Manipulating Genomes

Question 1

Techniques for studying genes

Answer 1

PCR
Cutting out dna fragments using restriction enzymes
gel electropheris

Question 2

Explain PCR

Answer 2

Used to = select a fragment of DNA and amplify it to produce millions of copies in hours
Step- a reaction mixture is set up that contains the dna sample,free nucleotides,primers and DNA polymerase. Primers are short pieces of dna that are complementary to the bases at the start of the fragment you want.dna polymerase is an enzyme that creates new dna strands.
Step2-dna mixture is heated to 95c to break the hydrogen bonds between the two strands of dna. DNA polymerase doesn’t denature even at this high temperature-this is important as it means many cycles of PCR can be carried out without having to use new enzymes each time.the mixture is then cooled to 50-65c so that the primers can bind (anneal) to the strands.
Step 3-reaction mixture is heated to 72c so dna polymerase can work so DNA polymerase can work. The dna polymerase lines up free dna nucleotides alongside each template strand .complementary base pairing means new complementary Starnes are formed.
Step4/2 new copies of the fragment of dna are formed and one cycle of PCR is complete. Then the cycle starts again-mixture is heated to 95c and this time all 4 strands (2 original and 2 new) are used as templates.

Question 3

PCR cycles

Answer 3

Each PCR cycle contains double the amount of dna eg 1st cycle =2x 2=4 DNA fragments
2nd cycle=4 x 2=8 DNA fragments

Question 4

Using restriction enzymes

Answer 4

Some sections of dna have palindromic sequences of nucleotide.
these sequences consist of anti parallel base pairs(base pairs that ready same in opposite directions)

Restriction enzymes recognise specific palindromic sequences(recognition sequences) and cut (digest) the dna at these places.
Difference restriction enzymes cut at different specific recognition sequences, because the shape of the recognition sequence is complementary to an enzymes active site.
If recognition sequences are present at either side of the dna fragment you can use restriction enzymes to seperate it from the rest of the dna. The dna sample is incubated with the specific restriction enzyme which cuts the dna fragment via a hydrolysis reaction . Sometimes the cut leaves sticky ends-small tails of unpaired bases at each end of the fragment. Sticky ends can be used to bind (anneal) the DNA fragment to another piece of DNA that has sticky ends with complementary sequences.

Question 5

Restriction enzymes

Answer 5

Question 6

What is electropheris and explain stage 1

Answer 6

Uses an electric current to seperate out dna fragments,rna fragments or proteins depending on size.

Stage 1- electjroheris is commonly performed using agarose gel that had been poured into a gel tray and left to solidly.a row of wells is created at one end of the gel. To perform electropherosis,firstly you need to put the gel tray into a gel box.you need t9 make sure the end of the gel tray with the wells is closest to the negative electrode on the gel box. Then add buffer solution to the resinous at the sides of the gel box so that the surface of the gel becomes covered in the buffer solution

Question 7

What is electropheris and explain stage 1

Answer 7

Uses an electric current to seperate out dna fragments,rna fragments or proteins depending on size.

Stage 1- electjroheris is commonly performed using agarose gel that had been poured into a gel tray and left to solidly.a row of wells is created at one end of the gel. To perform electropherosis,firstly you need to put the gel tray into a gel box.you need t9 make sure the end of the gel tray with the wells is closest to the negative electrode on the gel box. Then add buffer solution to the resinous at the sides of the gel box so that the surface of the gel becomes covered in the buffer solution

Question 8

What’s stage 2

Answer 8

Take your fragmented DNA samples and, using a micropipette, add the same volume of loading dye to each — loading dye helps the samples to sink to the sottom of the wells and makes them easier to see.
Next add a set volume (eg. 10 pl) of a DNA sample to the first well.
You have to be really careful when adding the samples to the wells - make sure the tip of your micropipette is in the buffer solution and just above the opening of the well (see Figure 5). Don’t stick the tip of the micropipette too far into the well or you could pierce the bottom of it.

Then repeat this process and add the same volume of each of your other DNA samples to other wells in the gel. Use a clean micropipette tip each time. Make sure you record which DNA sample you have added to each we’ll

Question 9

Stage 3

Answer 9

Put the lid on the gel box and connect the leads from the gel box to the power supply. Then turn on the power supply and set it to the required voltage, e.g.
100V. This causes an electrical current to be passed through the gel.
DNA fragments are negatively charged, so they’ll move through the gel towards the positive electrode at the far end of the gel (called the anode).
Small DNA fragments move faster and travel further through the gel, so the DNA fragments will separate according to size.
Let the gel run for about 30 minutes (or until the dye is about 2 cm from the end of the gel), then turn off the power supply. Remove the gel tray from the gel box and tip off any excess buffer solution. Wearing gloves, stain the DNA fragments by covering the surface of the gel with a staining solution then rinsing the gel with water. The bands of the different DNA fragments will now be visible

Question 10

Electrophoresis with rna fragments and proteins

Answer 10

Electrophoresis can be carried out on rna fragments following the same basic method as for dna fragments .

However proteins can be positively charged or negatively charged so before they undergo electrophoresis they’re mixed with a chemical that denatures the proteins so that they al have the same charge .
Electropheris of proteins has many uses eg to identify the proteins present in urine or blood samples which may help to diagnose diseases.

Question 11

What are dna profiles

Answer 11

some of an organism’s genome (all the genetic material in an organism) consists of repetitive, non-coding base sequences - sequences that don code for proteins and repeat over and over (sometimes thousands of times). the number of times these non-coding sequences are repeated differs from person to person, so the length of these sequences in nucleotides differs too.
The number of times a sequence is repeated at different, specific places (loci) in a person’s genome (and so the number of nucleotides there) can be analysed using electrophoresis. This creates a DNA profile. The probability of two individuals having the same DNA profile is very low because the chance of two individuals having the same number of sequence repeats at each locus in DNA is very low.

Question 12

Dna profile Uses in forensic science bf

Answer 12

Use in forensic science
Forensic scientists use DNA profiling to compare samples of DNA collected from crime scenes (e.g. DNA from blood, semen, skin cells, saliva, hair etc.) to samples of DNA from possible suspects, to link them to crime scenes. The DNA is isolated from all the collected samples (from the crime scene and from the suspects). PCR (see pages 206-207) is used to amplify multiple areas containing different sequence repeats - primers are used to bind to either side of these repeats and so the whole repeat is amplified. The PCR products are run on an electrophoresis gel and the DNA profiles produced are compared to see if any match (i.e. if they have the same pattern of bands on the gel). If the samples match, it links a person to the crime scene.

