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

Open the medicine fridge of any chemist's shop and you will probably find vials of human insulin. The insulin inside was not taken from a person. It was made by microbes that carry a copy of the human insulin gene, grown by the tonne in steel tanks. This chapter explains the two ideas that make such a product possible: how scientists move a chosen gene into a new host, and how they grow that host on a large scale and recover what it makes.

In the widest sense, biotechnology (जैव प्रौद्योगिकी) is any use of living things, or of enzymes taken from them, to make something we want or to do a job for us. By that yardstick, Indian kitchens have practised it for centuries: microbes turn milk into dahi and make idli batter rise. When scientists speak of biotechnology today, though, they usually mean work built on organisms whose genes have been deliberately changed, run on a factory scale. The modern field also takes in techniques such as IVF ('test-tube' babies), genes assembled in the laboratory, DNA vaccines and gene therapy, which repairs a faulty gene.

The European Federation of Biotechnology offers one definition broad enough to cover both kinds. In paraphrase, it describes biotechnology as bringing natural science together with organisms, their cells or cell parts, and molecular copies of them, in order to produce goods and services.

9.1 Principles of Biotechnology

Modern biotechnology stands on two techniques. One changes what an organism is; the other lets us grow that changed organism in huge numbers without letting anything else grow alongside it.

• Genetic engineering rewrites the genetic material. A gene (DNA, or sometimes RNA) is altered or moved into a host organism, and the host then shows a new character.
• Bioprocess engineering is the engineering of very large, germ-free cultures. It grows enormous numbers of one chosen cell type, microbial or eukaryotic, with no contaminating microbes, to manufacture antibiotics, enzymes, vaccines and similar products.

Why go to this trouble when breeders already cross plants and animals? Sexual reproduction reshuffles genes and so creates useful variety, whereas asexual reproduction keeps a genome unchanged. A cross, however, cannot pick genes one at a time. When a breeder crosses a crop with a wild relative to gain one helpful gene, long stretches of the wild chromosome come along with it, often carrying genes nobody wants (Fig. 9.1). Genetic engineering avoids this: it isolates just the gene, or small set of genes, that we want and puts only that into the target organism.

Why a foreign gene needs an origin of replication

A cell copies its DNA only from special starting points. Each chromosome and each plasmid carries such a sequence, called an origin of replication or ori, and copying of the whole molecule begins there. This has a direct consequence for genetic engineering. A loose fragment of foreign DNA has no ori, so a bacterium that picks it up does not copy it; after a few divisions it has been diluted out or broken down. Join the same fragment to a plasmid or a chromosome, and it is copied whenever its partner is copied and is passed to every daughter cell (Fig. 9.2). Producing many identical copies of a DNA segment in this way is called cloning it.

The first recombinant DNA

What Stanley Cohen and Herbert Boyer built in 1972–73 was, in effect, a plasmid carrying one extra gene: a gene that made bacteria resistant to an antibiotic. To get the gene out of the plasmid where it naturally sat, they used an enzyme that cuts DNA only at one particular sequence, a restriction enzyme (often nicknamed molecular scissors). The cut-out gene was then stitched into a small natural plasmid, which your textbook says came from Salmonella typhimurium, using DNA ligase, an enzyme that seals cut DNA ends. The product, the first artificial recombinant DNA, had been put together entirely in a test tube, yet it could copy itself like any natural plasmid.

In this experiment the plasmid served as a vector, a carrier for delivering DNA into a host; think of a courier van taking a parcel to the right address. The host chosen was Escherichia coli, a near cousin of Salmonella. Once inside, the recombinant plasmid needed no special help: the bacterium's own DNA polymerase copied it at every division, so the resistance gene was cloned in E. coli (Fig. 9.3).

Strip away the details and every genetic modification, of a microbe, a crop or an animal, needs three things to go right:

1. find the gene worth moving and isolate the DNA that carries it;
2. deliver it into the new organism;
3. keep it there, so that it stays in the host and is inherited by the host's descendants.

