What is genetic engineering?

Summary: Genetic engineering is used in research to study diseases, microbiology or the development of cell factories, among other things. Cell factories are cells that produce large quantities of valuable products, such as medicines.

In this material you will learn how to design your own cell factory with genetic engineering, and afterwards you can try out the entire process from DNA design to finished product in the virtual lab. You’ll also learn how DNA and genes function as instructions in all biological cells and much more about the modern tools of genetic engineering! The material includes a number of subpages describing specific genetic engineering methods – an overview can be found just below. The theory material consists of 6 articles. Several of the articles can be read independently, but it is recommended to read the first three articles chronologically. The material also includes a number of case studies on the actual use of genetic engineering.

Have a great time!

Theory:

Tap the buttons below to access the theory section. The section on genetic engineering tools provides a theoretical overview of a number of genetic engineering methods.

Cases:

Genetic engineering in plants and animals

Cases: Genetic engineering in plants and animals

Three cases describe examples of how genetic engineering is actually used to genetically modify plants and animals.

General introduction to genetic engineering in plants and animals
It’s not just the DNA of microorganisms that can be altered with genetic engineering. Today, scientists can create both plants and animals that are transgenic, i.e. with foreign genes inserted, either to aid in a research project or to give them entirely new properties.

Since the nineties, the world has seen various new genetically modified (GM) organisms, such as the Flavr Savr tomato that didn’t just rot, “golden rice” loaded with provitamin A and goats that secrete complex drugs in their milk. The consequences for the environment and farmers of growing GM crops can be both bad and good, but they are certainly not transparent, which is why there are very strict approval controls on new transgenic plants to be released into the wild. However, there are differences in the countries’ approval requirements, which is why GM cotton, corn and soy in particular are now widely used outside the EU, while the EU has approved fewer plants for cultivation (read more in ‘Ethics and legislation in genetic engineering’).

Despite consumer skepticism about GM food, especially in Europe, new species are still being developed, including the Golden Rice project, which aims to make rice plants produce provitamin A (β-carotene) in the rice grains. Vitamin A deficiency is found in around 140 million people in third world countries, and rice is a cheap, widely available food in many of these places.

Genetic engineering is also used in pharming, which instead of inserting enhancing properties, is the use of the advanced cellular machinery of plants and animals to produce complex medicinal proteins – essentially a kind of highly advanced cell factory (as introduced in ‘Cell Factories’). After cultivation by these living “medicine factories”, the substances can be harvested from, for example, thistle flowers, tobacco leaves or a goat’s milk.

Case: Insect tolerant cotton

Insect tolerance is one of the relatively few traits that have been successfully imparted to genetically modified plants, but there are still a number of challenges.

One of the few GM plants that has really taken off is the insect-tolerant Bt cotton, which is now found in large areas in the US and several developing countries, including China and India. The Bt cotton plant has genes from the bacterium Bacillus thuringiensis (Bt) inserted so that the bacterium’s natural insecticide Bt toxin is produced in the plant to the detriment of attacking insects. The mechanism of action of insect tolerance is relatively simple, as the plant takes over the bacteria’s protection, which can be conveniently encoded by one gene. Since 1996, insect-tolerant cotton has been in the fields in various countries – and the same principle is also used in genetically modified corn, soy and potatoes, among others. Bt maize (MON810) is one of the few genetically modified plants approved in the EU, but it is banned in some member states and is not cultivated in Denmark.

Figure 1 – Cotton field ready for harvest. Photo: David Nance

Bt cotton has become so widespread that different varieties with Bt toxin cover 45 percent of the world’s cotton area in 2009, and since the fields yield 30-40 percent more than normal, well over half of the world’s cotton production is now derived from this genetically modified (GM) cotton. Several developing countries have embraced the plant, and there have recently been reports of a significant improvement in the living conditions of women in cotton farming in India, among others, due to the plant. The higher yield of Bt cotton. So, clothes bought in Denmark may contain Bt cotton, but the Bt toxin does not bother humans.

The insecticidal Bt toxin

Bt toxin is a small crystal protein harmful to the cells in the digestive tract of many insects, including some that attack cotton fields. When the gut is thus destroyed, the insect dies. With Bt cotton, spraying with harmful pesticides can therefore be reduced. The Bt toxin is not new as a poison, and is in fact also used as a pesticide on conventional crops, and the Bt bacterium is also allowed to be released for pest control on organic crops. The disadvantage of spraying is that it only affects the insects that are present on the plant during spraying. Larvae that attack cotton often stay inside the plant, and spraying up to 15 times per crop may be necessary.

Criticism: Do tolerant plants create resistant insects?

GM plants with Bt toxin are criticized from several sides. As with other poisons, the affected insects can develop resistance so that they are no longer killed by the poison, which has been feared by some from the beginning; no one knows the long-term environmental effects of an altered plant.

In 2010, it therefore attracted attention when one of the companies behind the plants, Monsanto, reported that the pink bollworm in several Indian provinces had developed resistance to the Bt toxin (from thecry1Ac gene) and can therefore attack the plants again.

Because GM fields are hardly sprayed, it is also feared that the changed environment in the fields means that new pests are gaining a foothold in these fields. These pests were previously kept down by insects.

One of the methods used to contain the development of resistance is to cultivate fields with different variants and combinations of the Bt toxin inserted into the genes. The advantage is that even if an insect develops resistance to one of the toxin variants, it will likely still be killed by the other Bt toxin. The risk of resistance is therefore significantly lower. In the Indian case, the fields with resistance were cultivated with only one variant. Another contributing factor may also have been a lack of planting of the so-called Bt-free refugia.

Bt-free refuge in the fields

The development of resistance is a natural phenomenon that is also known from hospitals, where superbugs become resistant to antibiotics. As a safeguard against resistance in fields, users of Bt plants commit to planting a so-called refugium of the same crop, but without Bt toxin, on 20-50 percent of the field area. Ordinary pesticides can be used here, and the surviving insects will not be resistant to Bt toxin.