Eg
Below

Question 13

Uses in medical diagnosis

Answer 13

In medical diagnosis, a DNA profile can refer to a unique pattern of several alleles. It can be used to analyse the risk of genetic disorders. It’s useful when the specific mutation isn’t known or where several mutations could have caused the disorder, because it identifies a broader, altered genetic pattern.
- Example
Preimplantation genetic haplotyping (PGH) screens embryos created by IVF for genetic disorders before they’re implanted into the uterus. The faulty regions of the parents’ DNA are used to produce DNA profiles, which are compared to the DNA profile of the embryo. If the profiles match, the embryo has inherited the disorder. It can be used to screen for cystic fibrosis, Huntington’s disease etc.

Question 14

What is genetic engineering

Answer 14

Genetic engineering is the manipulation of an organism’s DNA.
Organisms that have had their DNA altered by genetic engineering are called transformed organisms. These organisms have recombinant DNA - DNA formed by joining together DNA from different sources.
Genetic engineering involves extracting a gene from one organism
and then inserting it into another organism (often one that’s a different species). Genes can also be manufactured (e.g. by PCR) instead of extracted from an organism. The organism with the inserted gene will then produce the protein coded for by that gene.
An organism that has been genetically engineered to include a gene
from a different species is sometimes called a transgenic organism.

Question 15

How to carry out genetic engineering

Answer 15

Part 1 - Obtaining DNA containing the desired gene
The first step is to get hold of a DNA fragment that contains the desired gene
(i.e. the gene you’re interested in). The fragment can be isolated from another organism using restriction enzymes.
Part 2 - Making recombinant DNA
The next step is to insert the DNA fragment into vector DNA - a vector is something that’s used to transfer DNA into a cell. Vectors can be plasmids (small, circular molecules of DNA in bacteria) or bacteriophages (viruses that infect bacteria). The vector DNA is isolated, then restriction enzymes and DNA ligase (an enzyme) are used to stick the DNA fragment and vector DNA together. Here’s how it works:

Question 16

How to make recombinant dna

Answer 16

Step 1
The vector DNA is isolated.
Step 2
The vector DNA is cut open using the same restriction enzyme that was used to isolate the DNA fragment containing the desired gene. This means that the sticky ends of the vector DNA are complementary to the sticky ends of the DNA fragment containing the gene.
Step 3
The vector DNA and DNA fragment are mixed together with
DNA ligase. DNA ligase joins the sugar-phosphate backbones of the two bits of DNA. This process is called ligation.
Step 4- the new combination of bases in the DNA (vector dna and dna ligament) is called recombinant dna

Question 17

Part 3 of genetic engineering

Answer 17

The vector with the recombinant DNA is used to transfer the gene into the bacterial cells (called host cells). If a plasmid vector is used, the host cells have to be persuaded to take in the plasmid vector and its DNA.
-Example
A suspension of the bacterial cells is mixed with the plasmid vector and placed in a machine called an electroporator. The machine is switched on and an electrical field is created in the mixture, which increases the permeability of the bacterial cell membranes and allows them to take in the plasmids. This technique is called electroporation.
With a bacteriophage vector, the bacteriophage will infect the host bacterium by injecting its DNA into it — see Figure 2,. The phage DNA (with the desired gene in it then integrates into the bacterial DNA.
Cells that take up the vectors containing the desired gene are
genetically engineered, so are called transformed

Question 18

How can plants be genetically modified

Answer 18

By having a gene inserted into their cells which makes the resistant to insect sects
Eg soybean yeild can be greatly reduced by insect pests that feed on the soybean plants
Scientists have successfully generically modified soybean plants to include. A gene og found in the bacteria BT. Gene codes for a protein that’s toxic to some of the insects that feed on soybean plants.
Step 1- to genetically modify a soybean plan the desired gene can be isolated from bt using resurrection enzymes and insteted into a plasmid taken from bacterium agrobacteriym tumefaciens.

Step2- the plasmid is put back into A.tumefaciens

Step3- soybean plants cells are the deliberately infected with transformed bacteria. The desired gene gets inserted into the soybean plants cells dna creating a genetically modified GM plant

Question 19

Ethical issues

Answer 19

There are positive ethical issues concerning GM soybean plants
- for example, they will reduce the amount of chemical pesticides
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that farmers use on their crops, which can harm the environment.
CM plants can also be designed to be more nutritious.
But there are also negative ethical issues to consider. For example, farming GM soybean plants may encourage monoculture (where only one type of crop is planted). Monoculture decreases biodiversity and could leave the whole crop vulnerable to disease, because all the plants are genetically identical. There is also a risk that GM soybean plants could interbreed with wild plants creating ‘superweeds’ — weeds that are resistant to herbicides.

Question 20

Producing drugs from animals

Answer 20

Many pharmaceuticals are produced using genetically modified organisms such as animals. This is called pharming.

Hereditary antithrombin deficiency is a disorder that makes blood clots more likely to form in the body. The risk of developing blood clots in people with this disorder can be reduced with infusions of the protein
protein using goats.

DNA fragments that code for production of human antithrombin in the mammary glands are extracted.
2. The DNA fragments are injected into a goat embryo.
3. The embryo is implanted into a female goat.
4. When the offspring is born it is tested to see if it can produce the antithrombin protein.
5. If it does, selective breeding is used to produce a herd of goats that produce antithrombin in their milk.

The protein is extracted from the milk and used to produce a drug that can be given to people with hereditary antithrombin deficiency

Question 21

Ethical issues of producing. Drugs from animals

Answer 21

There are positive ethical issues with ‘pharming’— drugs made
this way can be made in large quantities compared to other methods of production. This can make them more available to more people.
However, the creation of genetically modified animals raises negative ethical issues. For example, there is concern that manipulating an animal’s genes could cause harmful side-effects for the animal, and that using an animal in this way is enforcing the idea that animals are merely ‘assets’ that can be treated however we choose.

Question 22

Using pathogens for research

Answer 22

Scientists are carrying out research into genetically engineered pathogens (microorganisms that cause disease, such as viruses) in order to find treatments for disease.

