Biotechnology and Genetic Modification

• Three sentences hold the spine of the chapter: yeast turns glucose into ethanol and carbon dioxide; a fermenter controls temperature, pH, oxygen, nutrients and wastes; genetic modification changes DNA by removing, changing or inserting individual genes. Everything else hangs off those three.
• If a Topic 18 question is worth more than three marks, it is almost certainly asking for a sequence or a balance. Decide which before you write a word.
• If you fix only two things before the exam, fix these: the gas that raises bread is carbon dioxide, and a fermenter is cooled. Those two errors between them account for more lost Topic 18 marks than everything else on this page.
• If you can reproduce tables 4 and 5 from memory, you have most of the marks in Topic 18 already. Table 4 is the recall half of the topic; table 5 is the distinction the evaluation questions rest on.
• If drill 1 is the one you keep getting wrong, the fix is a single question, asked every time: has a gene been moved?
• Nine of these twenty come down to two habits: keeping enzyme and organism apart, and keeping possible and certain apart. Fix those two and most of the list disappears.
• If your mind goes blank on a fermenter question, ask: what problem does this part solve? Overheating, pH drift, no oxygen, uneven mixing, or invasion. Those five problems generate every feature on the diagram.
• Three visits, spaced a day, a week and a month apart, beat six visits in one evening. The forgetting between visits is what makes them work.

• Define biotechnology as the use of living organisms, cells or enzymes to make useful products, and give examples that are and are not biotechnology.
• Explain what fermentation means in yeast, and state that industrial fermentation may be aerobic or anaerobic depending on the organism and the product.
• State the definition of genetic modification in the syllabus wording: changing an organism's genetic material by removing, changing or inserting individual genes.
• Distinguish genetic modification from selective breeding, and explain why every biotechnology process is not genetic modification.
• Write the word equation for anaerobic respiration in yeast: glucose → ethanol + carbon dioxide.
• Describe the full mechanism of bread making, from mixing yeast with a sugar source to the effect of baking, naming carbon dioxide as the gas that raises the dough.
• Describe the use of yeast in ethanol production, including the limitation of oxygen and the control of temperature and pH.
• Explain the effect of low, suitable and excessive temperature on fermentation rate, using kinetic energy for the first and denaturation for the third.
• Give four reasons bacteria are useful in biotechnology, and connect each one to a production consequence rather than stating it alone.
• Describe a plasmid as a small circular DNA molecule that can receive a selected gene.
• Label an industrial fermenter and state the function of each labelled part.
• Explain why temperature, pH, oxygen, nutrient supply and waste products are each controlled, and what happens to yield if control is lost.
• Explain why cooling — not heating — is usually what a large fermenter needs.
• Explain the purpose of sterilisation, filtered air and inoculation, and describe three consequences of contamination.
• Explain how proteases and lipases in biological washing powders remove protein and fat stains, and why a moderate wash temperature is used.
• Explain how pectinase increases the yield and the clarity of fruit juice, naming pectin and where it is found.
• Explain how lactase is used to make lactose-free milk, and state the products of the reaction.
• Distinguish lactose intolerance from milk-protein allergy and explain why lactose-free milk helps only the first.
• Describe how a plasmid acts as a vector, and use the word vector in its genetic sense without confusing it with a disease vector.
• Outline the production of human insulin by genetically modified bacteria, in the correct order, from identifying the gene to purifying the product.
• Describe three crop modifications — herbicide resistance, insect-pest resistance and increased vitamin content — explaining the mechanism of each before its benefit.
• Explain why a herbicide-resistant crop does not itself kill weeds.
• Trace the gene-flow sequence from GM pollen to a wild relative, and say why it is possible rather than automatic.
• Explain the appearance of resistant pest populations as natural selection, without claiming the crop causes the mutation.
• Give balanced advantages and risks for a named GM application, each linked to its mechanism.
• Write a conditional conclusion — a judgement that states what it depends on — instead of an absolute one.
• Distinguish correlation from causation when reading supplied data on yields, insecticide use or pest frequency.
• Calculate a rate of production as quantity divided by time, with correct units.
• Calculate a percentage increase or decrease from supplied values and quote it to a sensible number of significant figures.
• Read a temperature–rate graph and explain the shape of both sides of the peak in enzyme terms.
• Identify an anomalous result and say what should be done about it.