The lack of Bt resistance in the refuge reduces the development of resistance in the closely growing Bt varieties, because the insects from the refuge will move in and mate with any Bt-resistant insects that have emerged. Here, they will have an offspring that has both an allele of the gene that leads to Bt resistance and an allele that does not; the insect is heterozygous (see figure 2). The heterozygous insects are not resistant to Bt because the resistance gene is recessive, and they are therefore likely to die from Bt toxin afterwards.

However, two heterozygous insects together can produce homozygous offspring that are Bt-resistant, and the use of refugia is certainly not a total protection against the development of resistance.

Second generation GM plants are now being developed to become tolerant to physical stresses such as cold, salt water and drought. The first drought-tolerant cotton plant is expected to reach the fields around 2012. Meanwhile, the question is raised whether the first generation of Bt plants may soon become obsolete because the insects may develop more resistance to the Bt toxin.

These reports suggest, according to some, that genetic engineering is hardly a miracle solution, but perhaps an aid, and that developers will have to continue the fight and perhaps perform new genetic engineering to maintain efficacy. If resistance becomes more widespread, cotton farmers may have to resort to using more old pest control products.

Working questions

How do insect tolerant cotton plants work?
What benefits do farmers get from using insect-tolerant cotton plants? Are there environmental benefits too?
What criticisms have been raised against Bt cotton?
How does the Bt cotton plant seem to be doing today?
Explain what things farmers can do to avoid developing resistant insects and how it works.
How does a Bt-free retreat work? Explain figure 2.
Discuss your own views on the use of genetic engineering in plants and relate it to the dilemmas you think governments have in developing countries like China and India with poor farmers and lack of resources.

Figure 2
– Overview of how crossing a Bt-resistant insect (red) with a non-resistant one from a refuge without Bt plants will lead to heterozygous, non-resistant offspring that can die from the Bt toxin. Since the resistance gene is recessive, two r alleles are required to create a resistant insect.

Case: Golden Rice 2.0

Golden rice is a genetically modified plant created to combat vitamin A deficiency, but 20 years after the project started, the plant is still a work in progress.

Many of the GM crops developed mostly make life easier for farmers, but the Golden Rice project has other, more humanitarian ambitions. The project was started by ETH researcher Ingo Potrykus

Zurich, Switzerland in 1992, and after seven years of work, his team presented a new, genetically modified rice variety designed to tackle the world’s vitamin A deficiency problem. An estimated 140 million people suffer from the deficiency, leading to blindness and 6,000 deaths daily. Despite the great attention, the project has faced a number of challenges, including obtaining the necessary approvals, and today Potrykus expects the rice to be ready for cultivation in 2012 at the earliest – twenty years after the start of the project.

Figure 1
– Plain and golden rice. Photo courtesy of Golden Rice Humanitarian Board. www.goldenrice.org

The golden rice is infused with two foreign genes that cause the white part of the rice grains (the seed white) to produce the orange-red β-carotene that is most commonly found in carrots. β-carotene is a provitamin that the body can convert into vitamin A. It was also the characteristic golden color of the rice that gave it the name golden rice.

Plant development

Rice plants do produce β-carotene naturally, but unfortunately not in the part of the rice that is eaten. So the genetic engineering task the researchers set themselves was to create the chemical production pathway in the rice seed white.

The rice naturally forms the substance geranylgeranyl-PP, which in a series of four steps can end up as β-carotene (figure 2) if the right enzymes are present. To create the enzymes, the researchers first inserted the gene psy , which they found in daffodils. It encodes the necessary enzyme phytoene synthase, which controls the first step in the reaction pathway. But the researchers also linked another small sequence to the gene, which cleverly directs the enzyme to the plastids.

Figure 2
– The novel reaction pathway in the genetically engineered rice plant to convert geranylgeranyl-PP to β-carotene. Each arrow is a reaction (a step) that is controlled by a necessary enzyme (in italics). The enzymes come from the psy, crt1 and lcy genes (mentioned in brackets). These are the three genes that need to function in the rice seed white.

For the next step in the transformation, the researchers found The crt1 gene from a soil bacterium(Erwinia uredovora). The gene encodes the next necessary enzyme phytoene desaturase, which catalyzes both the second and third steps in the conversion to β-carotene (figure 2). The final step from lycopene to β-carotene already had enzymes in the rice grain. The golden color of the genetically engineered rice indicated that the experiment was successful, and subsequent measurements proved them right: β-carotene was produced in the seed white.

Potrykus became world famous when his team published their results in 2000, but a number of problems emerged. Although the rice turned a beautiful golden brown, the β-carotene content was so low that you would have to consume unlikely amounts of rice to reach the recommended daily intake of vitamin A.

To solve the poverty problem that vitamin A deficiency primarily is, an additional problem was that Potrykus’ team had used a number of genetic engineering methods that biotech companies had the rights to because they had invented them. They would therefore need royalty money in license, which could make the rice too expensive. In addition, two companies had put money into the project, so they should also have a stake in it. The two companies created the company Syngenta, which was granted the patent rights to the golden rice. In return, they must, among other things, make the rice plants available license-free (i.e. equivalent to regular rice) for use by farming families earning less than USD 10,000 annually, so that Potrykus’ original dream of helping poor people can still be fulfilled.

Genetic optimization for version 2.0
However, there was still the problem of the rice’s low β-carotene content, so the researchers had to optimize the inserted genes. The Syngenta research team came up with the idea that the level could be increased by removing a so-called bottleneck at the first reaction step (thepsy gene) in the reaction pathway (see figure 2). It seemed that this first stage of transformation was much slower than the next steps.