Eg Scientists found that tumour cells have receptors on their membranes for the poliovirus — so the poliovirus will recognise and attack them. By Benetically engineering the poliovirus to inactivate the genes that cause poliomyelitis, scientists can use it to attack and kill cancer cells without causing disease. This may lead to a treatment for cancer.

Question 23

Ethical issues of using pathogens for research

Answer 23

The genetic modification of pathogens to help cure disease has obvious positive ethical issues - for example, it could mean that previousiv untreatable diseases can now be treated, reducing the suffering they would cause. However, there are many possible negative
ethical issues as well:
•Some people are worried that the scientists researching the pathogens could become infected with the live pathogen and potentially cause a
mass outbreak of disease.
•Some people are concerned that the genetically modified version of a pathogen could revert back to its original form and cause an outbreak
of disease.
•Some people worry that in the wrong hands, knowledge of how to genetically engineer dangerous pathogens could be used maliciously to create agents for biowarfare.

Researchers using live pathogens have to follow strict protocols, which makes the chance of any of these things happening very, very low.

Question 24

Ownership of GM organisms

Answer 24

Many scientists around the world are working on techniques to improve and advance genetic engineering. Scientists working for different institutions often share their knowledge and skills in this field so that, globally, beneficial genetically modified products can be created at a faster rate. The sharing of knowledge, skills and technology like this is called technology transfer.
Although they share information, a group of scientists or the company they work for may want to obtain legal protection for their genetically modified products, e.g. by getting a patent. This means, by law, they can control who uses the product and how for a set period of time.

Question 26

Ethical issues of ownership of GM organisms

Answer 26

This raises some positive ethical issues — it means that the owner HOW SCIENC WORK of the patent will get money generated from selling the product. This encourages scientists to compete to be the first to come up with a new, beneficial genetic engineering idea, so we get genetically engineered products faster. But the process raises many negative ethical issues too. For example, farmers in poorer countries may not be able to afford patenied genetically modified seeds. Even if they can afford seeds for one year, some patents mean that they are not legally allowed to plant and grow any of the seeds from that crop without paying again. Many people think this is unfair and that the big companies that own the patents should relax the rules to help farmers in poorer countries.

Question 27

How does gene therapy work

Answer 27

Genetic disorders are inherited disorders caused by abnormal genes or chromosomes, e.g. cystic fibrosis. Gene therapy could be used to cure these disorders — it isn't being used widely yet but there is a form of somatic gene therapy available, and other treatments are undergoing clinical trials. Gene therapy involves altering alleles inside cells to cure genetic disalers. How u do this depends on whether the the disorder is caused by a mutated dominant allele or two mutated recessive alleles. • If it's caused by two mutated recessive alleles you can add a working dominant allele to make up for them — you 'supplement the lauly ones, • If it's caused by a mutated dominant allele you can 'silence the dominant allele (eg. by sticking a bit of DNA in the middle of the allele so it doesn't work any more). To get the 'new allele (DNA) inside the cell, the allele is inserted into cells using vectors (see pages 212-213). A range of different vectors can be used, e.g. altered viruses, plasmids or liposomes (spheres made of lipid).

Question 28

Types of gene therapy

Answer 28

1. Somatic therapy This involves altering the alleles in body cells, particularly the cells that are most affected by the disorder. Eg Cystic fibrosis (CF) is a genetic disorder that's very damaging to the respiratory system, so somatic therapy for CF targets the epithelial cels lining the lungs. Somatic therapy doesn't affect the individual's sex cells (sperm or eggs) though, so any offspring could still inherit the disease. 2. Germ line therapy - This involves altering the alleles in the sex cells.This means that every cell of any offspring produced from these cells will be affected by the gene therapy and they won't inherit the disease. Germ line therapy in humans is currently illegal though.

Question 29

Ethical issues of gene therapy

Answer 29

There are positive ethical issues surrounding gene therapy: • Gene therapy could prolong the lives of people with life-threatening genetic disorders. • Gene therapy could give people with genetic disorders a better quality of life if it helps to ease symptoms. • Germ line therapy would allow the carriers of genetic disorders to conceive a baby without that disorder. • Germ line therapy could decrease the number of people that suffer from genetic disorders and cancer, which is beneficial for individuals and society as a whole (as fewer people will require treatment). Negative ethical issues of gene therapy There are also negative ethical issues surrounding gene therapy: -The technology could potentially be used in ways other than for medical treatment, such as for treating the cosmetic effects of ageing. There's the potential to do more harm than good by using the technology(risk of overexpression) • There's concern that gene therapy is expensive - some people believe that that health service resources could be better spent on other treatments that have passed health trials.

Question 30

Diadvanatages of gene therapy

Answer 30

There other potential disadvantages of gene therapy too: • The body could identify vectors as foreign bodies and start an immune response against them. • An allele could be inserted into the wrong place in the DNA, possibly causing more problems, e.g. cancer. An inserted allele could get overexpressed, producing too much of the missing protein, and so causing other problems. • The effects of the treatment may be short-lived in somatic therapy. The patient might have to undergo multiple treatments with somatic therapy. • It might be difficult to get the allele into specific body cells.

Question 32

Wat is gene and genome sequencingh

Answer 32

Finding out the order of bases in a gene Finding out the order of bases in all of an organisms dna

Question 33

How can dna be sequenced

Answer 33

Chain termination methods (one of first methods to determine order of bases in a gene Step 1 A mixture of the following is added to four separate tubes: A single-stranded DNA template — the DNA to be sequenced. • DNA polymerase — the enzyme that joins DNA nucleotides together. • Lots of DNA primer-short pieces of DNA (see page 206). • Free nucleotides - lots of free A, T, C and G nucleotides. • A fluorescently-labelled modified nucleotide — like a regular nucleotide, but once it's added to a DNA strand, no more bases are added after it. A different modified nucleotide is added to each tube (these are called A*, T*, C*, %. Step 2 The tubes undergo PCR (see pages 206-207), which produces many strands of DNA. The strands are different lengths because each one terminates at a different point depending on where the modified nucleotide was added. For example, look at Figure 1 below — in tube A (with the modified adenine nucleotide A*) sometimes A* is added to the DNA at point 4 instead of A, stopping the addition of any more bases (the strand is terminated). Sometimes A is added at point 4, then A* is added at point 5. Sometimes A is added at point 4, A again at point 5, G at point 6 and A* is added at point 7. So strands of three different lengths (4 bases, 5 bases and 7 bases) all ending in A* are produced.