9.2 Tools of Recombinant DNA Technology

Recombinant DNA work needs a small kit of tools, each with a single job. Nearly all of them were invented by microbes for their own ends, and we have simply borrowed them.

• Restriction enzymes cut DNA at chosen sequences.
• DNA polymerases copy DNA, in the cell or in a tube.
• DNA ligases join cut pieces of DNA.
• Vectors carry a gene into a host and get it copied there.
• A host organism, often a bacterium, takes up the DNA and multiplies it.

9.2.1 Restriction Enzymes

Bacteria live under constant attack from bacteriophages, viruses that inject their DNA into the cell. Their defence was traced in 1963 to a pair of enzymes from E. coli. One, a methylase, tags the bacterium's own DNA with methyl groups. The other is a nuclease that cuts DNA lacking those tags, so an invading phage genome is chopped up before it can take over. Because it restricts the growth of the virus, the cutting enzyme was named a restriction endonuclease.

The first restriction enzyme whose cutting was tied to a particular base sequence, Hind II, was isolated and studied about five years later. Whatever DNA it was given, it cut at the same kind of place: a stretch of six base pairs with a set sequence. Such a stretch is called the enzyme's recognition sequence. By the time your textbook was written, over 900 different restriction enzymes had been found in more than 230 bacterial strains, each with its own recognition sequence, and the list is still growing.

An enzyme's name is a compressed address. Take EcoRI. E stands for the genus, Escherichia, and co for the species, coli; these three letters are printed in italics. R identifies the strain, RY13, and the Roman numeral I says that this was the first enzyme isolated from that strain. Every restriction enzyme is named on the same plan: genus initial, two letters of the species, a strain code if needed, then the order of discovery.

Chemically, a restriction enzyme is a nuclease: it breaks the bonds that link one nucleotide to the next. What sets nucleases apart is where on the DNA they attack.

Palindromes and sticky ends

How does an enzyme find six particular base pairs among millions? It moves along the double helix, testing the sequence, and stops only where its recognition sequence occurs. Then it breaks the sugar–phosphate backbone of both strands at fixed positions. Most recognition sequences are palindromes. A number such as 12321 reads the same from either end; a DNA palindrome is subtler, because its two strands run in opposite directions. Read each strand from its 5′ end to its 3′ end, and both give the same sequence. For EcoRI the site is GAATTC on the top strand, and reading the bottom strand in its own 5′→3′ direction also gives GAATTC.

Look at where EcoRI breaks the backbone (Fig. 9.4). On each strand it cuts the bond between G and A, so the two breaks are four bases apart rather than opposite each other. When the halves separate, each end keeps a short single-stranded tail, AATT. Ends of this kind are called sticky ends (चिपचिपे सिरे). Any two ends produced by the same enzyme have complementary tails, so they find each other and pair by hydrogen bonds. Paired ends are held only loosely, with a break (a nick) still left in each backbone; DNA ligase closes the nicks with covalent bonds. A few enzymes, such as SmaI, cut both strands at the same point and leave blunt ends, which ligase can also join, though less efficiently.

A practical rule follows. Choose one enzyme (or a pair that leave identical tails) and use it on both the DNA that carries your gene and the vector. Tails from mismatched enzymes cannot pair, and the recombinant molecule will not form.

Separating and recovering the fragments

A digest leaves a mixture of DNA fragments of different lengths, and they are sorted by gel electrophoresis. Picture a slab of jelly made from agarose, a polysaccharide obtained from seaweeds. Inside, it is a tangled mesh of fibres. The DNA mixture is placed in small wells at one end and an electric field is switched on. Every fragment carries negative charges on its phosphate groups, so all of them head for the anode (+). Short pieces slip through the mesh easily; long ones keep getting caught. After a while the fragments have spread out by size, with the smallest farthest from the wells (Fig. 9.5).