The bottleneck was discovered because the researchers could measure that there was still plenty of the starter geranylgeranyl-PP present in the golden rice, but very little of the next substances, which were converted as quickly as possible by the next enzyme in line. Their solution was to find a faster variant of the slow first enzyme, because the formation of β-carotene will not be faster than the slowest step in the reaction pathway. The researchers therefore tested various other organisms’ versions of the psy gene instead of the original one from the daffodil. And when they tested the psy gene from corn, the result was a staggering 23-fold increase in initial yield; they achieved 31 micrograms of β-carotene per gram of rice. This boost in production was important because half of a 3-year-old child’s recommended daily intake of 300 micrograms of vitamin A could be obtained from 72g of dry rice. The body converts β-carotene to vitamin A in a ratio of approximately 12:1.

Environmental organizations such as Greenpeace have had a negative attitude towards GM golden rice from the beginning. Among other things, they criticize that β-carotene is to some extent lost during cooking, so rice is not a good source. Furthermore, Greenpeace notes that the long-term effects are not known, but they are not in favor of studying them either. In relation to the fact that the rice hasn’t saved anyone yet, they believe that the project has received far too much media hype.

Ingo Potrykus himself is still following the project and is today dissatisfied with the long time he feels he has “lost” in the project to obtain extensive test results and approvals from authorities. He now (2010) guesses that the rice could reach farmers in 2012, again depending on the authorities.

The golden rice may be the example that a more humanitarian GMO can be created, but it also shows that the authorities are firm in their requirements for approval.

Working questions

Some questions are based on theory from the rest of the material.

How can β-carotene benefit developing countries?
How is it possible to get a rice plant to produce β-carotene in the seed white?
What is the role of the psy, crt1 and lcy genes in golden rice? Are some of the genes also found in regular rice?
What criticisms have been made of golden rice?
Give an example of a genetic engineering tool that scientists could use to test that the new genes were actually inserted into the rice plant?
Explain why there was a need to create the new variant Golden Rice 2.0.
How did scientists come up with the idea of inserting a new psy-gene into golden rice 2.0?
Based on the information in the case, calculate how much vitamin A you would get in your body by eating 72 grams of golden rice 2.0.
Suggest at least two other optimization methods from 3 Genetic Tuning that could be used and explain your idea.
The local growing conditions for rice in Asia are highly variable, and different varieties of rice are popular depending on where you are. How could this be taken into account if you want to offer farmers ‘golden rice’ adapted to local conditions?
Is β-carotene a protein or a metabolite? Why?

Figure 3 – Rice paddy

Figure 4
– The yield of β-carotene in the original golden rice compared to the improved (2.0), where the researchers replaced the slow enzyme gene psy with one from corn.

Case: The goat as a modern pharmaceutical factory

Transgenic goats are engineered to produce human antithrombin and excrete it into milk.

Medical proteins, which a patient may no longer produce sufficiently on their own, can be complex. So complex that a simple E. coli bacterium or yeast cannot produce the substance. A more advanced organism that is more similar to humans could therefore be interesting to use. One of these is the goat, which scientists have successfully genetically engineered to secrete a substance in its milk to prevent blood clotting.

The valuable substance is called antithrombin. This suddenly made milking an important step in drug production, and the product is now approved by both the EU and the US Food and Drug Administration for treatment. Deficiency of the blood protein antithrombin (AT) is congenital in 1 in 5,000 people. They have previously obtained AT from human donor blood when needed, but extracting 1 kg alone requires 20,000 units of blood, making the treatment quite costly. Instead, on a specially designed farm in Massachusetts, USA, a company has a barn with some genetically modified goats, each capable of producing 1 kg of human antithrombin (rhAT) per year.

Figure 1
– Blood clots can be formed by thrombin, which converts the blood protein fibrinogen into the insoluble fibrin. Here you can see red blood cells in a vein.

The goats are milked and the protein is purified from the milk, which can be dissolved as a dry powder and given to needy patients in hospitals, reducing the risk of blood clots. Since donor blood can in principle carry diseases such as HIV and Creutzfeld-Jacobs from an undetected sick donor, large screening programs are needed to ensure that blood-derived drugs do not transmit such diseases – this need is not met by recombinant production.

Antithrombin in the blood: Prevents clotting

AT works in the blood by inhibiting the enzyme thrombin, which causes blood to clot. Thrombin converts the blood protein fibrinogen into the insoluble fibrin, allowing clotting to take place (see figure 2). Thrombin is therefore activated in wounds to prevent blood loss, but antithrombin is also important to set the balance that ensures blood clots only in the right place and doesn’t become too thick.

Patients who are deficient in antithrombin are more prone to blood clots, so during surgeries and births, patients can be given AT to restore their balance. AT is a larger protein (58 kDa) that is glycosylated (attached to sugar molecules). Glycosylation controls, among other things, the half-life of the substance in the blood, and therefore glycosylation must be as similar as possible to human glycosylation for recombinant AT to be effective.

Expression design

Of course, the researchers who developed the transgenic goat weren’t interested in just getting a goat that produced a lot of human antithrombin (rhAT) in all its cells. So, to localize production to the mammary glands, they inserted the gene for rhAT after a specific promoter for the milk protein β-casein. The promoter activates the transcription of its gene in the cells of the mammary glands, where β-casein is also produced. This way, the rhAT formed could be secreted into the goat’s milk just like β-casein (read more about promoters in Genetic Tuning)

Figure 2
– Antithrombin’s slowing effect on blood clotting. Antithrombin inhibits the enzyme thrombin, which causes blood to clot by converting fibrinogen into fibrin.

In the lab, the DNA was injected into goat egg cells, which were inserted into a female goat that acted as a surrogate mother. After the births, a hanged was found with the help of Southern blotting that carried the new gene. The male goat will not produce rhAT even though it carries the gene because it is activated in the mammary glands, so you could only look at the DNA itself.

After mating with a regular goat, female goats were born that produced rhAT in their milk. The concentration reached around 2 g/L milk in the best candidates that were taken forward. However, development was not without problems, and transgenic goats were born that were sterile or stopped producing milk prematurely.