Question 34

Step 3 of dna sequencing

Answer 34

The DNA fragments in each tube are separated by electrophoresis and visualised under UV light (because of the fluorescent label). The complementary base sequence can be read from the gel (see Figure 2). The smallest nucleotide you can build up the DNA sequence one base at a time. (eg. one base) is at the bottom of the gel. Each band after this represents one more base added. So by reading the bands from the bottom of the gel upwards,you can buried up the dna sequence one base at a time

Question 35

What is genome sequencing

Answer 35

The chain termination method can only be used for dna fragments about 750 bp long. So if you want to sequence the entire genome (all the DNA) of an organism using this method, you need to chop it up into smaller pieces first. The smaller pieces are sequenced and then put back in order to give sequence of whole genome It done by : 1) A genome is cut into smaller genome fragments (about 100 000 bp) using restriction enzymes. 2) The fragments are inserted into bacterial artificial chromosomes (BACs) - these are man-made plasmids. Each fragment is inserted into a different BAC. 3) The BACs are then inserted One BAC into one bacterium into bacteria - each bacterium contains a BAC with a different DNA fragment. Step4-The bacteria divide, creating colonies of cloned cells that all contain a specific DNA fragment. the different colonies tg make a complete genomic DNA library. Step 5- DNA is extracted from each colony and cut up using restriction enzymes,producing overlapping pieces of dna. 6)each piece of dna is sequenced using the chain termination method 7)pieces are put back in order to give the full sequence from that BAC Step8) dna fragments from all the BACS are put back in order by computers to complete this entire genome.

Question 36

Advances in sequencing

Answer 36

Continued research and improvements in modern technology have led to rapid advancements in the field of gene sequencing. The chain-termination technique is still commonly used but now more attinated and faster the tube now contains all modified nucleotides w different coloured fluorescent label and a machine reads the sequence for u. So instead of running a gel manually the defence is read automatically from computer . Further advances in the field have also led to high throughput seuwences - techniques that can seance a lot faster than original methods eg up to 1000x more bases in give time,at a fraction of cost. Eg chain termination technique has been made high throughput by new tech allowing upto 384 sequences to be run in parallel

Question 38

Other methods of hughtrhoughout sequenc8!( exists some which don’t use electrophoresis like …

Answer 38

Pyrosequencubg-a section of dna is cut into fragments and split in single strands and a strand from each fragment is attached to a small bead 2)PCR is used to amplify the DNA fragments on each bead. 3)Then each bead is put into a separate well. 4), free nucleotides added to the wells attach to the DNA strands via complementary base pairing. The four different types of nucleotides are added to the wells one after the other, over and over again for 100 cycles. 5) The wells also contain specific enzymes, which cause light to be emitted when a nucleotide is added to the DNA strand. More than one nucleotide can be added at a time if the bases are the same, so the intensity of the light can vary. 6) Computers analyse the occurrence and intensities of the light emitted in the different wells, after each type of nucleotide is added, and process this information to interpret the DNA sequence. This technique can sequence around 400 million bases in a ten-hour period (which is super fast compared to older techniques). With newer, faster techniques such as pyrosequencing available, scientists can , sequence whole genomes much more quickly.

Question 39

Sequencing and synthetic biology

Answer 39

You might remember from Module 2 that amino acids are coded for by triplets of bases in a gene. This means that by sequencing a gene, the sequence of amino acids that a gene codes for and so the primary structure of a polypeptide can be predicted. This has allowed us to create biological molecules from scratch and so has led to the development of an area of biology called 'synthetic biology. Synthetic biology is a large field that includes building biological systems from artificially made molecules (e.g. proteins) to see whether they work in the way we think they do, and redesigning biological systems to perform better and include new molecules. It also includes designing new biological systems and molecules that don't exist in the natural world, but could be useful to humans, e.g. energy products (fuels) and drug products. Example Artemisinin is an antimalarial drug — until recently we got artemisinin by extracting it from a plant. Using synthetic biology, scientists have created all the genes responsible for producing a precursor to artemisinin. They've successfully inserted these genes into yeast cells, so we can now use yeast to help produce artemisinin.

Question 40

Sequencing and comparing gencines

Answer 40

Gene sequences and whole genome sequences can be compared between organisms of different species and between organisms of the same species. This is a complicated process which is made easier with the use of computers — it involves computational biology (using computers to study biology, e.g. to create computer simulations and mathematical models) and bioinformatics (developing and using computer software that can analyse, organise and store biological data).

Question 41

Wha are the many reasons why biological research can involve comparison of gene sequences and genomes:

Answer 41

Studying genotype-phenotype relationships Sometimes it's useful to be able to predict an organism's phenotype by analysing its genotype. Example Marfan syndrome is a genetic disorder caused by a mutation of the FBN1 gene. The position and nature of the mutation on the gene affects what symptoms a person with Marfan syndrome will experience (e.g. they could get a number of problems associated with their vision, cardiovascular system or muscles). Scientists have sequenced the FBN1 gene of many people with Marfan syndrome and documented this along with details of their phenotype. Bioinformatics has allowed the scientists to compare all the data and identify genotype-phenotype correlations — this could help in the treatment of Marfan syndrome by using gene sequencing Epidemiology -the study of health and disease within a population - it considers the distribution of a disease, its causes and its effects. Some gene mutations have been linked to a greater risk of disease (e.g. mutations in the BRCA1 gene are linked to breast cancer). Computerised comparisons between the genomes of people that have a disease and those that don't can be used to detect particular mutations that could be responsible for the increased risk of disease. -Understanding evolutionary relationships All organisms evolved from shared common ancestors (relatives). Closely analysed using computer software to tell us how closely related different species are. E.g. the genomes of humans and chimpanzees are about 94% similar. Comparing the genomes of members of the same species can also tell us about evolutionary relationships. Example When different groups of early humans separated and moved to different parts of the world, their genomes changed in slightly different ways. By using computers to compare the genomes of people from different parts of the world, it's possible to build up a picture of early human migration.

Question 42

What is cloning

Answer 42

The process of producing genetically identical cells or organisms from the cells of an existing organism. Cloning can occur naturally in some plants and animals,but also carried out artificially

Question 43

Vegetable propagation

Answer 43

Some plants can produce natural clones by vegetative propagation - this Vegetative propagation is the production of plant clones from non-reproductive tissues, eg. roots, natural vegetative propagation methods used by plants: leaves and stems. It's a type of asexual reproduction.

