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16–19 Student activity sheet 2: Transformation
Feeding a growing world:
pGLO transformation
of E. coli
Student activity sheet
The issue
The Earth’s resources are limited, but the human population is growing
fast. How can we ensure food security – adequate safe, healthy food – for
everyone? Can we modify organisms quickly and safely to improve food
security?
How can we see easily which organisms have been successfully modified
and which have not?
The use of a plasmid vector, pGLO™, bearing a gene for a fluorescent
protein is one answer to these questions.
Introduction
Key terms
Aseptic technique: A protocol used in microbiology to prevent
contamination by unwanted microorganisms and ensure you only grow
the microbes that you want to investigate.
Gene: A length of DNA that may code for one or more proteins or for a
regulatory piece of RNA.
Genetic transformation: A change caused by genes; the take-up and
expression by a cell of a new piece of DNA, often producing a new visible
characteristic in that organism.
Phenotype: The observable characteristics of an organism.
Student activity sheet
Nutrient medium: A medium for growing bacteria that provides the
nutrient requirements of the bacteria. In this protocol the liquid nutrient
medium (broth) and solid nutrient medium (agar) are both LB (Luria
and Bertani), containing an extract of yeast and digested meat products,
thereby providing carbohydrates, amino acids, nucleotides, salts and
vitamins for bacterial growth. The solid medium can also contain
ampicillin and arabinose, called LB/amp/ara agar.
Plasmid: A small ring of DNA often found in bacteria in addition to their
one large circular piece of DNA. These plasmids contain genes for traits
that aid bacterial survival. In nature, bacteria may exchange plasmids,
thus sharing these beneficial genes. This aspect of their physiology can
be exploited in biotechnology to persuade bacteria to take up foreign
DNA, transforming them. The plasmids are vectors.
Recombinant DNA: A piece of DNA created by the combination of DNA
strands from two different sources, such as a jellyfish gene inserted into
a bacterial plasmid vector.
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Vector (in biology): An organism or agent that acts as a carrier. In
gene technology a vector carries and inserts foreign DNA into the host
genome. Examples include plasmids, viruses and some bacteria.
Use of the gfp gene
The gene for GFP was originally obtained from the bioluminescent
jellyfish Aequorea victoria. The gfp gene codes for GFP, a protein that
absorbs UV light and re-emits it as green light (fluorescence). Many
corals also contain this protein, which acts as a molecular sunscreen
by absorbing UV light and re-emitting it at a longer, lower-energy
wavelength, preventing damage to the organism by the UV light. The
fluorescence cannot be seen during daytime, as sunlight is too bright,
but it is visible when UV light is shone onto the organism in dim light.
Derric Nimmo & Paul Eggleston / Wellcome Images
In your first practical investigation, about reducing sugars in potatoes,
you learned that some genetically modified (GM) varieties of potato
have been developed. Your biology course covers the process of genetic
modification, and in this session you will see how the gfp gene for green
fluorescent protein (GFP) is used as a research tool. If the gene has been
taken up it is expressed in the phenotype, and the protein can be seen
under ultraviolet light as a green glow. Researchers can see whether a
plant has been modified (if gfp has been taken up, so have the other genes
in the plasmid) and can also see where exactly in the plant the genes are
being expressed and whether different environmental conditions affect
that expression. It is a widely used, safe and reliable research tool.
Genetically modified fruit flies (Drosophila
melanogaster) expressing GFP in their eyes.
Did you know?
The gfp gene has been around for over 160 million years in the jellyfish Aequorea victoria, which
now lives in the cold waters of the north Pacific Ocean.
Student activity sheet
Scientists in Sweden have harnessed a protein from Aequorea victoria to make miniature fuel cells
for electronics.
The gfp gene has been modified by a biotechnology company in California,
Bio-Rad, to include specific mutations that enhance the fluorescence of
the protein. In this practical protocol you will insert this gene into the
bacterium Escherichia coli, which will enable you to visualise the genetic
transformation as the bacteria express the gfp gene. You will be given the
modified form of the gfp gene, inserted into Bio-Rad’s pGLO™ plasmid.