The recombinant antithrombin from goats was also found to be slightly differently glycosylated than human antithrombin, but studies showed that it was still effective.

Use of recombinant antithrombing

AT is a protein, and any notion of providing AT-deficient patients with freshly made rhAT goat’s milk wouldn’t work, no matter how practically appealing it sounds. The protein, like other proteins, will simply be broken down in the stomach and not have its effect. AT must be administered intravenously (into the bloodstream) and therefore must first be purified from the milk. In addition, pharmaceutical production always places very high demands on quality, purity and the production itself. The company behind rhAT must also meet these requirements.

rhAT is now sold under the name ATryn and is the first approved drug produced in transgenic animals. ATryn will only be used in hospitals where surgeries pose a particular danger to patients lacking AT.

The consumption of antithrombin in the US and EU combined is relatively low for pharmaceuticals at around DKK 250 million/year. Drug development is also extremely costly, but the regulatory approval of ATryn has been seen as a green light for the safety of these drugs produced in transgenic animals, and development can continue towards drugs for more widespread diseases.

In addition to milk, researchers are now working on expressing human proteins for medicine in chicken eggs and thistle flowers. Perhaps the advanced drug factories of the future will be biological in a way that would have been unimaginable twenty years ago!

Discussion and working questions

Explain the importance of thrombin for blood to clot.
How does antithrombin affect blood clotting?
How could scientists get a goat to produce human antithrombin?
Why did the researchers use a promoter to activate the antithrombin gene in the mammary gland cells? (Consider what might happen if large amounts of antithrombin were produced in all the goat’s cells?)
What are the advantages/disadvantages of producing antithrombin recombinant in a goat vs. extraction from human donor blood?

Figure 3
– Farm goats, non-GMO. Photo: Fir0002/Flagstaffotos(GFDL 1.2).

Development of fat-degrading washing enzyme

Case: Developing a fat-degrading washing enzyme

Today, enzymes have emerged as biology’s solution to several challenges in the modern world. As is the case in cells, enzymes in everyday life and industry can make reactions possible that would otherwise not occur to any significant degree.

But how are these smart enzymes found and developed? Genetic engineering is part of the answer.

Why enzymes?

One branch of biotechnology is dedicated to discovering and developing enzymes that help improve baking and other foods, produce biofuels and process textiles. Enzymes can open up new opportunities or they can replace chemicals that, for example, do not have the biological advantage of being made up of amino acid chains that are easily degradable in nature.
Another big area is detergents, where enzymes catalyze the breakdown of dirt and have made it possible to wash clothes clean at just 20°C, as well as replacing some more environmentally harmful chemicals.

Figure 1. The search for new enzymes takes researchers far and wide. Photo: Novozymes.

Novozymes is the world’s leading developer and manufacturer of enzymes for detergents, among other things, and is headquartered in Bagsværd, north of Copenhagen. Novozymes’ washing enzymes help break down debris such as proteins, starches and fats, but they can take a long time to develop.
Enzymes developed by a company like Novozymes are first found in nature. It could be in microorganisms that live in harsh environments, similar to the rather alkaline conditions in a washing machine. But beyond the initial challenge of discovering an attractive enzyme, there’s also the genetic engineering task of creating a good cell factory to produce it in large quantities and possibly try to optimize the effect of the enzyme.

Enzyme action in a nutshell

An enzyme is a protein that has an active site region that can catalyze a chemical or biological reaction. Enzymes are often very specific in which reaction they catalyze because the reactants must fit spaciously into their three-dimensional structure in order to be converted into the product. Protein, starch and fat debris is broken down by proteases, cellulases and lipases, which are all hydrolytic enzymes. They break down the long chains of the substances into smaller molecules by adding_H2Oto their bonds, thereby splitting them into smaller parts. The smaller molecules can therefore be rinsed off the clothes much more easily (see reaction equation, figure 2).

Figure 2. The reaction equation for the enzyme lipolase’s cleavage of triglycerides (fat) into smaller molecules that can be flushed away more easily.

The structure and chemical groups of the enzyme interact with the reactants during the reaction and support the process. The reaction therefore requires less activation energy and often proceeds significantly faster. The enzyme is part of the reaction, but is not consumed and is therefore ready to catalyze the same reaction again afterwards. Remember that enzymes and other catalysts never affect the actual location of the equilibrium of the reaction – only how quickly it sets (the reaction rate).

In the fight against lipstick stains:

Development of a fat-degrading washing enzyme

One of the effective, fat-degrading lipase enzymes has been developed by Novozymes in Japan and Denmark for detergents that can remove stains from butter, oil or lipstick, for example. The enzyme is a biotechnological success story about a development process where, as is often the case, researchers suddenly had to think creatively under time pressure.
Fat-degrading lipases were already available in the 1970s for detergents, but they weren’t as effective back then.

To find better lipases, Steen Riisgaard, the current CEO of Novozymes, started a research lab with a small handful of people in Tokyo in 1982 for Novo Nordisk (before the enzyme part was later spun off into Novozymes). The lab’s first task was to analyze a bunch of microorganisms to see if they produced interesting lipases, a common strategy for finding new enzymes.
To get more focus on lipases, a lipase group of 15 people was established in 1984, and in the years 1985-86 they focused mostly on bacterial lipases, especially from the bacterial species Pseudomonas. However, they also found several lipases from fungi, but the question was how well they worked.

Working in Japan

Perhaps because Steen Risskov’s lab was located in Tokyo, the team started a collaboration with Japanese detergent manufacturer Lion, which was under pressure from competition in the market. Lion tested a number of Novo Nordisk’s lipases with their detergent to find the best candidates, and in the summer of 1987 they returned with the announcement that they had found a very good lipase from the thermophilic fungus Humicola lanuginosa.
The lipase was to be included in Lion’s new product, which they would introduce in April 1988. This was great news for the developers, but suddenly they also had to move fast if they were to deliver enough of the lipase to the large Japanese market.
The H. lanuginosa strain didn’t produce much lipase naturally, so the team set about optimizing the fermentations of the fungus in the classic way. This involved creating various random mutations that hopefully led to increased production, and it was not an easy task.

