host in rDNA technology. (b) Divalent calcium ions are added during transformation. (c) Bt sprays are less effective in the field than a Bt crop.
Explain why India allows a process patent and not a product patent. How does this distinction affect a rival company that wishes to make the same substance by a different method?
A patent was granted in the USA on the healing properties of turmeric, which had been known in India for centuries. On what ground was this patent finally cancelled? What does this case teach us about the documentation of traditional knowledge?
Distinguish between biopatent and biopiracy, giving one example of each from the chapter.
State the role of the GEAC. Why is such a regulatory body necessary even when a GMO has been shown to be scientifically sound?
Trace the path of a human insulin gene from its chemical synthesis to the purified hormone, naming the vector, the host, and the treatment used to release the insulin.
Look again at Fig. 12.5 (outline of rDNA technology), with the labels A to G removed. Write down what each of the seven stages represents, in order.
Name the vector which is used in the production of human insulin through recombinant DNA technology. (30–40 words)
Which cells of the islets of Langerhans of the pancreas produce the peptide hormone insulin? (30–40 words)
Give the role of \(Ca^{++}\) ions in the transfer of a recombinant vector into a bacterial host cell. (30–40 words)
Expand the following acronyms, which are used in the field of biotechnology: (30–40 words)
- YAC
- RE
- dNTP
- PCR
- GMO
- MAC
Explain the properties of a good and ideal cloning vector for rDNA technology. (80–100 words)
In a PCR machine the temperature can be raised up to 100 °C, but after that it is not able to lower the temperature below 70 °C automatically. Which step of PCR will be hampered first in this faulty machine? Explain why. (80–100 words)
In the process of rDNA technology, if two separate restriction enzymes are used to cut the vector and the donor DNA, then which problem will arise in the formation of rDNA or chimeric DNA? Explain. (80–100 words)
Define and explain the terms: (150–200 words)
- Biopiracy
- Biopatent
- Bioethics
Explain the steps in the process of rDNA technology with suitable diagrams. (150–200 words)
Explain gene therapy. Give two methods of the same. (150–200 words)
How are transgenic mice used in cancer research? (150–200 words)
Give the steps in PCR (polymerase chain reaction) with suitable diagrams. (150–200 words)
What is a vaccine? Give the advantages of oral vaccines or edible vaccines. (150–200 words)
Enlist the different types of restriction enzymes commonly used in rDNA technology. Write on their role. (150–200 words)
Enlist and write in brief about the different biological tools required in rDNA technology. (150–200 words)