Question 44

What are the many different natural vegetative propagation methods used by plants

Answer 44

Rhizomes - These are stem structures that grow horizontally underground away from the parent plant. They have 'nodes' from which new shoots and roots can develop. An example of a plant that uses rhizomes is bamboo. Stolons - Also called runners, these are pretty similar to rhizomes. The main difference is that they grow above the ground, on the surface of the soil. New shoots and roots can either develop from nodes (like in uses stolons is the strawberry (see Figure 1). rhizomes) or form at the end of the stolon. An example of a plant that • Suckers —These are shoots that grow from sucker buds (undeveloped shoots) present on the shallow roots of a parent plant. An example of a plant that uses, suckers is the elm tree. • Tubers — These are large underground plant structures that act as a food store for the plant. They're covered in 'eyes'. Each eye is able to is the potato. sprout and form a new plant. An example of a plant that uses tubers Bulbs These are also underground food stores used by some plants. New bulbs are able to develop from the original bulb and form new individual plants. An example of a plant that uses bulbs is the onion.

Question 45

Vegetative proration in horticulture and agriculture

Answer 45

Horticulturists (plant growers) and farmers can exploit a plant's natural ability to produce clones. By manipulating the way in which a plant grows, they can induce induce vegetative propagation, so they get natural clones of the parent plant. There are several different methods they can use to do this: They can take cuttings (see next page). • They can use grafting — joining the shoot of one plant to the growing stem and root of another plant. • They can use layering - bending a stem of a growing plant downwards so it enters the soil and grows into a new plant.

Question 46

How to produce clones from cutting

Answer 46

1. Use a scalpel or sharp secateurs to take a cutting, between 5 cm and 10cm long, from the end of a stem of your parent plant. 2.Remove the leaves from the lower end of your cutting (if there are any), leaving just one at the tip. 3. Dip the lower end of the cutting in rooting powder, which contains hormones that induce root formation. 4.Then plant your cutting in a pot containing a suitable growth medium (e.g. well-drained compost). 5. Provide your cutting with a warm and moist environment by either covering the whole pot with a plastic bag or by putting it in a propagator (a specialised piece of kit that provides these conditions - see Figure 3). 6. When your cutting has formed its own roots and is strong enough, you can plant it elsewhere to continue growing.

Question 47

How would you take cuttings from another party of the plant like root or leaf

Answer 47

To take a root cutting, cut a piece of root from the plant with a straight cut using a scalpel or secateurs. Then remove the uncut end of the root with a slanted cut. Dip the end of the cutting in rooting powder and plant it in a suitable growth medium. Then follow steps 5 and 6 from the method above. • A popular type of leaf cutting known as a split vein cutting) involves removing a complete leaf and scoring the large veins on the lower leaf surface using a scalpel (see Figure 4). You then put it on top of the growth medium with the broken veins facing down and then follow steps 5 and 6 from above. A new plant should ‹ form from each break in the veins.

Question 48

What’s an artificial way of cloning plants

Answer 48

Tissue culture - 1.Cells are taken from the original plant that's going to be cloned. Cells from the stem and root tips are used because they're stem cells — like in humans, plant stem cells can develop into any type of cell. 2. The cells are sterilised to kill any microorganisms — bacteria and fungi compete for nutrients with the plant cells, which decreases their growth rate. 3. The cells are placed on a culture medium containing organic nutrients (like glucose and amino acids) and a high concentration of plant hormones (such as auxins, see p. 98). This is carried out under aseptic conditions (see p. 246). The cells divide to produce a mass of undifferentiated cells. The mass can be subdivided to produce lots of plants very quickly (see below). 4. When the cells have divided and grown into a of the medium and planted in soil - they'll de genetically identical to the original plant.

Question 49

Uses of tissue culture

Answer 49

Tissue culture is used to clone plants that don't readily reproduce or are endangered or rare. Example A number of British orchid species are now endangered in the UK. It's very difficult to reproduce orchids using seeds because it can take a long time for the plants to produce flowers, they have a very specialised mechanism of pollination and the seeds usually need a specific fungus present in order to germinate. But many have been successfully reproduced using tissue culture. It's also used to grow whole plants from genetically engineered plant cells.

Question 50

Micropropogation

Answer 50

Micropropagation is when tissue culture is used to produce lots of cloned plants very quickly. Cells are taken from developing cloned plants and subcultured (grown on another fresh culture medium) - repeating this process creates large numbers of clones (see Figure 8). This technique is used extensively in horticulture and agriculture, eg. to produce fields full of a crop that has been genetically engineered to be pest-resistant.

Question 51

Arguements for and against artificial cloning

Answer 51

-Desirable genetic characteristics (e.g. high fruit production) are always passed on to clones. This doesn't always happen when plants reproduce environment is controlled. -Tissue culture allows plants to be reproduced in any season because the the same number of plants by conventional growing methods. grow them from seeds. -it produces lots of plants quickly compared to the time it would take to Less space is required by tissue culture than would be needed to produce Arguments against -Undesirable genetic characteristics (e.g. producing fruit with lots of seeds) are always passed on to clones. -Cloned plant populations have no genetic variability, so a single disease could kill them all. • Production costs of tissue culture are very high due to high energy use and the training of skilled workers, so it's unsuitable for small scale production. • Contamination by microorganisms during tissue culture can be disastrous and result in. complete loss of the plants being cultured.

Question 52

Natural animal clones

Answer 52

Animal clones can be produced naturally as a result of sexual reproduction. Durine cexual reproduction, once an egg has been fertilised, it's possible for in to split durine the very early stages of development and develop into multiple embryos with the same genetic information. The embryos can develop as normal to produce offspring that are all genetically identical - they are clones. For example, identical twins are natural clones.

Question 53

Ways to do Artificial animal clones

Answer 53

Artificial embryo twinning- Somatic cell clear transfer

Question 54

Artificial animal cloning

Answer 54

Artificial embryo twinning- This type of artificial cloning is similar to what happens when animal clones form naturally. Figure 1 shows how this is done in cows, but the same technique can be used for other animals: 1. An egg cell is extracted from a female cow and fertilised in a Petri dish. 2. The fertilised egg is left to divide at least once, forming an embryo in vitro (outside a living organism). 3. Next the individual cells from the embryo are separated and each is put into a separate Petri dish. Each cell divides and develops normally, so an embryo forms in each Petri dish. 4. The embryos are then implanted into female cows, which act as surrogate mothers. 5. The embryos continue to develop inside the surrogate cows, and eventually the offspring are born. They're all genetically identical to each other.