At this point in the late eighties, genetic engineering was new but rapidly developing, and the year before (1986) other researchers at Novo Nordisk had succeeded in producing recombinant human insulin using yeast as a cell factory.
That’s why some believed that genetic engineering should also be used for this challenge. A small group therefore worked on inserting the gene encoding the lipase into an expression vector. The idea was to put the expression vector into Aspergillus oryzae, which is a good fungus for production. Initially, the genetic engineering project did not go well, but in November 1987, with a good promoter in front of the H. lanuginosa lipase gene, high lipase yields were achieved in A. oryzae.

The first production

At that time, Denmark had passed the world’s first law on genetic engineering. Therefore, it was difficult to get a quick production approval for a mushroom that had been genetically modified. Luckily, Novo Nordisk had just built a factory in Japan, so the fermentations were done there. The problem was that enzymes come in a powdered granular form, which the Japanese factory couldn’t make. The germ-filtered (without live material) fermentation liquid had to be sent to Denmark to be granulated, after which it was sent back to Japan and Lion. In 1988, Lion came to market as planned with Hi-Top detergent, which contained the world’s first recombinantly produced washing enzyme – Lipolase.

Optimizing the enzyme(Protein engineering)

Lipolase was soon selling well around the world, and in the early 90s Novo Nordisk wanted to improve the lipase so that it worked even better during washing. One of the problems was that some of the grease was broken down after the wash itself, so the stains didn’t disappear until the second wash!

To improve the enzyme, the amino acid sequence itself had to be changed, and to do that, the gene had to be altered. Since bacteria are easy to work with, the plan was to use them to produce a number of different variants of the lipase with one or more amino acids altered. Unfortunately, lipase could not be produced in reasonable quantities in bacteria, but it turned out that lipase could be produced by the yeast Saccharomyces cerevisae. This yeast is also easy to genetically engineer, which made it possible to create the many different variants of the lipase and see if the variants improved in washing powder.

Surprisingly, the researchers discovered that lipase produced from yeast itself worked better in washing powder than the same lipase produced in A. oryzae. However, the yeast could not produce enough of the lipase to make it commercially viable. The researchers had to find out what was causing this difference between lipase produced in A. oryzae and in yeast. It turned out that more positively charged amino acids were added to the end (N-) of the lipase produced in yeast, which resulted in the increased activity of the lipase in the washing powder. Many attempts were made to create an equally good lipase that could be produced in large quantities by A. oryzae. Fortunately, an X-ray structure of the lipase had been created, which made it possible to see the lipase in 3D.
Therefore, it was possible to say exactly where the extra positive amino acids were located in the three-dimensional structure of lipase. A number of lipase variants were made by changing the amino acids in the same region of the lipase to positively charged amino acids. One of these variants turned out to be really good. It could even be produced in large quantities by A. oryzae and has since become Novozymes’ washing enzyme Lipex.

Work questions:

It’s a good idea to look up ‘Genetic Engineering Tools’ along the way.

What functions are industrial enzymes able to help with? And how are they typically found?
What does it mean that an enzyme is hydrolytic in its function?
In this case study, we hear how researchers enlisted the help of three different organisms to develop the fat-degrading enzymes. Explain which and what each organism was used for in brief.
Suggest a genetic engineering method that researchers could use to assemble the DNA to genetically engineer the mushrooms?
When different pieces of DNA are put together, pieces of the wrong length can be formed. Suggest how researchers could clean up the real thing.
Suggest how the researchers could tell if the genetically engineered fungus had actually taken up and contained the new inserted DNA. Briefly explain how

Development of fast-acting insulin

Case: Development of fast-acting insulin

Several biological medicines are produced in cell factories using genetic engineering, including certain hormones, vaccines, antibodies and the bactericidal drug penicillin, whose natural production in a mold has been significantly optimized with genetic engineering. Another example is insulin for diabetes.

For many years, the pharmaceutical company Novo Nordisk has used genetic engineering to develop and manufacture medicines used in the treatment of diabetes, among other diseases. This case begins with an overview of diabetes and how the disease is treated with insulin produced in genetically engineered yeast cells. It then describes how the researchers used genetic engineering and creative thinking to develop a new type of insulin and the challenges they faced along the way.

Insulin for the treatment of diabetes

Diabetes is a common disease in which the body’s own production of insulin is reduced or completely stopped. Insulin binds to the body’s cells, allowing them to take up glucose, which is the cells’ primary source of energy. A lack of insulin means that the glucose circulating in the blood cannot be absorbed by the cells and therefore the glucose concentration increases. A prolonged elevated blood sugar concentration can cause late complications, which will be described below in the section on the consequences of diabetes. Worldwide, 5% of adults suffer from diabetes. The number of people with diabetes is increasing dramatically globally.

Fortunately, diabetes has long been treated with insulin injections. Treatment with human insulin makes the disease much less troublesome and dangerous because patients can continuously fuel the body’s cells and lower the concentration of glucose in the blood to the right level, reducing the risk of developing late complications. However, insulin treatment can’t exactly mimic the natural, continuous insulin release of a healthy person, partly because the effect doesn’t kick in until some time after the injection. Using genetic engineering, Novo Nordisk researchers have therefore developed both fast-acting and slow-acting insulin (insulin variants) that can be combined to provide near-normal blood sugar levels throughout the day, thereby reducing symptoms and preventing late complications of diabetes.

The consequences of diabetes (late complications)

Diabetes patients who live with even slightly elevated blood sugar levels for many years are at risk of blindness, poor wound healing and blood clots. At the same time, too low blood sugar due to too high doses of insulin can lead to fainting and, in the worst case scenario, death. Diabetes is therefore a disease that requires careful treatment.