A gel straight out of the tank looks blank, because DNA is colourless. It is soaked in a dye such as ethidium bromide, which slips in between the base pairs, and placed under ultraviolet light; now every band glows orange. A band you need can be sliced out with a blade, and the DNA in that slice is washed out and purified. Recovering DNA from a gel slice in this way is called elution. The recovered fragment is ready to be joined to a cloning vector.

9.2.2 Cloning Vectors

A carrier for genes is most useful if the host copies it often. Plasmids and bacteriophages both qualify, because a bacterium copies them separately from its own chromosome. Each has a typical copy number, the number of copies per cell. For phages it is very high. Plasmids range widely: some are kept at a single copy or two, while others reach 15 to 100 copies or more. Splice a gene into such a carrier and the gene reaches the same copy number. Today's cloning vectors (संवाहक) are plasmids or phages redesigned for the job: they make it easy to insert foreign DNA and easy to find the cells that end up with an insert.

Four features make a vector useful.

(i) Origin of replication. Copying of the vector starts at its ori, so a gene linked to the vector is copied too. The ori also decides how many copies of the vector each cell keeps. To harvest a gene in large amounts, therefore, clone it in a vector with a high-copy ori.

(ii) Selectable marker. When bacteria are mixed with a vector, only a small fraction take it up. The process of taking up DNA is called transformation (रूपांतरण), and the cells that succeed are transformants. To get rid of all the others, the vector carries a selectable marker, a gene that lets only the cells containing it survive on a particular medium. In E. coli the favourite markers are antibiotic-resistance genes, for example against ampicillin, kanamycin, tetracycline or chloramphenicol. Ordinary E. coli has no such protection, so the antibiotic kills every cell that lacks the vector.

(iii) Cloning sites. Foreign DNA is inserted where a restriction enzyme opens the vector. If the enzyme cut the vector in two or three places, the vector would fall apart into pieces that would then have to be put back in the right order, a near-impossible task. Good vectors therefore have just one recognition site, or at most very few, for each commonly used enzyme. The classic E. coli vector pBR322 shows these features (Fig. 9.7).

How do you find the colonies that carry an insert? pBR322 has two resistance genes, and its BamHI site lies inside the tetracycline-resistance gene ($te{t}^R$). A gene inserted at BamHI breaks $te{t}^R$, but leaves the ampicillin-resistance gene ($am{p}^R$) working. So the transformed bacteria are first spread on a plate with ampicillin, where only cells that took up a plasmid, with or without an insert, form colonies. A velvet pad then copies the colonies, each in its own position, onto a plate with tetracycline. Colonies that appear on both plates have the empty plasmid. Colonies that grew on ampicillin but are missing on tetracycline are the recombinants (Fig. 9.8). In short, one marker selects the transformants, and the other, switched off by the insert, reveals the recombinants.

On some plates the colonies come in two colours, blue and white. This is a quicker trick that needs only one plate. Here the vector's cloning site lies inside the gene for the enzyme β-galactosidase, and the medium contains a colourless chromogenic substrate that the enzyme turns blue. A colony whose plasmid has no insert makes the enzyme and turns blue. In a colony whose plasmid gained an insert, the gene is broken by insertional inactivation, no enzyme is made and the colony stays white. You simply pick the white colonies (Fig. 9.9).

Vectors for plants and animals

To get genes into plants and animals, scientists copied the experts: pathogens. Agrobacterium tumefaciens is a soil bacterium that causes crown gall in many dicot plants. It carries a large Ti plasmid (tumour-inducing plasmid), and part of this plasmid, the T-DNA, actually travels into the plant cell and becomes part of a plant chromosome. Genes on the T-DNA make the cell divide into a tumour, the crown gall, and make it produce special chemicals, opines, on which the bacterium feeds (Fig. 9.10).