Where you meet this outside biology. Cloudy apple juice and clear apple juice usually start as the same pressed liquid; the clear version has been treated with pectinase. The same chemistry works the other way round in a kitchen: pectin is what makes jam set, which is why fruits naturally rich in pectin set easily and others need pectin added. Same molecule, opposite intention.

Yeast in ethanol production

The use of yeast to make ethanol on an industrial scale. A sugar source such as grain or fruit is prepared as a solution and yeast is added to a closed vessel, so that oxygen is limited or excluded and the yeast respires anaerobically. Glucose is converted to ethanol and carbon dioxide; the carbon dioxide escapes through a gas outlet while the ethanol accumulates in the liquid and is collected. Temperature and pH are held near suitable values throughout so that the yeast's enzymes work efficiently without denaturing. The ethanol produced may be used for beverages, for industrial purposes or as a biofuel.

Fermentation

The use of microorganisms to make a useful product. In yeast, fermentation is anaerobic respiration: glucose is converted to ethanol and carbon dioxide, and energy is released, without oxygen being used. The same reaction supports two industries that want opposite products from it - bread making, which uses the carbon dioxide to raise dough, and ethanol production, which collects the ethanol. In industrial usage the word also describes the growth of microorganisms in a fermenter, a process that may be aerobic or anaerobic depending on the organism and the product required.

Genetically modified crop

A crop plant into which one or more selected genes have been inserted to give it a new characteristic. Three examples are required at Cambridge O Level. Herbicide resistance allows the crop to survive being sprayed with a particular herbicide, so that herbicide can be used to control competing weeds without killing the crop; the crop itself does not kill weeds. Insect-pest resistance means the crop carries a gene that gives it resistance to particular insect pests, so crop losses fall and less insecticide may be needed. Increased vitamin content means inserted genes raise the production or accumulation of a vitamin or its precursor, improving the nutritional value of the crop. Each modification works against particular herbicides or particular pests, never against all of them.

Pectinase

An enzyme used in fruit-juice production that breaks down pectin. Pectin is a substance found in plant cell walls and in the middle regions between neighbouring cells, where it acts as a cement holding the tissue together and holding the cell sap inside. When crushed fruit is treated with pectinase, the pectin is broken down, the cells separate and their contents are released, so a greater volume of juice is obtained from the same mass of fruit. Because fewer suspended fragments of tissue remain in the liquid, the juice is also clearer and easier to filter. Pectinase acts specifically on pectin; it does not digest cellulose.

Human insulin production

The manufacture of human insulin using genetically modified bacteria. The human gene that controls insulin production is identified and cut out of human DNA, a bacterial plasmid is cut open and the gene is joined into it to form a recombinant plasmid, and that plasmid is introduced into bacteria. The modified bacteria are then cultured in a fermenter under controlled conditions; because the genetic code is shared between organisms, they read the human gene and produce human insulin. The insulin is separated from the culture and purified before being prepared for medical use. The bacteria do not learn to make insulin - their DNA has been changed - and live modified bacteria are never administered to patients.

Biological washing powder

A washing powder containing enzymes that break down the substances in stains. Proteases hydrolyse the large insoluble protein molecules in stains such as blood, egg and grass into small soluble amino acids; lipases hydrolyse the fats and oils in greasy stains into fatty acids and glycerol. Because the products are small and soluble they dissolve in the wash water and are carried away, so the stain is removed. The enzymes work effectively at moderate temperatures, so a lower wash temperature can be used and less energy is needed for heating; at very high temperatures the enzymes denature and the powder loses its effect. The powder contains isolated enzymes, not living microorganisms, and the enzymes catalyse reactions rather than eating anything.

Genetic modification

Changing the genetic material of an organism by removing, changing or inserting individual genes. The change is made directly to DNA rather than by choosing which organisms reproduce, and it can move a gene between species because the genetic code is essentially the same in all organisms. A modified organism may express the inserted gene and produce a protein, and therefore a characteristic, that it could not produce before. Genetic modification is one technique within biotechnology, not a synonym for it, and it is not the same as selective breeding, in which parents with desirable features are chosen and bred over many generations without any gene being moved.