Both type 1 and type 2 diabetes can be treated with insulin injections. In type 1, hardly any insulin is produced in the body, while in type 2, the body’s cells no longer respond adequately to insulin. Lifestyle changes such as dietary changes and increased exercise can help treat type 2 diabetes.

Insulin on a molecular level

Insulin is a protein that acts as a hormone in the body. It is released into the blood when blood sugar levels are high, which is especially the case after meals as food is broken down into glucose, among other things.

Each insulin protein consists of two amino acid chains held together by disulfide bridges. Insulin is produced in the pancreas by the same mechanism as all other proteins: transcription of the insulin gene from DNA to mRNA and subsequent translation of the mRNA into the protein.

Cell factories produce human insulin in large quantities

Insulin is one of the first proteins produced by a cell factory by inserting the insulin gene. This breakthrough meant that since the 1980s, both yeast(S. cerevisiae) and the bacterium E. coli have been used as cell factories for the production of human insulin to treat diabetes. Because the human insulin gene has been inserted into the cell factory, the insulin is identical to the insulin produced in the human pancreas. Until the mid-1980s, insulin for medical use was extracted from the pancreases of cows and pigs, but animal insulin is not completely identical to human insulin: some amino acids have been replaced.

In addition to reducing the risk of allergic reactions by using human insulin instead of animal insulin, patients now also avoid the theoretical risk of disease transmission from the animals. If the world’s current demand for insulin drugs were to be met by pig pancreases, 500 million pigs would be needed. So cell factory production also solves a major practical challenge.

In large fermentation plants in e.g. In Kalundborg, Denmark, yeast cells are cultivated to produce insulin drugs for large parts of the world, as Novo Nordisk is one of the leading providers of treatment for diabetes today.

Researchers’ challenge: Fast-acting insulin

When a diabetic eats, it’s important to keep blood sugar levels low enough after the meal, for example by injecting insulin. However, human insulin injected into the subcutaneous tissue is slowly absorbed into the bloodstream, which is why a meal must be planned well in advance to avoid dangerous situations. A development team at Novo Nordisk wanted to address this challenge in patients’ everyday lives by designing a new, fast-acting insulin variant.

Improving the speed of insulin action

Insulin must be injected into the subcutaneous tissue and is slowly absorbed into the bloodstream because the individual insulin molecules bind to each other in pairs in so-called dimers (figure 1). At the high concentrations found in the injection fluid, three dimers even tend to cluster around a zinc atom to form a hexamer. These hexamers are too large to pass through the blood vessel membrane, so only when the hexamers are slowly broken down into individual molecules over time can they be absorbed into the bloodstream and regulate blood sugar levels.

The development team at Novo Nordisk therefore worked on the idea of making it harder for the dimers to assemble into hexamers in the first place, so they could be absorbed into the bloodstream faster.

Studies of the chemical attraction between the individual insulin molecules showed that there are some particularly attractive amino acids in the two chains of insulin (B9, B12, B13, B16 and B23-28, see figure 2).

Figure 1. Insulin (red and green) collects in pairs in dimers. Three dimers assemble into a hexamer that is too large to be absorbed into the blood vessel membrane after an injection under the skin.

Figure 2. Human insulin consists of an A and B chain with 21 and 30 amino acids respectively. Disulfide bridges between cysteines hold the chains together. The important amino acids for insulin’s binding with other insulin molecules are highlighted.

The developers therefore bet that absorption would be accelerated if some of the chemically attractive amino acids were replaced with amino acids that could repel each other. Conversely, the effect of insulin itself should obviously not be destroyed by it; there is no benefit from a rapidly absorbed protein that no longer has any effect on the body. As insulin works by binding to the surface membrane of cells, the developers looked at insulin’s contact with these so-called cell receptors in cell membranes, because this contact needed to be maintained. It was previously discovered that insulin actually binds to the receptor at amino acids B12, B16 and B23-25, so naturally, these should not be changed.

The first rapid-acting insulin candidate produced by the development team back in the early 1990s had replaced the amino acid B10 to prevent the formation of the insulin hexamer, and it seemed to be a very effective change.

Figure 3. The two amino acid chains in insulin aspart, where insulin’s natural amino acid at B28 proline (Pro) is replaced by aspartic acid (Asp).

Expectantly, X10 was produced for chemical tests on cell cultures, which yielded positive results. However, as often happens with potential drugs in development, the project had to be discontinued when tests in rats showed poor results. The study showed an increased risk of mammary gland cancer in the rats that received X10 compared to those that did not. rats that, in comparison, were only injected with saline.

The road from idea to finished, approved drug is long and very expensive. In the very last phase of development of all new medicines, the substances must be tested in humans through a series of clinical trials to gain knowledge about the function and safety of the medicine. During this multi-year period, medicines are detected and stopped if they are found to have unwanted side effects.

The idea now needed to be put into practice, so yeast cells were genetically engineered with an expression vector containing the gene for the new variant called insulin aspart. The only difference in the gene was the codon for B28, which now encoded aspartic acid. Once the yeast cells’ newly produced insulin aspart was purified, the researchers were excited to examine the results.

Testing insulin aspart

Of course, insulin aspart also had to go through a lengthy testing procedure to ensure knowledge of its efficacy and safety. Much to the delight of the developers, after many trials, the drug proved safe to use and the absorption of insulin aspart into the bloodstream was twice as fast as human insulin. Insulin aspart was still able to bind to the receptor on the cells, so it had not lost its effectiveness with the new speed of action.

Insulin aspart’s rapid absorption after injection is more similar to healthy people’s secretion of insulin into the bloodstream with meals Therefore, insulin aspart was an interesting improvement in diabetes management for many patients.

In 1999, insulin aspart was approved for sale in the EU under the name NovoRapid and the following year in the US under the name NovoLog.

The fast-acting insulin, NovoRapid, is an example of how genetic engineering can be used not only to produce drugs identical to nature’s, but also more advanced variants where parts of the gene have been altered. As mentioned, genetic engineering has also been harnessed to develop slow-acting insulin, which helps maintain insulin levels over a longer period of time.