To use this delivery system safely, the Ti plasmid has been disarmed. The tumour-causing genes were taken out and the transfer machinery left in, so any gene placed between the T-DNA borders now enters plant cells without causing disease. Animals have their own natural gene couriers, retroviruses, which can make normal cells cancerous; disarmed retroviruses are used to bring useful genes into animal cells. Whatever host we need, bacterium, plant or animal, there is now a vector that can carry a cloned gene into it.

9.2.3 Competent Host (For Transformation with Recombinant DNA)

Why won't a bacterium simply soak up DNA from its surroundings? DNA is hydrophilic and carries many negative charges, while the core of a cell membrane is oily and water-repelling, so DNA cannot pass through it on its own. Bacteria are therefore made competent first. The usual trick is to soak them in a solution of a divalent cation, typically calcium from calcium chloride, at a carefully chosen concentration; this makes it much easier for DNA to get in through pores in the cell envelope.

The rest is a matter of temperature. The competent cells and the recombinant DNA are mixed and chilled on ice. The tube is then given a heat shock: about 42 °C for a short time, typically 30–90 seconds, before it goes straight back onto ice. During this brief jolt the plasmid enters some of the cells (Fig. 9.11).

Other hosts need other methods. Animal cells can receive DNA by micro-injection: a glass needle far finer than a hair delivers the recombinant DNA straight into the cell's nucleus. Plant cells, with their tough walls, are often hit with biolistics (the gene gun): microscopic gold or tungsten beads are coated with DNA and fired into the cells at high speed. A third route sends in a disarmed pathogen, such as Agrobacterium or a modified virus, which delivers the recombinant DNA while it infects the cell.

9.3 Processes of Recombinant DNA Technology

With the tools in hand, a recombinant DNA project runs through a fixed sequence of steps, from raw tissue to a bottled product.

9.3.1 Isolation of the Genetic Material (DNA)

Restriction enzymes are fussy: they need clean DNA, not a soup of cell contents. Yet in a living cell the DNA sits behind a membrane, and in bacteria, plants and fungi behind a wall as well. So the cells are first broken open, which spills out everything at once: DNA, but also RNA, proteins, lipids and polysaccharides. (DNA is the genetic material of almost every organism; only some viruses use RNA instead.)

Each kind of wall has its own enzyme: lysozyme opens bacterial cells, cellulase plant cells and chitinase fungal cells. The unwanted molecules are then removed one class at a time. Ribonuclease (RNase) digests the RNA. Protease digests proteins, including the histones around which the DNA is wound. Further treatments remove the remaining contaminants. Last, ice-cold ethanol is added. DNA is insoluble in it, so the purified DNA comes out of solution as a mass of fine white threads, which can be wound out on a glass rod; this is called spooling (Fig. 9.12).

9.3.2 Cutting of DNA at Specific Locations

Each restriction enzyme has its favourite working conditions, a particular buffer, salt level and temperature (often 37 °C). The purified DNA is incubated with the enzyme under those conditions. How do you know when the cutting is done? Take a little of the mixture now and then and run it on an agarose gel: being negatively charged, the DNA moves towards the anode, and as the digest proceeds the single band of uncut DNA gives way to the bands of the fragments (Fig. 9.5). The vector is opened in the same way, normally with the same enzyme.

Now the two cut DNAs are brought together. The fragment carrying the gene of interest is mixed with the opened vector and DNA ligase is added; matching sticky ends pair, ligase seals them, and recombinant DNA is formed.

9.3.3 Amplification of Gene of Interest using PCR

Sometimes the gene you need is present in only a few copies, too few to cut and clone. The polymerase chain reaction (PCR) solves this by copying a chosen stretch of DNA again and again in a test tube. The reaction contains the template DNA, two primers (short, chemically made single strands of DNA that match the two ends of the target region), the four deoxynucleotides and a DNA polymerase. Starting from the end of each bound primer, the polymerase adds nucleotides one by one, each chosen to pair with the template strand.

A cycle runs through three temperatures, controlled by a machine called a thermal cycler (Fig. 9.13).