Question 55

Somatic cell nuclear transfer

Answer 55

-A somatic (any cell that isn't a reproductive cell) is taken from sheep. -An oocyte (immature egg cell) is taken from sheep B. Its nucleus is removed to form an enucleated oocyte. -The nucleus from sheep A is inserted into the enucleated oocyte - the nocyte from sheep B now contains the genetic information from sheep A. The nucleus and the enucleated octe are fused topether and stimulated to divide (e.g. by electrofusion, where an electrical current is applied). This produces an embryo. -Then the embryo is implanted into a surrogate mother and eventually a lamb is born that's a clone of sheep A.

Question 56

Used of animal cloning

Answer 56

Animal cloning has many uses. Here are a few to take a look at: • Scientists use cloned animals for research purposes. - Example In the field of medicine they can test new drugs on cloned animals. They're all genetically identical, so the variables that come from genetic differences (e.g. the likelihood of developing cancer) are removed. • Cloning can be used in agriculture so farmers can increase the number of animals with desirable characteristics to breed from. Example A prize-winning cow with high milk production could be cloned. Animals that have been genetically modified (see page 215) to produce a useful substance that they wouldn't normally produce could be cloned to produce lots of identical animals that all produce the same substance. Example A goat that has been genetically modified to produce a beneficial protein in its milk could be cloned. • Cloning can also be used to save endangered animals from extinction by cloning new individuals. Example The European mouflon is a species of wild sheep which is currently endangered. Scientists have successfully cloned a European mouflon and it's hoped that this could help save the species.

Answer 57

Cloning doesn't have to be used to make whole animals. Sometimes scientists only want the cloned embryonic stem cells. These cells are harvested from young embryos and have the potential to become any cell type, so scientists think they could be used to replace damaged tissues in a range of diseases, eg. heart disease, spinal cord injuries, degenerative brain disorders like Parkinson's disease. If replacement tissue is made from cloned embryonic stem cells that are genetically identical to the patient's own cells, it won't be rejected by their immune system.

Question 58

Arguments for and against animal cloning

Answer 58

Arguements for Desirable genetic characteristics are always passed on to clones (e.g high milk production in cows). This doesn't always happen with sexual reproduction because of processes such as independent assortment and crossing-over, which generate genetic variation during meiosis. So if a farmer had a cow that produced a lot of milk, the only way he could guarantee that his calves would also produce a lot of milk would be to clone the cow. • Infertile animals can be reproduced, so if a farmer's prize winning cow was infertile for any reason, they could still reproduce it. • Animals can be cloned at any time — you wouldn't have to wait until a breeding season to get new animals. • Increasing the population endangered species helps to preserve biodiversity. • Cloning can help us develop new treatments for disease, which could mean less suffering for some people. Arguments against Animal ning is very difficult, time-consuming and expensive. • There's no genetic variability in cloned populations, so undesirable genetic characteristics (eg. a weak immune system) are always passed on to clones. This means that all of the cloned animals in a population are susceptible to the same diseases. Potentially, a single disease could wipe them all out. Some evidence suggests that clones may not live as long as natural offspring pop ring. Some may think it’s unethical. Example: Example Dolly the sheep was a clone generated by somatic cell nuclear SCIENCE transfer. It took 277 nuclear transfer attempts before Dolly was WORKS finally born, which shows just how difficult it is to successfully cone an animal. The average life expectancy of sheep the same breed as Dolly is 11-12 years but Dolly had to be put down at the age of six after developing a lung disease and arthritis. -Using cloned human embryos as a source of stem cells is controversial. The embryos are usually destroyed after the embryonic stem cells have been harvested - some people believe that doing this is destroying a human life.

Question 59

What is bio technology

Answer 59

The industrial use of living organisms to produce food drugs and other products

Question 60

What are the living organisms used for bio technology and why

Answer 60

microorganisms (bacteria and fungi). -Their ideal growth conditions can be easily created - microorganisms will generally grow successfully as long as they have the right nutrients, temperature. pH, moisture levels and availability of gases (eg som • Due to their short life-cycle, they grow rapidly under the right conditions, need oxygen). so products can be made quickly. • They can grow on a range of inexpensive materials - this makes them economical to use. • They can be grown at any time of the year.

Question 61

Enzymes in biotechnology

Answer 61

Biotech also uses enzymes as well to make products Eg lactase (breaks down lactose) prepared from aspergillus fungi and is used in the production of lactose free products Enzymes used in industry can be contained within the cells of microorganisms -intracellular enzymes . within the cells of microorganisms — these are called intracellular enzymes. Enzymes are also used that aren't contained within cells — these are called isolated enzymes. Some are secreted naturally by microorganisms (called extracellular enzymes), but others have to be extracted. Naturally secreted enzymes are cheaper to use because it can be expensive to extract enzymes from cells.