Today, Novo Nordisk’s researchers continue to work on improving the treatment of diabetes using genetic engineering. One of the big bets is stem cells (the Biotech Academy has another project on this), which may be able to cure diabetes, but like all medical research, the development has many challenges. The improved insulin variants such as insulin aspart are therefore also expected to be a great help in diabetes treatment for many years to come.

The virtual lab:

This teaching material is accompanied by laboratory exercises in the Virtual Laboratory.
Here you can try your hand at experimental genetic engineering in a lab set up just like a professional biotech lab!

The exercises associated with “Modern Genetic Engineering” are:

Production of insulin
Enzymes for detergent
Production of antibodies

Supplementary material:

Reference list
Below are the most important books and other publications on which this material is based and which can provide further inspiration.

Essential references for the theory material and inspiration for further reading

Please note that most materials are in English and are generally not freely available on the internet.

1. What is DNA and genes?

Basic textbooks in molecular biology:

Life: The Science of Biology. 2006. Sadava, D; Heller, HC; Orians, GH; Purves, WK; Hillis, DM. 8th edition. W. H. Freeman

Molecular Cell Biology. 2007. Lodish, H; Berk, A; Kaiser, CA; Krieger, M; Scott, MP; Bretscher, A; Ploegh, H; Matsudaira, P. 6th edition. W. H. Freeman

Molecular Genetics of Bacteria. 2007. Snyder, L; Champness, W. 3rd edition. ASM Press

2. Cell factories – The biotech route from DNA to protein

Basic textbooks as before, as well as specific ones:

Principles of Fermentation Technology. 1999. Stanbury, PF; Hall, S; Whitaker, 2nd edition. A. Butterworth-Heinemann. Explanation of fermentation and purification.

Wurm, FM. 2004. Production of recombinant protein therapeutics in cultivated mammalian cells. Nature biotechnology, vol:22,iss:11.pp 1393-1398.

Nielsen, J. 2001. Metabolic engineering. Applied microbiology and biotechnology. vol:55, iss:3,pp:263-283. Introduction to the enhancement possibilities of cells with metabolic engineering.

Life: The Science of Biology. 2006. Sadava, D; Heller, HC; Orians, GH; Purves, WK; Hillis, DM. 8th edition. W. H. Freeman. Chapter 14?

You take a gene…, 1994. Novo Nordisk.

Beekwilder, et al. 2006. Production of resveratrol in recombinant microorganisms. Applied and environmental microbiology, vol:72,iss:8,pp:5670-5672.

Cleetham, JC et al. 1998. NMR structure of human erythropoietin and a comparison with its receptor bound conformation. Nature structural biology. Vol:5,iss:10,pp:861-866.

3. Genetic tuning of cell factories

Hannig, G; Makrides, S. 1998. Strategies for optimizing heterologous protein expression in Escherichia coli. Trends in biotechnology. vol:16,iss:2,pp:54-60.

Sørensen, HP; Mortensen, KK. 2005. Advanced genetic strategies for recombinant protein expression in Escherichia coli. Journal of biotechnology. vol:115,iss:2,pp:113-128.

Hengen, PN. 1995. Purification of His-Tag fusion proteins from Escherichia coli. Trends in biochemical sciences. vol:20,iss:7,pp:285-286.

4. Genetic engineering tools

Basic textbook for genetic engineering tools:

Molecular Biotechnology – Principles and Applications of Recombinant DNA. 2002. Glick, BR; Pasternak, JJ. 3rd edition. ASM Press

Molecular Biotechnology – Principles and Applications of Recombinant DNA. 2009. Glick, BR; Pasternak, JJ; Patten, CL. 4th edition. ASM Press

DNA sequencing:

Metzker, ML. 2010. Sequencing technologies – the next generation. Nature reviews genetics, vol:11,iss:1,pp:31-46.

Synthetic biology:

Panke, Sven. 2008. Synthetic Biology – Engineering in Biotechnology, ETH Zurich. Highly recommended.

Alper, J. 2009. Biotech in the basement. Nature Biotechnology. vol:27,iss:12,pp:1077-1078

Focus issue, Dec 2009. Synthetic Biology. Nature Biotechnology, vol:27, iss:12, Especially pp:1091-1111. Link.

What Is Life? Investigating the Nature of Life in the Age of Synthetic Biology. 2008. Regis, Ed. 1st edition. Farrar, Straus and Giroux.

Ethics and legislation in genetic engineering

Phillips, T. 2008. Genetically modified organisms (GMOs): Transgenic crops and recombinant DNA technology. Nature Education vol:1,iss:1

Ministry of Higher Education and Science. 2003. Genetically modified and cloned animals. Chapter 3. Version 1.0 Link.

Ministry of Food, Agriculture and Fisheries. 02-10-2014. Website about Genetically Modified Organisms (GMO). Link.

Cases:

Insect tolerant cotton

Subramanian, A et al. 2010. GM crops and gender issues. Nature biotechnology, vol:28,iss:5,pp:404-406.

Damgaard, C et al. 2005. Genetically modified plants. Danish Environmental Research Institute and the publisher Hovedland. 2nd edition. Link

Lu, Y et al. 2010. Mirid Bug Outbreaks in Multiple Crops Correlated with Wide-Scale Adoption of Bt Cotton in China. Science, vol:328,iss:5982,pp:1151-1154

Sharma, DC. 2010. Bt cotton has failed admits Monsanto. India Today. Link

Golden rice 2.0

Paine, JA et al. 2005. Improving the nutritional value of Golden Rice through increased pro-vitamin A content. Nature biotechnology, vol:23,iss:4,pp:482-487.

Potrykus, I. 2010. Regulation must be revolutionized. Nature, vol:466,iss:7306,pp:561.

Meyer, G. 2001. Golden rice has a long journey ahead of it. News from the Center for Bioethics and Risk Assessment, KVL. 2nd year, 3. no. Link.