Biotechnology

The use of living organisms, cells or enzymes to make useful products or to carry out useful processes. Biotechnology is a broad field that includes baking bread and brewing with yeast, growing bacteria in industrial fermenters, and the industrial use of isolated enzymes such as proteases, pectinase and lactase. Genetic modification is one technique used within biotechnology, not a synonym for it: many biotechnological processes, including all of the yeast and enzyme applications required at Cambridge O Level, involve no change to genetic material at all.

Sterilisation

The killing or removal of all microorganisms from the fermenter vessel, the growth medium and the incoming air before a culture is started. Sterilisation is carried out first, so that the vessel contains no living organisms at all; the chosen microorganism is then deliberately added in a separate step called inoculation. The two must not be confused: sterilisation removes everything, inoculation adds one thing. Sterilisation matters because unwanted microorganisms entering the fermenter would compete with the chosen organism for nutrients, reduce the yield, alter or spoil the product, and in some cases create a safety risk.

Fermenter

A large vessel, usually of stainless steel, in which microorganisms are grown on a commercial scale under controlled conditions so that a useful product can be collected. A fermenter allows temperature, pH, oxygen supply, nutrient supply and waste products all to be controlled at once. Typical features include a cooling jacket to remove the heat released by respiration, temperature and pH probes, inlets for nutrients and for acid or alkali, a sterile air inlet through a sparger for aerobic processes, a motor-driven impeller to distribute heat, nutrients and oxygen, an inoculation port, a sampling port, an exhaust outlet and a product outlet. Whether air is supplied depends on the organism and the product: an anaerobic process limits or excludes it.

Gene flow

The movement of alleles from one population into another. Where a genetically modified crop is grown, gene flow is the possibility that pollen from the crop reaches a compatible wild relative growing nearby, cross-pollination occurs, fertilisation succeeds, and offspring carry the inserted allele, so that the feature may spread into the wild population. Every step in that sequence is a condition rather than a certainty: the two plants must be able to breed together, the pollen must reach a stigma, and fertilisation must succeed. Gene flow is therefore a genuine possibility whose likelihood depends on the crop, the wild relatives present and the conditions - not an automatic consequence of growing a GM crop, and not something that happens between unrelated species in ordinary field conditions.

Evaluating genetic modification

The structured comparison of the advantages and disadvantages of a particular genetic modification, ending in a conclusion that states the conditions it depends on. A complete evaluation names the modification, gives the biological mechanism, states a possible benefit and a possible risk that each follow from that mechanism, identifies the evidence that would be needed to decide, and reaches a conditional judgement. Risks are stated as possibilities rather than certainties, and benefits are tied to the specific application rather than claimed for the technology as a whole. Absolute statements such as all genetic modification being unsafe, or having no risks, are not supported by evidence about any single application.

Cooling jacket

An outer water jacket surrounding an industrial fermenter, used to remove excess heat from the culture. Microorganisms release thermal energy as they respire, and in a large vessel containing an enormous number of cells this heat would raise the temperature far above the optimum, denaturing enzymes and killing cells. Cold water is circulated through the jacket and leaves warmer, carrying the excess heat away, while a temperature probe monitors the culture so the cooling can be adjusted. A large fermenter therefore needs cooling rather than heating, which is the opposite of most students' expectation.

Impeller

A motor-driven stirrer inside an industrial fermenter. Turning the impeller distributes the microorganisms, the nutrients, the dissolved oxygen where air is supplied, and the heat produced by respiration, so that conditions are the same throughout the vessel rather than varying from place to place. Without stirring, cells settle out at the bottom, warm liquid collects at the top, nutrients are used up locally and oxygen reaches only the region above the sparger, so parts of the culture are starved while others overheat. Stirring distributes what is already present; it does not create nutrients or oxygen.

Lactase

An enzyme that hydrolyses lactose, the sugar naturally present in milk, into the simpler sugars glucose and galactose. Milk treated with lactase therefore has a much reduced lactose content and is sold as lactose-free milk. It is suitable for many people with lactose intolerance, who do not produce enough lactase of their own to digest lactose properly. Lactose intolerance is not the same as an allergy to milk protein: removing the lactose does not remove the protein, so lactose-free milk does not help with a milk-protein allergy. The enzyme name ends in -ase and the sugar it acts on ends in -ose, which is the reliable way to keep the two words apart.

