Cloning and biotechnology (6.2.1)

Many plants naturally produce clones:

• Bulbs – plants such as daffodils produce bulbs underground.
• Runners – plants such as strawberries and spider plants produce runners. These are shoots that can root and produce a new plant.
• Rhizomes – plants such as mint or bamboo produce specialised stems underground.
• Stem tuber – plants such as potatoes produce tubers underground.

Many plants can be easily cloned by taking cuttings.

A section of the stem of the plant is taken. Rooting hormone can be added to the bottom of the stem, and then the stem can be placed in soil.

The stem will grow roots, producing a separate but genetically identical new plant.

Growing from cuttings is faster than growing from seed, but can lead to a lack of genetic variation and susceptibility to infection.

Many invertebrates, such as starfish, flatworms and sponges, make clones naturally when fragmented. This makes artificial cloning of many invertebrates relatively easy.

Some insects, such as certain stick insect species, are able to produce offspring without mating. Sometimes, the offspring have genetic differences due to mutations, which will lead to some genetic variation, but the differences are not due to two individuals combining their DNA.

In vertebrates, natural cloning is mostly due to monozygotic (identical) twins. Monozygotic twins are genetically identical, but may appear different due to environmental factors.

Some female amphibians can produce offspring without a male present. These offspring are usually male, so they are not true clones, but all the genetic information originates from the mother.

Artificially cloning vertebrates is more difficult than cloning invertebrates.

Vertebrates can be cloned via artificial twinning or somatic cell nuclear transfer.

After an egg is fertilised, a zygote is formed. The zygote will divide to form a ball of cells. These cells can be separated to produce clones in a process called artificial twinning.

This process is often used in agriculture to create organisms with desirable characteristics.

Artificial twinning of a cow involves the following steps:

1. A cow is given hormones that cause the cow to produce more eggs (ova) than normal.
2. Ova can be fertilised naturally or by artificial insemination. Fertilisation can occur in the cow’s uterus, with the zygotes later removed, or the eggs can be removed and fertilised in a lab (in vitro fertilisation).
3. The fertilised cell replicates to form an early embryo, which is then split up into multiple cells.
4. These cells are left to divide, then are implanted into different cows.
5. Different cows will birth the cloned offspring.

Adult vertebrates can be cloned via somatic call nuclear transfer (SCNT). An adult somatic (body) cell is used. The nucleus from the somatic cell is inserted into an enucleated egg cell; an egg cell where the nucleus has been removed.

The genetic material will be identical to the somatic cell, but the mitochondrial DNA will be the same as that of the egg cell donor.

The cell is given a pulse of electricity and the cell divides, forming an embryo.

The embryo is transferred into a surrogate mother who carries the pregnancy and gives birth to the clone.

Note that SCNT can be combined with artificial twinning by splitting the embryo.

Biotechnology involves using enzymes or microbes to produce, transform or break down materials.

The term biotechnology includes both traditional methods, such as producing fermented products and the use of genetically modified organisms to produce food or drugs.

Biotechnology can be used within food production:

• Baking – yeast can be used to produce carbon dioxide during anaerobic respiration.
• Brewing – yeast can be used to produce ethanol during anaerobic respiration.
• Making cheese – bacterial cultures are usually used. Chymosin enzyme is sometimes added, which is often produced by genetically modified organisms.
• Making yoghurt – bacterial cultures Lactobacillus bulgaricus and Streptococcus thermophilus are used to ferment milk, to produce ethanal and lactic acid. Both species also make yoghurt thicker.

Biotechnology can be used to directly produce food.

Protein can be produced by microorganisms, known as single-cell protein, such as Quorn^TM.

Quorn^TM is a type of mycoprotein made by the bacterial culture Fusarium venetatum in fermenters using glucose syrup. The mycoprotein is mixed with egg proteins and formed into meat substitutes.

The antibiotic penicillin is produced by the mould Penicillium notatum and was the first antibiotic to be discovered. Penicillium notatum produces penicillin slowly and is not used industrially to produce penicillin.

A different microculture, Penicillium chrysogenum, is used to produce penicillin industrially, as this mould produces penicillin at a much faster rate.

To produce penicillin, Penicillium chrysogenum requires a nutrient–rich medium, high oxygen levels and a carefully controlled and temperature.

A semi–continuous batch process is used to produce penicillin industrially due to the mould’s requirements.

• Small fermenters of approximately 100 are used so oxygen levels can be kept high. The mixture is continuously stirred to expose it to oxygen.
• A nutrient–dense medium is used to ensure the mould has adequate nutrition.
• The growth medium contains a buffer to maintain optimum .
• An optimal temperature of approximately 26 is maintained.

Insulin is used to treat type 1 diabetes and occasionally type 2 diabetes.

Historically, insulin was extracted from the pancreases of slaughtered animals. This method of obtaining insulin led to infections and allergic reactions as the insulin was not identical to human insulin.

There were also ethical and religious issues with using insulin from slaughtered animals such as pigs and cows.

Insulin is now produced using genetically modified bacteria. This allows the gene for human insulin to be inserted into a bacterial plasmid. The plasmid is then taken up by bacteria, which produce human insulin.

This method reduces the risk of allergic reaction and allows insulin to be produced on demand.

This also allows different modified versions of the insulin protein to be produced, such as long–acting insulin.