The goat as a modern medicine factory

Edmunds, T et al. 1998. Transgenically Produced Human Antithrombin: Structural and Functional Comparison to Human Plasma-Derived Antithrombin. Blood, vol:91,iss:12,pp:4561-4571.

Nielsen, RH. 2006. The way is paved for new, cheap medicines produced from animals. The Engineer, Link

Choi, C. 2009. ATryn, on Old MacDonald’s Pharm. Scientific American. Link
Glossary of terms

List of important terms and explanation of what they mean.

Activation energy
Activation energy is a chemical term that describes how fast a reaction can proceed in relation to temperature and other factors. The less activation energy, the faster the reaction. Enzymes can often lower the activation energy of a reaction.

Allele
An allele of a gene is a specific version of the gene. Many organisms have two alleles of each gene.

Amino acid
An amino acid is a specific chemical group of substances. The 20 biological amino acids are combined in different combinations in the long chains that make up a protein. All amino acids contain an amine group (-NH3) and carboxy group (-COO-).

Antibiotic
An antibiotic is a substance that acts against specific microorganisms, either killing or inhibiting their growth.

Base pairing
Base pairing is the chemical attraction that causes two DNA strands to pair if T is opposite A and G is opposite C.

Cell factory
A cell factory is a cell that produces a valuable product, usually as a result of genetic engineering.

Codon
A codon is three consecutive nucleotides in a gene. Each of the 64 possible codons codes for an amino acid, which becomes part of the protein that the codon’s gene encodes. Codons are also called triplets.

The genetic code
The genetic code is the translation of which amino acids the 64 possible codons lead to. See figure 1.3 in 1 What is DNA and genes?

DNA
DNA stores the genetic material (genes) of all cells in long double strands of nucleotides A, T, C and G. The order of these determines which protein the gene codes for.

DNA polymerase
A type of enzyme that can copy DNA in a process called replication.

Expression
In biology, expression is when a gene is activated and therefore leads to the creation of mRNA and then protein.

Expression vector
An expression vector is DNA that contains the auxiliary DNA elements (promoter, terminator etc) needed to express the gene in an organism. Read much more in 3 Genetic tuning.

Enzyme
Enzymes are proteins that catalyze certain biochemical reactions and processes, i.e. make them happen.

Fermentation
Fermentation is the cultivation of microorganisms in containers (fermenters). Read more.

Gel electrophoresis
Gel electrophoresis is a technique to separate different DNA or protein in a sample. Read more here.

Genome
A genome is an organism’s total amount of DNA sequence, i.e. all genes and gene regulatory regions etc.

Glycosylation
Proteins can be glycosylated by cells, which is an attachment of different kinds of sugars. Glycosylation can affect the half-life of a drug in the blood or, for example, provide specific signals about the protein to the cell.

Half-life
The half-life is the time it takes for the concentration of a substance to halve. Often used to refer to medication in the blood.

Heterozygous
A heterozygous organism has two different alleles of a gene, and the dominant form will be seen in the phenotype (how the organism turns out).

Homozygous
A homozygous organism has two identical alleles of the gene in question. If the alleles are the recessive (declining) form, they will, like the dominant form, be visible in the phenotype (how the organism turns out).

Catalysis
Catalyzing a reaction means increasing its ability to proceed (preferably significantly). The catalysis is done by a catalyst (e.g. an enzyme) that is not consumed in the reaction itself.

Complementary nucleotides
Nucleotides that fit together by base pairing. I.e. A-T and C-G.

Chromosome
Chromosomes are long strands of DNA. In normal (mitotic) cell division, one copy of each chromosome is transferred to the new cell.

mRNA
mRNA (messenger RNA) is a type of RNA. It is formed by transcription of the genes that are active and is used in translation to form protein.

Mutation
A mutation is a change in the DNA sequence that can occur to the advantage or disadvantage of the cell, e.g. due to UV light. Cells that are genetically altered are called mutants.

Nucleotide
The chemical structure that makes up the DNA and RNA strands. There are A, T, C and G. In RNA, T is replaced by the nucleotide U.

Protein
A protein is chemically a long chain of amino acids that are folded in a specific way to perform a function in the cell.

Recombinant production
Production with genetically modified organisms.

Replication
Replication is a cell biological process where a DNA strand is copied in two. A DNA double strand is separated along the way into two single strands, each of which has the complementary nucleotides attached. The result is two double strings.

Restriction enzyme
A restriction enzyme (or restriction endonuclease) is able to cut a DNA double strand at a specific sequence.

Screening
Screening is a method that can be used to distinguish specific genetic variants from the others.

Selection
Selection is a method of establishing conditions for organisms that allow them to survive and thus be selected. Read more.

Southern blot
Southern blotting is used to search for the presence of a specific DNA sequence in a DNA sample by base pairing with a probe. Read more.

Transgenic organism
An organism that contains genes from a foreign organism.

Transcription
Transcription is a process where active genes are transcribed from the DNA into mRNA, which can be used in the cell for translation.

Translation
Translation is a process where mRNA is translated into protein in a ribosome. Each codon (three nucleotides) is translated into an amino acid according to the genetic code.

Expressions
View expression

Source reference:

This project was published in April 2011. It was created by the Biotech Academy and has been updated regularly.

The project was prepared by Peter Rugbjerg.
Peter is a civil engineer from DTU.

Peter Rugbjerg

The Ministry of Science, Technology and Innovation has supported the project with funds from the Tips/Lotto pool.

Ministry of Science, Technology and Innovation...

Novo Nordisk is one of the world’s leading biotech pharmaceutical companies in diabetes treatment. Has been a sponsor and partner on this project.

Novo Nordisk

Novozymes is a world leader in the research and production of enzymes and has been a partner and sponsor on the project.

Novozymes

Jesper Vind is Senior Science Manager and works on improving enzymes at Novozymes. Jesper has been a sparring partner on this article.