When the bacteria take up this plasmid they express the gene, making the
protein GFP that fluoresces very brightly, so you can see the green glow
of the bacterial colonies when they are illuminated with a hand-held UV
lamp or a UV pen light. Hence this protocol allows you to observe gene
expression in real time.
Practical investigation: pGLO transformation of E. coli
Aims
In this investigation you will:
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move a gene (gfp) taken from a jellyfish into an E. coli bacterium using
a plasmid as the vector
find out how heat shock treatment helps the process
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understand the importance of using aseptic (sterile) technique
develop an understanding of a technology that may help improve food
security.
Method
Safety
Carry out a risk assessment with your teacher. What hazards
do you predict, and how will control them, that is, reduce the
risks from them to a minimum?
Ampicillin is a penicillin-type antibiotic. It is present in some of
the agar plates in this practical investigation. If you are allergic
to penicillin you should avoid any contact with ampicillin.
The E. coli in this kit belong to the strain HB101 and are nonpathogenic. This strain is selected for work of this kind and
must be grown in an enhanced nutrient medium, therefore
it is not able to ‘escape’ into the environment. However, you
still need to use aseptic technique and observe normal safety
procedures for carrying out practical investigations using
microbes. This will reduce the chances of contamination of
your plates (petri dishes), the lab and yourself, as well as
teaching you best practice.
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When taping plates shut it is important to use just two
pieces of tape, one on each side of the plate. Do not seal the
plate completely, as this creates an anaerobic environment,
which can allow dangerous microorganisms to grow, and
allows drops of condensation to form, which can carry
microorganisms out of the plate.
Report spillages to your teacher/technician.
Follow your teacher’s instructions about disposal of material
(once material is collected, it must be autoclaved before
disposal).
Cover all cuts. Wash your hands with soap before and after
carrying out the protocol and before leaving the lab.
No eating, drinking or applying cosmetics.
Take care when handling disinfectant solution – it can
damage skin.
Student activity sheet
●●
Thoroughly disinfect work surfaces (which must be impervious)
with disinfectant before and after carrying out the protocol.
Work in such a way as to minimise the production of aerosols.
UV light can damage your eyes. Ensure that you point the UV
source downwards and away from yourself and other people,
and do not look into it.
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16–19 Student activity sheet 2: Transformation
Equipment
1 × E. coli starter plate (LB)
4 × poured agar plates (1 LB, 2 LB/amp, 1 LB/amp/ara)
●● 1 mL (cm3) transformation solution (sterile calcium chloride,
CaCl2, solution)
●● 1 mL LB nutrient broth
●● 7 × sterile inoculation loops
●● 5 × sterile micropipettes
●● 2 × microcentrifuge tubes
●● 1 × foam microtube holder
●● 1 × polystyrene cup full of crushed ice
●● 1 × marker pen
●● timer
●● sticky tape
●● container of disinfectant solution for disposal of used
pipettes and loops
●● laminated paper mat pre-soaked in disinfectant solution
●●
●●
Access to:
rehydrated pGLO™ plasmid DNA
●● water bath at 42 °C
●● UV light
●● incubator set at 37 °C (or 28 °C or 21 °C – see step 17 of
procedure)
●●
Student activity sheet
1. Microbiological techniques
If you have not carried out any microbiological practicals before you will
spend one lesson on basic microbiological techniques (aseptic technique,
preparing plates, plating out and using micropipettes). These techniques
– for example never putting a loop down on a surface, disinfecting the
work surface before and after work – are designed to ensure that no
microbes from the environment can get onto a plate, and no microbes
from your work can escape into the environment. In your next lesson
you will carry out the genetic transformation. Note that although in this
protocol you open the starter plate in order to transfer a colony of E. coli,
you should normally leave all plates taped shut.
