Download Genetic engineering NOTES File

Genetic engineering and bacteria
Read pages 176-177 of your text book
Q
What do we call a plasmid that has incorporated extra DNA? [1]
Q
How can bacterial cells be treated so that they take up plasmid DNA? [2]
Genetic Markers
Genetic markers are used to detect whether recombinant plasmids have been produced and
incorporated into bacteria. Plasmids such as pBR322 carry genes for resistance to the antibiotics
tetracycline and ampicillin. During plasmid preparation, a restriction enzyme is used that has a
restriction site within the tetracycline gene. Consequently, if a gene is inserted at this site and a
recombinant plasmid is produced, resistance to tetracycline is lost. As the ampicillin gene is not
affected, bacteria transformed by a recombinant plasmid are resistant to ampicillin but not
tetracycline. In contrast those that have picked up an unaltered plasmid will be resistant to both
ampicillin and tetracycline.
Transformed bacteria are identified by growth on ampicillin – this includes bacteria that have
acquired either a recombinant plasmid or a non-recombinant plasmid. Those that have been
transformed with a recombinant plasmid are identified by replica plating (page 179 of your text
book) onto tetracycline where they will not grow. The process is outlined in the diagram. Add brief
notes to explain what is happening.
Non-recombinant plasmid
Recombinant plasmid
ampR
ampR only
and tetR
Tetracycline resistance
Ampicillin resistance
Plasmid
Inserted gene, e.g. HGH
Identification of bacteria containing the recombinant plasmid using replica plating (see page 179)
GCE Biology BC
Q
Suggest why some scientists are concerned about the use of antibiotic resistance genes as
genetic markers [2]
Look at Figure 2 on page 177
Q
Describe bacterial conjugation using diagrams if appropriate [3]
Q
Outline the advantages to micro-organisms of taking up DNA from the environment [1]
Q
Explain how Griffith’s experiment on pneumonia in mice showed that bacteria can acquire
and incorporate DNA from their surroundings [2]
Q
Are Griffith’s experiments on mice an example of bacterial conjugation? Explain your
response [2]
Genetic Engineering case studies
The following case studies illustrate the techniques used and some of the issues involved in genetic
engineering of organisms. Insulin and Golden Rice™ are the examples specified. I actually
completed the insulin procedure as an undergraduate practical when at university in 1998 so you
see how long the technique has been around!
The others are extension material to further illustrate some of the techniques (the basic
techniques are similar, though the fine detail may vary). Bear in mind that you may be given a
synoptic question in the exam where you use your knowledge of the case studies to answer Qs in a
novel context.
Human Insulin
Type 1 diabetes is caused by an inability of the pancreas (specifically β cells of the Islets of
Langerhans) to make insulin, with consequent lack of control over blood sugar level and ensuing
serious health problems. Traditionally diabetes has been treated with insulin preparations obtained
from the pancreases of slaughtered farm animals (cattle for bovine insulin and pigs for porcine
insulin). Unfortunately such animal insulin is not identical to human insulin, is costly to prepare and
purify and some people object on religious or moral grounds. Consequently biotechnologists saw
insulin as a suitable candidate for genetic modification.
In the 1980s, the options were a little limited – the human genome had not been sequenced so
finding a gene of 153 bases (51 amino acids x 3 bases per amino acid) really was needle in a
haystack territory; constructing a DNA base sequence based on the amino acid composition of
insulin was possible, but impractical; isolating mRNA from β cells and reverse manufacturing DNA
using reverse transcriptase was the best option.
Relatively large quantities of mRNA are present in β cells of the pancreas and this was isolated
from the other mRNA based on size. Reverse transcriptase was used to make a complementary
copy (equivalent to the base sequence in the original template DNA strand). From this single-
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stranded DNA, a double-stranded DNA was made by adding DNA polymerase and a supply of
nucleotides.
The resulting cDNA is a copy of the original gene that only needed sticky ends adding. Plasmids
from Escherichia coli were cut with a restriction enzyme, producing sticky ends. When these
plasmids and cDNA were mixed, some took up the gene (hydrogen bonding between the
complementary sticky ends), leaving a small nick that was sealed with DNA ligase to produce a
recombinant plasmid. Note: Some plasmids reseal without taking up the insulin gene, so a way of
identifying recombinant plasmids, or rather the E. coli cells transformed with recombinant
plasmids is required.
When plasmid is mixed with E. coli, some take up the plasmid in the presence of Ca2+ ions. The
transformed bacteria, i.e. those capable of producing insulin were identified using an antibody
reaction.
Q
What cells provided the original mRNA? [1]
Q
Why were these cells suitable? [1]
Q
How is cDNA produced? [2]
Q
How can we tell which bacterial cells have taken up a recombinant plasmid? [5]
Complete the following diagram to describe the main steps in genetic modification of E. coli with
the human insulin gene.
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Isolation of mRNA and production
of cDNA
Isolation of plasmid and cutting
with restriction enzyme
Gene splicing to produce a
recombinant plasmid
Genetic modification of Escherichia coli so that it can synthesise human
insulin
Golden Rice™
Worldwide many people are at risk of disease as a result of dietary deficiency caused by
restricted diet and reliance upon a staple food. The solution may appear simple – do not rely upon a
staple and have a varied diet that includes a wide range of foods from plants and animals. This may
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be impractical as it is too costly for many in underdeveloped countries. A more viable alternative
might be to use a more nutritious crop variety, or if one is not available, to improve existing
varieties. This approach has been taken with rice.