Question 62

Uses of microorganisms in biotechnology

Answer 62

Brewing-To make beer, yeast (eg. Saccharomyces cervisiae) is added to a type of grain (such as barley) and other ingredients. The yeast respires anaerobically using he glucose from the grain and produces ethanol (alcohol) and Co, Wher anaerobic respiration produces ethanol, the process is called fermentation.) Baking-Yeast is also the organism that makes bread rise. The CO, produced by fermentation of sugars in the dough makes sure it doesn't stay flat. Many flat breads, like tortillas, are made without yeast. Cheese making- Cheese production used to rely on a substance called rennet. Rennet contains the enzyme chymosin, which clots the milk a key process in cheese making. Traditionally we used to get chymosin by extracting rennet from the lining of calves' stomachs, but now chymosin can be obtained from yeast cells that have been genetically modified to produce the enzyme. Cheese making also involves lactic acid bacteria (e.g. Lactobacillus and Streptococcus). These bacteria convert the lactose in milk into lactic acid, which makes it turn sour and contributes to it solidifying. The production of blue cheeses also involves the addition of fungi to make the characteristic blue veins. Yoghurt production- Just like cheese making, yoghurt production involves the use of lactic acid bacteria to clot the milk and cause it to thicken. This creates a basic yoghurt product and then any flavours and colours are added. Penicillin production- In times of stress, fungi from the Penicillium genus produce an antibiotic, penicillin, to stop bacteria from growing and competing for resources. Penicillin is one of the most common antibiotics used in medicine, so we produce it on a massive scale. The fungus (usually Penicillium chrysogenum) is grown under stress in industrial fermenters (see page 242) and the penicillin produced is collected and processed to be used in medicine. Figure 2: Rennet being added to milk to make cheese. Figure 3: Penicillium fungus - this fungus produces penicillin. Insulin production- Insulin is a hormone that's crucial for treating people with Type 1 diabetes. Insulin is made by genetically modified bacteria, which have had the gene for human insulin production inserted into their DNA. These bacteria are grown in an industrial fermenter on a massive scale and the insulin produced is collected and purified. Bioremediation- Bioremediation is a posh name for the process of using organisms (usually microorganisms) to remove pollutants, like oil and pesticides, from contaminated sites. Most commonly, pollutant-removing bacteria that occur Tip: Bioremediation can also be carried out by introducing new bacteria to the environment that needs cleaning up, although this is less common. naturally at a site are provided with extra nutrients and enhanced growing conditions to allow them to multiply and thrive. These bacteria break down the pollutants into less harmful products, cleaning up the area.

Question 63

What is single-cell protein and give examples of microorganisms used to produce it.

Answer 63

Single-cell protein is protein produced from microorganisms as a food source for humans and animals. Examples include the fungus Fusarium venenatum (used to make Quorn) and the bacterium Methylophilus methylotrophus (used for animal feed).

Question 64

Advantages and disadvantages of using microorganisms in food production

Answer 64

Microorganisms used to make single-cell protein can be grown using many different organic substrates, including waste materials such as molasses (a by-product of sugar processing). Production of single-cell protein could actually be used as a way of getting rid of waste products. • Microorganisms can be grown quickly, easily and cheaply. Production costs are low because microorganisms have simple growth requirements, can be grown on waste products and less land is required in comparison to growing crops or rearing livestock. Microorganisms can be cultured anywhere if you have the right equipment. This means that a food source could be readily produced in places where growing crops and rearing livestock is difficult (e.g. very hot or cold climates). This could help tackle malnutrition in developing countries. Single-cell protein is also often considered a healthier alternative to animal protein. Disadvantage: Because the conditions needed to grow the desired microorganism are also ideal for other microorganisms, a lot of effort has to go into making sure that the food doesn't get contaminated with unwanted bacteria, which could be dangerous to humans or spoil the food. waste products. People may not like the idea of eating food that has been grown using • Single-cell protein doesn't have the same texture or flavour as real meat. If single-cell protein is consumed in high quantities, health problems could be caused due to the high levels of uric acid released when the large amounts of amino acids are broken down.

Question 65

Culturing micro organism

Answer 65

Biotechnology uses cultures of microorganisms. A culture is a population of one type of microorganism that's been grown under controlled conditions. Cultures are grown in large containers called fermentation vessels (see Figure 1) to either obtain lots of the microorganism (e.g. for production of single-celled protein - see page 240) or to collect lots of a useful product that the microorganism makes. There are two main methods for culturing microorganisms - batch fermentation and continuous fermentation. Batch fermentation is where microorganisms are grown in individual batches in a fermentation vessel — when one culture ends it's removed and then a different batch of microorganisms is grown in the vessel. This is known as a closed culture — see next page. Continuous fermentation is where microorganisms are continually grown in a fermentation vessel without stopping. Nutrients are put in and waste products taken out at a constant rate. The conditions inside the fermentation vessels are kept at the optimum for growth — this maximises the yield of microorganisms and desirable products. The factors that need to be controlled in a fermentation vessel are explained below and on the next page.

Answer 66

pH The pH is monitored by a pH probe and kept at the optimum level. This increases the product yield because enzymes can work efficiently, so the rate of reaction is kept as high as possible. Temperature The temperature is kept at the optimum level by a water jacket that surrounds the vessel. This increases the product yield because enzymes can work efficiently, so the rate of reaction is kept as high as possible.

Question 67

Interpreting data values from a log scale

Answer 67

When you see microbial growth data presented on a logarithmic scale, the units on the y-axis may tell you the total number of cells rather than the log This makes it easier to work out how many cells are present at a given are not evenly spaced. ime, but you have to be careful because the smaller increments on the y axis are not evenly spaced. If only the log values are shown on the y-axis, it's still possible to work out how many cells are present at a given time by finding the antilog. To do this you need to use the 10: button on your calculator, Simply press this, then enter the log value at your chosen time. When you press equals, you'll get the number of cells.

Question 68

Culturing microorganisms in the lab

Answer 68

Cultures of microorganisms can be grown in the lab. A common way to do this is on an agar plate — a sterile Petri dish containing agar jelly. Nutrients can be added to the are likely to be provided in a liquid broth la mixture of distilled water and agar to help improve the growing conditions. The microorganisms you use nutrients). To culture the microorganisms, use a sterile implement like a wire inoculation loop to transfer some of the sample to the plate and gently spread to allow the microorganisms to grow.

Question 69

Aseptic technique

Answer 69

Aseptic techniques An important part of culturing microorganisms is using aseptic techniques. These are used to prevent contamination of cultures by unwanted microorganisms, which may affect the growth of the microorganism being cultured. Contaminated cultures in laboratory experiments give imprecise results and may be hazardous to health. Contamination on an industrial scale can be very costly because entire cultures may have to be thrown away. Below are some important aseptic techniques that you should follow when culturing microorganisms in the lab: Tie long hair back to prevent it from falling into anything. • Work near a Bunsen flame. Hot air rises, so any microorganisms in the air should be drawn away from your culture. • Regularly disinfect work surfaces to minimise contamination. Sterilise the instrument used to transfer cultures before and after each use, e.g. sterilise a wire inoculation loop by passing it through a hot Bunsen burner flame for 5 seconds. This will kill any microorganisms on the instrument. • If you're using broth, briefly pass the neck of the broth container through a Bunsen burner flame just after it's opened and just before it's closed — this causes air to move out of the container, preventing unwanted organisms from falling in. Minimise the time that the agar plate is open and seal the lid on as soon as possible. This reduces the chance of airborne microorganisms contaminating the culture. You could even work in an inoculation cabinet (a chamber that has a flow of sterile air inside it). Sterilise all glassware before and after use, e.g. in an autoclave (a machine which steams equipment at high pressure).