2. The transformation
Bio-Rad’s pGLO™ plasmids contain:
●● the gfp gene
●● a gene (bla) that codes for the enzyme beta-lactamase, which
confers resistance to the antibiotic ampicillin
●● a gene regulation system that can control the expression of the
gfp gene, which directs the synthesis of GFP, in the transformed
bacterial cells.
In order to get the modified plasmids into the bacterial cells, you will:
●● treat the bacterial cells with transformation solution (calcium chloride)
●● subject the bacteria to heat shock.
The calcium ions neutralise the negative charges of the phosphate
groups in the DNA backbone and in the phospholipids of the cell
surface membrane. This reduces the normally repulsive forces
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between the DNA of the plasmid and the cell surface membrane of the
bacterial cells, allowing the plasmids to enter the cells.
Heat shock increases the permeability of the cell surface membrane
to the DNA of the plasmid.
3. Ampicillin resistance
To enable the transformed cells to grow in the presence of ampicillin
you will:
●● provide them with nutrients, including arabinose
●● incubate them at a suitable temperature long enough for them to
start expressing their newly acquired gene and synthesise the GFP.
When ampicillin is included in the nutrient agar it kills most E. coli –
only transformed bacteria (those that have taken up the plasmid) can
grow, as only the transformed bacteria contain a plasmid that includes
a bla gene to confer ampicillin resistance.
When the sugar arabinose is added to the nutrient medium it switches
on the gfp gene in transformed bacteria, so they can make the GFP
and will glow green under UV light. Transformed bacteria grown on
nutrient agar without arabinose will appear white.
4. Results and further work
In a follow-up lesson you will observe the results and assess the
success of your work, as well as discussing some of the applications
and uses of the gfp gene in real life and considering the use of GM
animals and plants.
Did you know?
Repeated or prolonged exposure to antibiotics may
disrupt the balance of the types of microbes within the
gut microbiota. Such disruptions could lead to a person
not being able to properly regulate appetite and could
contribute to obesity. Similarly, overexposure to antibiotics
may upset the balance of microbes in the gut such that
one type, Clostridium difficile (C. diff) is no longer kept in
check and can cause an infection, leading to persistent and
severe diarrhoea. This infection can be fatal and is often very
difficult to treat. However, faecal transplants (faeces from
a healthy donor) into the colon of the recipient are proving
effective in 90% or more of cases. The donated faeces has to
be screened for pathogens first.
Student activity sheet
E. coli bacteria are part of your gut microbiota. We each have
many species of bacteria and Archaea living inside our gut
lumen (so not inside our body tissues). These organisms help
us to digest food and produce some vitamins, hormones and
other chemicals that help regulate appetite and other aspects
of our metabolism. We may have more microbial cells in the gut,
mouth and vagina and on the skin than we have cells in our
body. However, since prokaryotic cells are much smaller than
eukaryotic cells, the total mass of the microbiota of a human is
estimated to be as much as 2000 g, or 1–3% of body mass.
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16–19 Student activity sheet 2: Transformation
Step-by-step procedure
1. Ensure that your group has all the equipment listed before you start.
Wash your hands. Work over a disinfected, impervious surface. Label
one closed micro test tube ‘+pGLO’ and label the other ‘−pGLO’. Label
the tubes with your group’s name and place them in the rack.
2. Open the tubes and use a sterile transfer pipette to transfer 250 µL
(0.25 mL) of transformation (calcium chloride) solution into each tube.
+pGLO
3. Place the tubes (still open and still in the rack) on crushed ice.
Figure 2 Dispersing colonies in a solution
in a microtube.
4. Using a sterile loop pick up 2–4 large colonies of bacteria from your
starter plate. The bacteria in colonies are actively dividing, hence
actively growing. Choose colonies that are uniformly circular and
have smooth edges. Pick up the ‘+pGLO’ tube and immerse the loop
into the transformation solution. Spin the loop between thumb and
index finger until the entire bacterial colony is dispersed in the
transformation solution (Figure 2). Place the tube back on the ice.
Drop the loop into the disinfectant solution (it will be disposed of
later).