1. Denaturation (about 94 °C): heat separates the two strands of the DNA.
2. Annealing (about 50–60 °C, depending on the primers): the two primers bind to their matching sequences, one on each strand, on either side of the target.
3. Extension (72 °C): the polymerase builds a new strand from each primer, copying the template.

Why doesn't the heat of every denaturation step destroy the polymerase? Because PCR uses a thermostable enzyme, Taq polymerase, from Thermus aquaticus, a bacterium that lives in hot springs. It survives being heated again and again, so one dose lasts the whole run. Each new strand made in one cycle serves as a template in the next, so the number of copies roughly doubles every cycle, and about 30 cycles turn a single molecule into roughly a billion. If the gene is to be cloned, these copies can be joined into a vector like any other DNA fragment.

9.3.4 Insertion of Recombinant DNA into the Host Cell/Organism

Picture the next step as a sorting problem. A tube of competent E. coli is mixed with ligated DNA, using one of the methods of Section 9.2.3, and only some cells take a plasmid in. Suppose the plasmid carries an ampicillin-resistance gene. Spread the whole tube on agar that contains ampicillin: cells without a plasmid die, while every cell with one, now resistant, grows into a colony. That gene is doing the job of a selectable marker. A second test, such as plating on tetracycline or blue–white screening, then separates recombinants from cells carrying an empty vector (Fig. 9.8, Fig. 9.9).

9.3.5 Obtaining the Foreign Gene Product

Copying the gene is rarely the end goal; nearly always the aim is the protein it codes for. For that, the host must express the gene, that is, transcribe it and translate the message. Expression in a foreign host is a technical art: the gene needs suitable control signals, and the growth conditions must switch it on. A protein produced from a gene expressed in a different species, a heterologous host, is a recombinant protein. Human insulin made in E. coli or in yeast is an example.

In the laboratory, a flask of these cells yields enough protein to extract, purify and study. A medicine, however, may be needed by millions of patients, and flask-sized cultures cannot come close to that. Production has to be scaled up.

One way to raise the yield is continuous culture. Fresh medium flows in all the time, and an equal volume of spent medium, carrying some cells, flows out. The cells never run out of food or build up waste, so they stay in their most productive, exponential (log) phase of growth and give more biomass, and more protein, than a single batch left to run its course.

Scaling up needs bioreactors. A bioreactor is a vessel in which a culture of living cells, bacteria or yeast or cells from plants, animals or humans, converts cheap raw materials into a chosen product such as a protein or an enzyme. Your textbook describes vessels holding 100–1000 litres; industrial tanks are often much larger. Above all, a bioreactor must keep conditions ideal for the cells: enough oxygen and nutrients (substrate, salts and vitamins), and the right pH and temperature.

Most bioreactors are of the stirred-tank type (Fig. 9.15). The tank is a cylinder, or has a rounded bottom, because curved surfaces leave no dead corners where the culture could stagnate. A motor turns impellers on a central shaft, keeping the broth uniform and carrying oxygen to every part of it. In a sparged stirred tank, sterile air is also blown in through a perforated ring (a sparger) at the bottom; many small bubbles offer a huge total surface through which oxygen dissolves. Around the vessel sit the systems that keep the cells comfortable: the agitator and the air (oxygen) supply, a water jacket that holds the temperature steady, a pH probe linked to pumps that add acid or alkali, a foam breaker, and sampling ports through which small volumes of culture can be drawn off without letting microbes in.

9.3.6 Downstream Processing

At the end of a run the tank holds the product mixed with cells, spent medium and by-products, nowhere near ready for a pharmacy shelf. The work that follows, chiefly separating the product and purifying it, is called downstream processing (अनुप्रवाह संसाधन). The purified product is then formulated, for instance made into an injection with preservatives that keep it stable. Like any other drug, a new medicine has to pass clinical trials, and every batch must clear strict quality control tests. No single recipe fits all products: each one needs its own downstream steps and tests.