Pest resistance

The increase in frequency of an allele giving resistance, in a pest population exposed to an insect-resistant crop or to an insecticide. Variation already exists in the pest population before the crop is planted, and a few individuals happen to carry an allele that lets them tolerate it. The crop then acts as a selection pressure: susceptible individuals die or reproduce less, while resistant individuals survive, reproduce and pass the allele to their offspring. Over generations the proportion of resistant individuals rises until the crop no longer controls the pest effectively. The crop selects among variation that already existed; it does not cause the mutation, and individual pests do not become resistant during their lifetimes.

Yeast in bread making

The use of yeast to raise bread dough. Yeast is mixed into dough containing a sugar source and respires anaerobically, converting glucose to ethanol and carbon dioxide. The carbon dioxide is released as bubbles that become trapped in the stretchy dough; as more gas is produced and the bubbles expand, the dough rises. Baking then kills the yeast, evaporates most of the ethanol and sets the structure so that the risen shape is permanent. The gas responsible for the rise is carbon dioxide - not ethanol, which is a liquid, and not oxygen, which is not a product of respiration.

• “Biotechnology means genetic modification.” Why wrong It collapses a whole field into one of its techniques, and makes it impossible to answer a question about bread or washing powder.
• “All industrial fermentation is anaerobic.” Why wrong Many fermenters have a sterile air inlet precisely because the organisms inside them respire aerobically. If oxygen were never wanted, the inlet would not exist.
• “Selective breeding inserts genes.” Why wrong Nothing is inserted. Parents are chosen, they reproduce, and their alleles recombine as they would in any cross. The change is in which combinations survive, over generations.
• “Enzymes are microorganisms.” Why wrong An enzyme is a protein molecule — thousands of times smaller than a bacterium, not alive, and incapable of reproducing. Microorganisms may be used to make the enzyme; the enzyme itself is the isolated product.
• “Microorganisms make these products for us.” Why wrong Yeast is not baking bread. It is respiring, because respiring releases the energy it needs to live, and the carbon dioxide is a waste product it has no use for. Human beings arrange conditions so that a by-product of the organism's own metabolism becomes useful. Writing as though the organism intends the outcome is treated as a real error, not a stylistic one.
• “The bacteria learn to make insulin.” Why wrong Bacteria do not learn anything. Their DNA has been changed: a gene they did not have has been inserted, and they read it as they read any other gene.
• “The modified bacteria are injected into the patient.” Why wrong The purified protein is what is used. The bacteria stay in the fermenter, and separating the insulin from them is step 7 for exactly this reason.
• “The whole human genome is put into the bacterium.” Why wrong One gene is transferred. The definition of genetic modification says individual genes, and a plasmid could not carry a genome in any case.
• “The insulin comes out of the fermenter ready to use.” Why wrong It is mixed with bacteria, culture medium and everything else in the vessel. It must be separated and purified, and its quality checked, before it is prepared for medical use.
• “All biotechnology is genetic modification.” Why wrong It collapses a whole field into one technique. Bread, brewing and every enzyme application in this chapter involve no gene transfer at all. Say instead Biotechnology is the use of living organisms, cells or enzymes to make useful products. Genetic modification is one technique used within it.
• “All fermentation is anaerobic.” Why wrong True of yeast fermentation, but an industrial fermenter is a vessel, not a process, and many processes run in one are aerobic — which is why the sterile air inlet exists. Say instead In yeast, fermentation is anaerobic respiration. In a fermenter, whether oxygen is supplied depends on the organism and the product.
• “Yeast produces oxygen, which makes bread rise.” Why wrong Oxygen is not a product of any kind of respiration. It is a reactant in aerobic respiration and absent from anaerobic respiration altogether. Say instead Yeast respires anaerobically, producing carbon dioxide and ethanol. The carbon dioxide is trapped as bubbles and makes the dough rise.
• “The ethanol makes the bread rise.” Why wrong Ethanol is a liquid. It cannot form the bubbles that expand in the dough. Say instead Carbon dioxide, a gas, raises the dough. Most of the ethanol evaporates during baking.
• “Baking kills the yeast enzymes.” Why wrong Enzymes are protein molecules and are not alive, so they cannot be killed. Say instead Baking kills the yeast and denatures its enzymes.
• “Fermenters should be kept as hot as possible.” Why wrong Above the optimum, enzymes denature and cells die. And a large fermenter is already overheating from the heat its own microorganisms release. Say instead The culture is held near a suitable temperature, and cold water in the cooling jacket removes the excess heat produced by respiration.