5. Now use another sterile loop and repeat step 4 for the ‘−pGLO’ tube.
6. Examine the solution of pGLO™ plasmid DNA under the UV light.
Note your observations.
7. Immerse a new sterile loop into the stock tube of rehydrated
plasmid DNA. Withdraw a loopful and make sure that there is a film
on the loop (it should resemble the soapy film you get when using
soap solution to blow soap bubbles). Mix this loopful into the cell
suspension of the ‘+pGLO’ tube. Alternatively, use a new sterile
micropipette to transfer 10 µL (0.01 mL) plasmid solution. Again, drop
the loop or micropipette into the disinfectant.
8. Close both tubes (the ‘+pGLO’ and the ‘−pGLO’) and leave them both
on ice for 10 minutes. Make sure the tubes are pushed all the way
down into the foam rack so their bases are in the crushed ice.
Student activity sheet
9. During this 10 minutes label your four agar plates, on their bases, not
their lids, as follows. The agar type will already be labelled:
●● −pGLO on LB agar/your group name
●● −pGLO on LB/amp agar/your group name
●● +pGLO on LB/amp agar/your group name
●● +pGLO on LB/amp/ara agar/your group name.
10. Double-check the temperature of the water bath with the
thermometer. It should be 42 °C. At the end of the 10 minutes, transfer
the foam rack with the two tubes from the ice into the water bath at
42 °C for exactly 50 seconds. This is the heat shock treatment.
11. Then place both tubes, in their rack, back on the ice for 2 minutes.
The times and temperature here are critical. The rapid transfer from
ice to heat and back to ice gives the heat shock, and any time less or
greater than 50 seconds at 42 °C reduces the ability of the bacteria to
take up plasmids.
12. After 2 minutes remove the rack and tubes from the ice and place on
your bench.
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Wash your hands and
disinfect the surface.
3
Add 0.25 mL
transformation
solution to
each tube.
+pGLO
1 ml
750 µL
500 µL
250 µL
100 µL
–pGLO
4
Place open tubes
on crushed ice.
Transfer 2–4
starter colonies
into +pGLO tube
with a loop.
–pGLO
2
Label closed
micro tubes
+pGLO, –pGLO.
+pGLO
1
ice
250 µL
transformation
solution
6
With a fresh loop,
repeat step 4 for
–pGLO tube.
7
Examine the pGLO
plasmid DNA
N
NA
solution under UV.
8
Transfer 0.01 mL
pGLO plasmid into
+pGLO tube with
a new loop.
plasmid
DNA
–pGLO
9
10
Label your four
agar plates with
your group name.
11
Check that water
bath is at 42oC.
Warm tubes
for 50 s.
ice
Close tubes, push
their bases into
the ice and leave
for 10 min.
+pGLO
5
rack
ice
12
Put tubes and
rack on your
workbench.
Place tubes back
on ice for 2 min.
ice
water bath
42oC for 50 sec
14
With a sterile
pipette, add 0.25
mL LB broth
to +pGLO
With a new
pipette, add 0.25
mL LB broth
to –pGLO.
15
Pipette 0.1 mL of
the correct
suspension to
each plate.
+pGLO
–pGLO
+pGLO
Use a fresh sterile pipette
each time.
100 µL
17
Streak each
suspension over
its plate with a
new loop.
18
Tape the plate closed,
stack them and
incubate them at
37°C for 24 h.
19
Disinfect your
work surface and
wash your hands.
+pGLO +pGLO
LB/ampLB/amp/ara
–pGLO
LB/amp
–pGLO
LB
Student activity sheet
–pGLO on LB agar
–pGLO on LB/amp agar
+pGLO on LB/amp agar
+pGLO on LB/amp/ara/agar
250 µL
LB-broth
16
Incubate both
tubes for 10 min at
room temperature.
–pGLO
13
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16–19 Student activity sheet 2: Transformation
13. Open the + pGLO tube, use a new sterile pipette to add 250 µL
(0.25 mL) of LB nutrient broth to the tube and then reclose the tube.