Question 70

Investigating factors that affect growth of microorganism

Answer 70

you can investigate the effects of different factors on the growth of ACTIVITY nicroorganisms by growing them on agar plates under different conditions. The example below shows how you can investigate the effect of temperature on the growth of bacteria (although the same method can be used for other microorganisms such as fungi) 1. You should be supplied with a sample of bacteria (eg. E. coll in broth Using a sterile pipette, add the same volume (e.g. 0.1 cm) of vour sample to each of six agar plates. Discard your pipette safely after use (eg. if it's a glass pipette, put it into a beaker of disinfectant while you are working and then into an autoclave once you've finished) 2. Spread the broth across the entire surface of the agar using a wire inoculation bacteria loop (see Figure 9). Make sure that you sterilise the inoculation loop before and after each use by passing it through a hot Bunsen burner flame for 5 seconds. Give it a few seconds to cool before use. 3. Put the lids on the agar plates and tape them shut. 4. Place three plates in a fridge at 4°C and put three in an incubator at 25°C. If you don't have access to an incubator, just leave the plates at room temperature, somewhere where the temperature is most likely to remain constant. The plates should be incubated upside down. This stops any condensation forming on the lid from dropping onto the agar. 5.Put another lidded agar plate in each of the two different temperature locations - these plates should be uncultured (i.e. you shouldn't have added any bacteria to them. These plates will act as negative controls. 6. Leave all the plates for the same amount of time (e.g. 24 hours) then observe the results. 7. If bacterial growth has occurred, you should see colonies of bacteria on the surface of the agar (see Figure 10). 8. Count the number of colonies that have formed the naked eye. on each plate and record your results in a table. 9. Work out the mean number of colonies formed at each temperature. You might find that you have so many colonies that they overlap and you can't count them. If this happens, try making serial dilutions of your Tip: Serial dilutions are a set of dilutions that decrease in concentration by the same factor each time. bacteria in broth and plate them on agar — this should give you a more manageable number of colonies because there will be fewer bacteria present in the solution to begin with.

Question 71

How to adapt the experiment to investigate the effects of different factors on the growth of microorganisms

Answer 71

investigate the effect of pH by adding buffers at different pH levels to the broth. • investigate the effects of nutrient availability by using different preparations of agar, which contain different nutrients. You could also investigate the growth of microorganisms directly in broth without the need to plate the broth on agar using a spectrophotometer. This is a machine that measures the turbidity (cloudiness) of the broth. Higher turbidity means that more cells are present and, therefore, more replication has taken place.

Question 72

Immobilised enzymes

Answer 72

Immobilising isolated enzymes Isolated enzymes used in industry can become mixed in with the products of reaction. The products then need to be separated from this mixture, which can be complicated and costly. This is avoided in large-scale production by using immobilised enzymes —enzymes that are attached to an insoluble material so they can't become mixed w the products.

Question 73

What are the three main ways enzymes are immobilised

Answer 73

1)Encapsulated in jelly-like alginate beads, which act as a semipermeable membrane. 2)trapped in silica gel matrix 3) Covalently bonded to cellulose or collagen fibres. In industry, the substrate solution for a reaction is run through a column of immobilised enzymes (see Figure 1). The active sites of the enzymes are still available to catalyse the reaction but the solution flowing out of the column will only contain the desired product

Question 74

Uses of immobilised enzymes

Answer 74

Conversion of lactose to glucose and galactose- Some people are unable to digest lactose (a sugar found in milk) because they don't produce enough (or any) of the enzyme lactase. Lactase breaks lactose down into glucose and galactose via a hydrolysis reaction. Industrially, fresh milk can now be passed over immobilised lactase to produce lactose-free milk for use in the production of lactose-free dairy products. Production of semi-synthetic penicillins- Penicillin is a useful antibiotic, but some bacteria have become penicillin resistant. Semi-synthetic penicillins can now be produced, which have the same antibiotic properties as natural penicillin, but are effective against penicillin-resistant organisms. Immobilised penicillin acylase enzyme is used in their production. Conversion of dextrins to glucose- Glucose and glucose syrup are used in massive amounts in industry eg food to sweet n thicken it. Glucose can be derived from starchy goods such as corn and potatoes, with the help of immobilised enzymes. Starch breaks down into dextrins (carbohydrate products ) which are then broken down into glucose by the immobilised enzyme glycoamylase. Conversion of glucose to fructose - Fructose is a sugar that's much sweeter than glucose. It's used as a sweetener in food - using fructose rather than glucose means that less sugar is needed to obtain the same level of sweetness in our foods. Immobilised glucose isomerase is used to convert glucose to fructose on an industrial scale. Production of pure samples of L amino acids -Amino acids have two chemical forms (isomers) - L or D. Most amino acids utilised by the body need to be in the L form. Scientists are able to chemically synthesise amino acids, but end up with a mix of L and D forms. The enzyme aminoacylase separates them. Immobilised aminoacylase is used for the industrial production of pure samples of L-amino acids, which can be used for many purposes in the production of animal and human food, as well as in dietary supplements. Conversion of acrylonitrile to acrylamide- Acrylamide is a chemical that is typically used in industry to produce synthetic polymers (e.g. plastics), which have a wide range of uses. For example, acrylamide is involved in the production of the polymer that's used in disposable nappies to make them super-absorbent. In industry, immobiliser nitrilase is used to convert acrylonitrile (a man-made chemical) to acrylamide

Question 75

Advatnages an disadvantages of immobilising isolated enzymes

Answer 75

Columns of immobilised enzymes can be washed and reused - this reduces the cost of running a reaction on an industrial scale because you don't have to keep buying new enzymes. -The product isn't mixed with the enzymes - no money or time is spent separating them out • Immobilised enzymes are more stable than free enzymes - they're less likely to denature (become inactive) in high temperatures or extremes of pH. Disadvantages : Extra equipment is required, which can be expensive to buy. - e.g. for an advar • Immobilised enzymes are more expensive to buy than free enzymes, so coupled with the equipment costs, they're not always economical for use more economical free enzymes, gi in smaller-scale production. • The immobilisation of the enzymes can sometimes lead to a reduction in the enzyme activity because they can't freely mix with their substrate.