• “The fermenter is heated to make the reactions go faster.” Why wrong The control problem in a running fermenter is removing heat, not adding it. Say instead Respiration releases thermal energy, so the culture is cooled to keep it near the optimum for its enzymes.
• “Stirring gives the bacteria more oxygen.” Why wrong Stirring cannot create oxygen. The oxygen came from the sparger; the impeller only moves it about. Say instead Stirring distributes the oxygen, nutrients, microorganisms and heat evenly through the culture.
• “Sterilisation means adding antibiotics to the culture.” Why wrong Sterilisation happens before the culture starts and removes all microorganisms, including the one you are about to add. Say instead The vessel and medium are sterilised and the incoming air is filtered, and then the chosen microorganism is inoculated into the sterile vessel.
• “Enzymes are tiny microorganisms.” Why wrong An enzyme is a protein molecule, thousands of times smaller than a bacterium. It does not respire, grow or reproduce. Say instead An enzyme is a protein that catalyses a reaction. Microorganisms may be used to produce it, but the enzyme itself is an isolated molecule.
• “The enzymes in washing powder eat the stains.” Why wrong Eating is something an organism does. An enzyme catalyses a reaction on a substrate. Say instead Protease hydrolyses protein in the stain into amino acids, and lipase hydrolyses fats into fatty acids and glycerol. The small soluble products dissolve and wash away.
• “Pectinase digests the whole plant cell wall.” Why wrong It acts on pectin. The cellulose of the cell wall is a different substance and needs a different enzyme. Say instead Pectinase breaks down the pectin in the cell walls and between the cells, so the cells separate and their contents are released.
• “Lactose-free milk is suitable for anyone who reacts badly to milk.” Why wrong It addresses lactose only. A milk-protein allergy is a response to the protein, which is entirely unchanged by lactase. Say instead Lactase-treated milk is suitable for many people with lactose intolerance. It does not help with milk-protein allergy, because the protein is still present.
• “Lactase is the sugar in milk.” Why wrong One letter, two different kinds of molecule. The sentence has an enzyme being digested by a sugar. Say instead Lactose is the sugar; lactase is the enzyme that hydrolyses it into glucose and galactose.
• “The bacteria learn to make insulin.” Why wrong Bacteria do not learn. Their DNA has been changed. Say instead The human insulin gene has been inserted into the bacterial DNA, and the bacteria express it as they would any other gene.
• “The modified bacteria are injected into patients.” Why wrong The product is a purified protein. Separating the insulin from the bacteria is an explicit step in the process. Say instead The insulin is separated from the culture and purified before being prepared for medical use.
• “The whole human genome is inserted into the bacterium.” Why wrong The definition of genetic modification specifies individual genes, and a plasmid could not carry a genome anyway. Say instead One selected gene — the gene that controls insulin production — is inserted.
• “Selective breeding is a kind of genetic modification.” Why wrong No gene is moved in selective breeding. The population changes because of which organisms reproduced, over many generations. Say instead Selective breeding chooses parents with desired features and breeds them; genetic modification directly inserts, removes or changes a gene.
• “Herbicide-resistant crops kill the weeds around them.” Why wrong The crop does nothing to the weeds. The herbicide kills them; the modification only lets the crop survive the spray. Say instead The crop survives being sprayed with a particular herbicide, so that herbicide can be applied to the whole field to control the weeds.
• “Insect-resistant crops mean pests can never be a problem again.” Why wrong The pest population is still present and now under selection. Resistant individuals survive and reproduce, and resistance spreads. Say instead Crop losses fall, but the crop acts as a selection pressure, so resistant pests may become more common over generations.
• “The GM crop causes the pests to mutate.” Why wrong Variation existed before the crop was planted. Selection changes how common an allele is; it does not create it. Say instead A few pests already carried a resistance allele. The crop acted as a selection pressure, so those individuals survived and reproduced and the allele became more common.
• “GM genes spread to every plant and animal nearby.” Why wrong An allele moves the way any allele moves — by successful reproduction between compatible plants. Transfer between distant species is exactly what requires a laboratory. Say instead Pollen may reach a compatible wild relative; if cross-pollination and fertilisation occur, the offspring may carry the allele and the feature may spread in that population.
• “All GM is unsafe.” — and — “GM has no risks.” Why wrong Both are absolute claims about a whole technology on the evidence of one application, and each ignores everything on the other side of the balance sheet. Say instead Name the application, give the mechanism, give a benefit and a risk that follow from it, and conclude with what the judgement depends on.
• Yeast produces oxygen.
• Fermenters are heated as hot as possible.
• Enzymes are killed / enzymes eat stains.
• Herbicide-resistant crops kill weeds.
• All GM is unsafe / GM has no risks.