White rice is a staple food for many people. It contains carbohydrate and protein, but is lacking in
some essential micronutrients such as β-carotene or pro-vitamin A. Lack of vitamin A is associated
with blindness, increased disease risk and early death in children – the WHO estimate that as a
result of vitamin A deficiency, between 250,000 and 500,000 children go blind each year and that
the immune system is compromised in up to 40% of children under five, making them more
susceptible to childhood disease. Rice has all the genes necessary to synthesise β-carotene in its
endosperm (the bit that is eaten), but some of the genes are switched off so that some of the key
enzymes in the synthesis pathway are not expressed.
Golden Rice™ is a genetically modified variety of rice that is able to produce β-carotene in its
endosperm. A recombinant plasmid from Agrobacterium tumefaciens containing genes coding for
phytoene synthase (source: daffodils) and carotene desaturase (source: Erwinia uredovora) was
used to infect rice embryos. These were grown on and self-pollinated. The genes in the
recombinant plasmids were moved into the chromosomes of the rice embryos and all resulting cells
had the genes which were then expressed, meaning that the endosperm was able to produce and
store β-carotene, giving it a yellow-orange colour.
Numeracy alert!
Q. The vitamin A RDA for infants is 300 μg, although only 150 μg provides adequate for normal
health. The vitamin A equivalency ratio of β-carotene is 12:1, i.e. it takes 12 units of β carotene
to make one unit of vitamin A. Assuming that the first generation Golden Rice™ contained 1.6 μg
β-carotene g-1, how much rice would need to be consumed to satisfy the RDA? Show your working
out.
Q. A second generation of Golden Rice™ was produced using genes from maize and rice. This had a
much improved β-carotene content of 31 μg g-1. How much rice would need to be consumed to
satisfy the RDA? Show your working out.
Q. Children in target countries typically eat 100–200 g of rice a day-1. Will this provide sufficient
vitamin A?
Additional example: Human Growth Hormone
Human growth hormone is a peptide hormone, 191 amino acids long. It is made and secreted by the
pituitary gland and, as its name implies, it is involved in growth. It stimulates height growth in
children and adolescents and has a number of other effects on the body:
 Strengthens and increases the mineralisation of bone
 Increases muscle mass
 Increases protein synthesis
 Affects blood sugar level, by reducing glucose uptake by the liver and increasing
gluconeogenesis
It is used clinically to increase growth in children with short stature; in the treatment of musclewasting conditions; to promote weight loss in obese patients. It has also been used by athletes and
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body builders, though it is banned by the IOC (International Olympic Committee) and other
sporting bodies. Originally it was obtained from the pituitary glands of cadavers but genetically
engineered HGH became available in the 1980s.
Complete the following diagram, which summarises the main stages in genetic modification of E. coli
to produce human growth hormone.
pBR322 has a restriction
site for BamH1 within the
tetR gene
Gene splicing to produce
recombinant plasmids
Q. Suggest why recombinant HGH is preferable to that sourced from the pituitary of human
cadavers.
Vegetable Rennin
Rennin is an enzyme produced by the stomach of young mammals. It helps coagulate milk protein so
that it is retained in the stomach for digestion. Traditionally rennet, an extract of calf stomach
containing rennin, is used is cheese production – the combined action of the rennin and lactic acid
bacteria help curd production and its separation from the whey. The fungus Aspergillus niger has
been genetically modified so that it now carries and expresses the gene for chymosin (= rennin).
This genetically engineered enzyme is widely used in vegetarian cheese production.
GCE Biology BC
Q. Suggest advantages of using a genetically modified organism such as A. niger as a source of
rennin.
Genetically Engineered Farm Animals
The possibility of using genetically modified animals to produce proteins became a reality in the
mid to late 1980s. Tracy the sheep was the first transgenic farm animal and produced milk where
more than 50% of the protein was human alpha 1 antitrypsin. Clinical trials using sheep-derived
alpha 1 antitrypsin are now underway for the treatment of cystic fibrosis (in the USA and UK) and
congenital emphysema (in the UK). Another sheep (Polly) has been genetically modified to produce
the blood clotting protein Factor IX (note: there are many different blood clotting factors, not
just Factor VIII).
Q Suggest the advantage of using a genetically modified farm animal to produce proteins such as
alpha 1 antitrypsin and blood clotting factors.
Herman the bull (1990–2004) was the first transgenic
bovine. Early embryo cells were microinjected with the
human gene for lactoferrin – a protein that improves
resistance to the bacteria that cause mastitis (an
udder infection that reduces milk yield). The gene was
successfully passed into calves sired by Herman.
Q Suggest why the expression of the lactoferrin
gene in cattle might be an advantage.
Herman the bull, the first transgenic bovine.
GCE Biology BC
Q a.
Complete the following description by inserting appropriate words or phrases in the spaces.
In the 1980s bacteria were genetically modified to produce human insulin. The gene could
not be extracted directly from the human genome. Instead mRNA coding for insulin was
extracted from __ _____ of the pancreas. This mRNA was used to make a complementary
DNA copy using the enzyme ___________ _______________. The base sequence in the
resulting single-stranded DNA is equivalent to the base sequence in the original _________
DNA strand.
From the single-stranded DNA, double-stranded DNA was made by adding the enzyme ____
_______________ and a supply of activated nucleotides. Sticky ends were added to the
cDNA so that they could hydrogen bond to complementary sticky ends on prepared plasmids
taken from Escherichia coli. A _______ enzyme was used to seal the small nick in the DNA to
produce a recombinant plasmid. E. coli bacteria that took up the recombinant plasmid were
able to produce human insulin. (5)
b. How is mRNA different from DNA? (3)
c. Genetic markers are often used to determine if bacteria have taken up recombinant plasmids.
i. What is a genetic marker? (2)
ii. Describe how replica plating is used to identify bacteria that have been transformed with a
recombinant plasmid.(5)
iii. Are there any concerns about the use of genetic markers in genetic engineering? Explain (2)
(Total = 17)
GCE Biology BC