Name:
Per:
Bacterial Transformation:
Creating E. coli that Glow
PA Standards:
3.3.10C Describe how genetic information is inherited and expressed
3.3.12C Explain gene inheritance and expression at the molecular level
Introduction
Advances in biotechnology have given us the power to create organisms that would normally not
occur in nature by manipulating DNA. In this lab, you will use a gene found in a jellyfish
(Aequoria Victoria) that gives these jellyfish the ability to fluoresce and glow in the dark. You
will take this gene and put it into cells of the common bacteria, E. coli. If the E. coli take up the
gene and express it by making the protein, they will also glow in the dark under ultraviolet light!
This process is called genetic transformation: the insertion of a new gene into an organism, in
order to change the characteristics of the organism. Remember from your studies of DNA that:
New DNA → New RNA → New Protein Produced → New Trait
Why use E. coli?
 To genetically transform an entire organism, you must insert the new gene into every cell
in the organism. Which organism would be most convenient for us to use?
o One composed of many cells or one composed of a single cell? _____________
 Once an organism has been genetically transformed, it should be able to pass on the new
DNA (and the new trait) to all of its offspring. To determine if this is true, which type of
organism would be most convenient for us to use?
o Each new generation develops and reproduces quickly (hours or days) or each new
generation develops and reproduces slowly (months or years)? _________________
 Safety is another important consideration in choosing an experimental organism. If we
are not sure what the effects of our experimental treatment will be, what characteristics
would be important for the experimental organism to have (or not have)?
_______________________________________________________________________
 Based on the above considerations, which would the best choice for a genetic
transformation?
○ Bacterium ○ Fruit fly ○ Mouse ○ Fish
Explain your choice:
______________________________________________________________________
Bacteria are small and easily contained in petri dishes on growth medium called agar. They
reproduce quickly; their population size doubles every 20 minutes under ideal conditions. They
are also relatively inexpensive to obtain and keep alive. These cells can be killed with chemicals
such as alcohol and bleach, as well as antibiotics such as
penicillin and ampicillin. All of these characteristics make
them ideal for use in a genetic transformation experiment.
Petri dish with agar
*Adapted from the pGLO™ Bacterial Transformation Kit, 166-0003EDU, Biotechnology Explorer (TM)
instruction manual, Rev. E. copyright 2005 by Bio-Rad Laboratories, Life Science Education. 1-800-4BIORAD (800-424-6723), www.explorer.bio-rad.com
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Bacterial DNA
Bacteria such as E. coli have no nucleus, but they do have a large, circular strand of DNA. In
addition, they have tiny circles of “extra” DNA called plasmids. Plasmids usually contain a few
genes that are beneficial for the bacterium, such as genes that allow the cell to resist and survive
treatment with antibiotics. Bacteria are able to exchange plasmids, so they can pass these
genes to other bacteria – even to bacteria of different species. This is one of the reasons bacteria
can quickly develop resistance to antibiotics.
DNA
PLASMIDS
BACTERIAL
CELL WALL
& MEMBRANE
FLAGELLUM
E. coli bacterium
Plasmids make useful tools for genetic engineering
Scientists discovered that they could use these plasmids as a vehicle to carry genes into bacterial
cells, if they engineered the plasmids to contain the desired genes. They cut open the plasmids
using restriction enzymes, add the chosen genes, then splice the plasmids closed. Under certain
conditions, we can induce bacteria to take up the plasmids through their cell walls, and adopt the
plasmids (along with the genes they carry) as part of their own genetic material. This is the basic
procedure for genetic transformation, also known as genetic engineering.
In this lab, you will use a plasmid called pGLO, which has been engineered to contain several
genes that will be useful to us. Below is a “map” of the plasmid, showing the three genes that
interest us.
Ampr
Gene for
antibiotic resistance
pGLO
plasmid
ARA
Gene for turning on GFP when
arabinose sugar is present
GFP
Gene for Green Fluorescent
Protein; glows green under
UV light
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Ampr Gene
Genetic selection refers to the process whereby colonies can be selected for their ability to grow
in various conditions. The Ampr gene codes for a protein that enables bacteria to survive
treatment with the antibiotic ampicillin. Any bacterium that has this gene will be able to grow in
the presence of ampicillin (ampicillin resistance), while bacteria without this gene will not.