• Reading the command word. Notice that the last statement in the table says discuss, not state. Discuss means both sides, each with a mechanism, ending in a judgement that depends on something. A list of four advantages will not reach the top band however accurate it is.
• A timing note. The evaluation question is usually worth 5 or 6 marks and tempts students to write a page. It does not need one. Three clearly separated advantages with mechanisms, three risks with mechanisms, and two sentences of conditional conclusion will reach the top band and take about eight minutes. The extra page costs time you need elsewhere in the paper and earns nothing.
• Two words worth spelling carefully. Write fermenter, not fermentor, and write plasmid, not plastid — a plastid is a plant organelle and belongs in a different chapter entirely.
• The word that earns the mark. On the cold side write kinetic energy. On the hot side write denatured and active site. Those three phrases are what mark schemes look for, and an answer that gets the biology right but uses neither of them often scores below one that is less well written but uses both.
• How this is usually asked. Very often as a diagram with three or four lines drawn to unlabelled parts, and the instruction “name the part and state its function”. That is two marks per line, and the function mark is the one candidates miss. Practise saying the function out loud while pointing at the diagram until the pairing is automatic.
• Give the products by name. “Lactase breaks down lactose” is one mark at best. “Lactase hydrolyses lactose into glucose and galactose” is a complete answer, and naming both products is the detail that separates it from a vague one.
• Two-mark questions are usually one mechanism and one benefit. If you find yourself writing two benefits, you have almost certainly missed the mechanism mark. Go back and add what the enzyme does to the molecule.
• Mechanism before benefit, every time. All three modifications follow the same two-sentence shape: a gene is inserted that does X; because of X, the benefit is Y. Candidates who write only Y score about half.
• A sentence that gains a mark in an evaluation. “This risk is not unique to genetic modification: repeated use of a single insecticide selects for resistance in the same way.” It shows you are comparing the technology with the realistic alternative rather than with a perfect one, which is what evaluate asks for.
• Significant figures. Give your answer to about the same number of significant figures as the data you were given. If a table shows 46 cm³ and 10 minutes, answer 4.6 cm³/min, not 4.600000 cm³/min. Writing more figures than the data support is treated as a misunderstanding of the measurement, not as extra care.
• One habit that lifts every answer in this topic. Before writing, look at the mark allocation and decide how many separate statements you owe. Then write that many, each as its own clause. Topic 18 is unusually generous to candidates who do this, because almost every question in it is a sequence or a balance.
• Marking yourself honestly. Award a mark only where the marking point actually appears in your own words. In this topic the commonest way to over-mark yourself is to credit an answer that gives the benefit but never gave the mechanism — look for the because in your own sentence before you tick.

• Chapter 18 behaves differently from the physiology topics around it. There is very little to draw from memory, almost no calculation that is unique to the topic, and a great deal of writing that has to be structured rather than merely correct. Knowing the shape of the questions is worth as much here as knowing the content.
• Typically tests a single clean distinction: which gas raises dough, which enzyme acts on which substrate, which condition a fermenter cooling jacket controls, what genetic modification means. The distractors are almost always the misconceptions in this chapter's mistake clinic, written as though they were facts.
• Usually one longer part on the fermenter or on insulin production, and one shorter part on an enzyme application. Expect a labelled fermenter to complete, a sequence to put in order, or a table of data on yield to interpret before you explain something.
• The one that separates grades. A named GM application is given, and you are asked to discuss advantages and disadvantages. Marks come from mechanisms and from balance — not from the number of points listed.
• Three habits do most of the work in this topic, and they are all about structure rather than recall.
• Name the mechanism before the benefit. “Pectinase increases juice yield” is one statement. “Pectinase breaks down pectin in the plant cell walls, so more juice is released” is two, and the second one earns the first.