14. Using a new sterile pipette, repeat step 13 for the – pGLO tube.
15. Incubate both tubes for 10 minutes at room temperature. This allows
the bacteria time to recover and to express the beta-lactamase
enzyme that gives it resistance to ampicillin, so that transformed
bacteria can survive on the LB/amp and LB/amp/ara plates.
16. Now gently flick the closed tubes with your finger to mix the contents
within each tube. Using a new sterile pipette for each tube, pipette
100 µL (0.1 mL) from each of the tubes onto the corresponding plates.
17. Use a new sterile loop for each plate and spread the suspensions
evenly over the surface of the agar jelly. To do this quickly skate the
flat surface of the loop back and forth and criss-cross over the agar
surface (Figure 3).
Figure 3 Streaking a plate.
18. Replace the lids on the agar plates and tape them in two places, then
stack the plates and tape them with a different-coloured tape, as
shown in Figure 4. Incubate the plates upside down (this prevents
any condensation from running onto the bacterial colonies) in the
incubator set at 37 °C for 24 hours (or 28 °C for 48 hours or 21 °C for
72 hours, depending on when your next lesson is).
Student activity sheet
19. Leave your disposable swabs or loops and micropipettes soaking in
the disinfectant and thoroughly disinfect your work surface. When
you have finished, wash your hands.
Figure 4 Agar plates labelled, taped closed,
stacked upside down and taped together.
20. Before you observe your incubated plates, predict what you expect
to see.
(a) On which plate(s) would untransformed E. coli bacteria grow?
Explain your prediction.
(b) On which plate(s) would transformed E. coli bacteria grow?
Explain your prediction.
(c) On which plate(s) would bacterial colonies glow green under
UV light? Explain your prediction.
21. Observe the colonies on your incubated plates, without and with
UV light.
●●
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●●
Record your observations by drawing what you see on each plate
and adding notes on your observations.
Compare the relative bacterial growth on each plate.
Record the colour of the colonies and record the number of
colonies on each plate.
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Questions
1. Some of your bacteria (those that have been transformed) glow green
under UV light. There are two possible sources of this fluorescence
– the gfp gene and the protein GFP. How can you tell that it is not the
gene that directly fluoresces?
2. Explain your observations recorded in stage 20 above.
3. For an organism to be genetically transformed, a copy of the new gene
has to be inserted into each of its cells. Explain why populations of
bacteria are well suited to being totally genetically transformed.
4. Suggest why bacteria are useful organisms to enable scientists to see
if the new gene (or trait) is passed to subsequent generations.
5. Why is the gfp gene a useful gene for allowing scientists to see from
the phenotype whether the bacteria have taken up this gene and
become transformed?
6. An organism’s traits (phenotype) are usually caused by a combination
of its genes (genotype) and its environment. Which two factors in the
environment must be present for you to see the green fluorescence of
the transformed bacteria?
7. In most organisms genes can be turned on or off in response to
certain conditions. (You may have learned about the lac operon.)
What is the advantage to an organism in being able to switch genes on
or off?
Did you know?
For most of us our gut is colonised with microbes at birth.
Babies are born head-down so they pick up microbes from
the mother’s anus. They will also take in microbes from the
mother’s skin during suckling and cuddling, and from contact
with other surfaces. Babies born by caesarean section do
not acquire the mother’s gut microbes at birth so their gut
microbiota take a little longer to become established.
Student activity sheet
The hundreds of different species of microbes in the human
gut together have more genes than are present in the human
genome. Many of these microbial genes encode proteins that
we use and rely on for healthy functioning of our metabolism.
Some scientists say we should therefore consider ourselves
to be superorganisms. Other examples of superorganisms
include bee colonies, ant colonies and termite colonies.
Certain foods, such as live yoghurt, are recommended
because they contain ‘friendly’ or ‘good’ bacteria that we
need in our gut. However, if you do not eat a healthy diet,
these bacteria will not thrive. Diets with lots of vegetable
matter, such as leeks, promote the growth of the bacteria we
need in our gut.
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