ARA and GFP Genes
These genes work together to create the “glow-in-the-dark” effect. Gene expression in all
organisms is carefully regulated to allow for adaptation to differing conditions and to prevent
wasteful overproduction of unneeded proteins. When arabinose sugar is present in the
bacterium’s environment, the ARA gene will be turned on. In normal bacteria, this gene initiates
production of a protein that enables them to digest arabinose as a food source. For our plasmid,
scientists have inserted this gene as an “on” switch for the GFP gene; when arabinose sugar is
present, instead of initiating production of an arabinose enzyme, ARA activates the GFP gene so
that the glow-in-the-dark protein is manufactured by the bacteria. If there is no arabinose sugar
present in the bacteria’s environment, the GFP gene will not be transcribed and no glowing
protein will be made. This mechanism is referred to as gene regulation.
Adding genes to bacteria
In order to get the engineered pGLO plasmid (and the gene to produce the Green Fluorescent
Protein) into the E. coli bacteria, we have to perform several steps:
Add pGLO plasmids to a CaCl2 solution containing E. coli cells. It is thought that the
Ca2+ cation of the transformation solution neutralizes the repulsive negative charges of
the phosphate backbone of the DNA and the phospholipids of the cell membrane,
allowing the DNA to enter the cells.
Heat shock the cells which increases the permeability of the cell membrane so the
plasmids will pass through.
Revive the cells that survive the heat shock process by incubating with nutrient broth to
allow the bacteria to express the new genes.
Plate the cells onto nutrient agar so the cells that received the plasmid can reproduce
(passing on the new DNA to their offspring) and be seen.
When we perform these steps, we will end up with millions of bacteria that survive the process,
and only a few bacteria that have successfully received the pGLO plasmid. How might we
separate the genetically modified bacteria from the ones that didn’t take up the new DNA? (hint:
read over the features of the pGLO plasmid)
______________________________________________________________________________
______________________________________________________________________________
Guiding Questions
How can we use molecular biology to change the characteristics displayed by an organism?
______________________________________________________________________________
______________________________________________________________________________
What ways do you think this technology could be applied in our daily life?
______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
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Vocabulary
Genetic transformation: the insertion of a new gene into an organism, in order to change the
characteristics of the organism
Plasmid: circles of “extra” self-replicating DNA usually containing a few genes that are
beneficial for the bacterium; often convey antibiotic resistance
Genetic selection: the process whereby colonies can be selected for their ability to grow in
various conditions; antibiotic resistance is a type of genetic selection
Agar: medium containing a nutrient mixture of carbohydrates, amino acids, nucleotides, salts, and
vitamins that provides a solid matrix to support bacterial growth
Green fluorescent protein (GFP): protein originally isolated from a bioluminescent jellyfish; the
unique three-dimensional conformation of GFP causes it to resonate when exposed to ultraviolet
light and give off energy in the form of visible green light
Lawn: the appearance of bacterial colonies when all the individual colonies on a petri-dish agar
plate merge together to form a field or mat of bacteria
Gene regulation: the control of gene expression in all organisms to allow for differing conditions
and to prevent wasteful overproduction of unneeded proteins; an example is the arabinose operon
Materials
Per lab group:
1 starter LB plate of E. coli strain: HB101
1 LB agar plate
2 LB/amp agar plates
1 LB/amp/ara agar plate
1 microtube sterile transformation buffer
(CaCL2 solution)
1 microtube LB nutrient broth
2 sterile microtubes
7 sterile inoculation loops
1 100-1000 μl micropippettor
1 rack sterile pipette tips
1 foam microtube holder
1 container of crushed ice
1 waste container lined with biohazard bag
1 Sharpie marking pen
1 UV lamp
1 stopwatch
1 roll masking tape
Per class:
1 vial rehydrated pGLO plasmid (on ice)
1 heat block set to 42° C
1 incubator set to 37° C
Safety
Students will employ proper sterile technique in the handling of bacteria. Proper hand washing at
the beginning and conclusion of the laboratory is important. All materials coming in contact with
bacteria should be placed in biohazard bags for decontamination.