Microorganisms can be used for bioremediation, which involves breaking down pollutants and contaminants.

Naturally occurring microorganisms can catalyse the breakdown of pollutants, such as oil spills. If the conditions support the growth of the microorganisms, then the bioremediation will occur faster.

Genetically modified (GM) organisms have been developed to carry out bioremediation, such as bacteria which can remove mercury from water sources. GM organisms can be bred for specific purposes, but are often slower than naturally occurring bacteria, which carry out bioremediation.

Culturing microorganisms requires aseptic techniques. This prevents contamination and ensures only the desired microorganism is cultured.

Aseptic conditions require a sterilised growth medium, usually sterilised broth or sterilised agar.

The desired bacteria are added to the sterile growth medium; this process is called inoculation.

Broth can be inoculated by adding a bacterial sample to sterile broth with an inoculating loop or micropipette.

1. The inoculating loop is sterilised using a Bunsen burner.
2. The loop tip is submerged, and the bacterial sample is added to the broth.
3. The mouths of the bottles are also sterilised after opening and before sealing to ensure no unwanted bacteria enter the broths.
4. The inoculated broth is then partially sealed, either using cotton wool or by loosely placing a lid on the bottle.

Agar can be inoculated using many different methods.

In a simple benchtop method:

1. The bacterial sample is added to a sterile agar plate using an inoculating loop.
2. The lid of the agar plate is opened as little as possible to prevent airborne microorganisms from accessing the agar.
3. The inoculating loop is sterilised using a Bunsen burner, added to the bacterial sample, and then gently zig-zagged across the surface of the sterile agar.
4. The agar plate is then partially sealed using tape.

Industrially, microorganisms can be used to produce products which can be sold:

• The microorganism itself can be sold, e.g., baker’s yeast.
• Primary metabolites can be produced. These are substances produced as an essential part of the microorganism’s function, e.g., ethanol produced by yeast.
• Secondary metabolites can be produced. These are substances which are not produced as part of a microorganism’s essential functioning, and are usually produced under specific conditions, e.g., penicillin.

Batch fermentation involves inoculating a fixed volume of growth medium with microorganisms, which grow and replicate. As the microorganisms grow and replicate, the bacterial population will eventually stabilise. Batch fermentation is often used when the desired end products have specific potencies, such as penicillin.

Continuous fermentation involves inoculating a growth medium with microorganisms, which grow and replicate. The nutrient medium is continuously added while the broth is continuously removed. This enabled the conditions to be kept consistent and the product to be consistently removed.

The conditions of fermenters are controlled to increase the yield of the product:

• Temperature: the optimum temperature is used to speed up the rate of microorganism growth without denaturing enzymes.
• Nutrients and oxygen: these can be added to ensure the microorganisms can grow and respire at a fast rate, and aerobically (if this is desired).
• Stirring: bioreactors are stirred constantly, as reactors are large, and stirring allows nutrients and oxygen to be dispersed evenly.
• Asepsis (a sterile environment): microorganisms in the air could contaminate the bioreactor, and genetically modified organisms from the bioreactor could contaminate the air. Bioreactors are sealed to prevent this, and any air or nutrients added must be sterile.

The number of bacteria in a colony can be determined by the formula:

Where:
= number of bacteria
= bacteria at the start
= number of replications

This formula assumes the bacteria are in the log phase of the growth curve.

Isolated enzymes can be used to carry out processes rather than using whole microorganisms.

Extracellular enzymes tend to be used as they tend to be more robust and able to cope with changes in conditions.

Intracellular enzymes are less able to cope with changes in conditions because they are intended to catalyse reactions inside the cell’s cytoplasm, where conditions are very stable.

Extracellular enzymes are excreted by microorganisms, so are easier to extract than intracellular enzymes. Microorganisms also produce fewer types of extracellular enzymes, making it easier to isolate the desired enzyme.

There are many advantages to using isolated enzymes rather than whole microorganisms:

• Isolated enzymes are more cost-efficient than whole microorganisms; enzymes do not require nutrients to be maintained.
• Enzymes are specific in the reaction they catalyse and produce one product, whereas microorganisms produce a range of primary and secondary metabolites. This makes the product purification simpler.
• Enzymes can be used in higher concentrations than whole microorganisms, increasing the rate of reaction.

Isolated enzymes can be easily immobilised. Enzymes can be attached to a surface or held within a matrix. When used in industrial processes, this allows the enzyme to be easily recovered from the end product.

Some examples of immobilised enzymes used in industry:

• Semi-synthetic penicillin production: immobilised enzymes chemically alter penicillins, allowing a wider variety of antibiotics to be produced (reducing instances of antibiotic resistance).
• Fructose production: immobilised glucose isomerase enzymes catalyse the conversion of glucose to fructose (which is sweeter than glucose, meaning less is used).
• Lacto-free dairy production: immobilised lactase enzyme catalyses the breakdown of the lactose into galactose and glucose, producing milk that people who cannot digest lactose can drink.
• L-amino acid production: immobilised aminoacylase produces pure L-amino acid samples for use in food, organic synthesis and cosmetics.
• Glucose production: amylase catalyses the breakdown of starch to produce short–chain polysaccharides called dextrins. Immobilised glucoamylase catalyses the breakdown of dextrins into glucose.