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Procedure
1. Label one closed micro test tube +pGLO and another –pGLO. Place them in the foam rack.
2. Open the tubes and transfer 250 µl of transformation solution (CaC12) to each tube using the
micropipettor and a sterile tip.
3. Use a sterile loop to pick up a single colony of bacteria from your starter plate. Pick up the
+pGLO tube and immerse the loop into the transformation solution at the bottom of the tube. Spin
the loop between your index finger and thumb until the entire colony is dispersed in the
transformation solution (with no floating chunks of agar). Place the tube back in the tube rack.
Using a new sterile loop, repeat for the –pGLO tube.
4. Place the tubes on ice.
5. Immerse a new sterile loop into the plasmid DNA stock tube. Withdraw a loopful. There
should be a film of plasmid solution across the ring. This is similar to seeing a soapy film across a
ring for blowing soap bubbles. Mix the loopful into the cell suspension of the +pGLO tube. Close
the tube and return it to the foam rack on ice. Also close the –pGLO tube. Do not add plasmid
DNA to the –pGLO tube. Why not?
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6. Incubate the tubes on ice for 10 minutes. Make sure to push the tubes all the way down in the
rack so the bottoms of the tubes stick out and make contact with the ice.
7. While the tubes are sitting on ice, examine the pGLO plasmid DNA solution with the UV
lamp. Note your observations in Laboratory Observations: Day 1. Also label your four agar
plates on the bottom (not the lid) as follows: Label one LB/amp plate: +pGLO; Label the
LB/amp/ara plate: +pGLO; Label the other LB/amp plate: –pGLO; Label the LB plate: –pGLO.
8. Heat shock. Place both the +pGLO and –pGLO tubes directly into the heat block, set at 42 °C,
for exactly 50 seconds. When the 50 seconds are done, place both tubes back in the foam rack on
ice. For the best transformation results, the change from the ice (0°C) to 42°C and then back to be
rapid. This step is critical for success! Then incubate tubes on ice for 2 minutes.
Ice
42°C Heat Block
50 seconds
Ice
9. Remove the rack containing the tubes from the ice and place on the bench top. Open a tube
and, using a new sterile pipet tip, add 250 µl of LB nutrient broth to the tube and reclose it.
Repeat with a new sterile pipet tip for the other tube. Incubate the tubes for 10 minutes at room
temperature.
10. Tap the closed tubes with your finger to mix. Using a new sterile pipet tip for each tube, pipet
100 µl of the transformation, +pGLO, and control, –pGLO, suspensions onto the appropriate agar
plates you labeled in step 7.
11. Use a new sterile loop for each plate. Spread the suspensions evenly around the surface of the
agar by quickly skating the flat surface of a new sterile loop back and forth across the surface.
12. Stack up your plates and tape them together. Put your group name and class period on the tape
and place the stack upside down in the 37°C incubator overnight.
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Bacterial Transformation: Creating E. coli that Glow
Laboratory Observations: Day 1
1. Examine the E. coli bacteria growing on the stock culture plate. Describe:
distribution of colonies on the plate:
color of the colonies in room light:
under UV light:
size of the colonies:
number of bacterial colonies (1 spot = 1 colony) on the plate:
2. Why do we heat shock the bacteria in the transformation solution?
3. Why do we add nutrient broth to the cells after heat shocking them?
4. Which plates serve as the control? What purpose does a control serve?
5. Predict what will happen on each of the plates: Will there be bacterial growth? Which plates
will contain genetically transformed bacterial cells? Which plates will glow?
-pGLO
LB
-pGLO
LB+AMP
pGLO Bacterial Transformation
+pGLO
LB+AMP
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+pGLO
LB+AMP+ARA
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Laboratory Observations: Day 2
Observe the results on your plates under normal room lighting; then hold the UV lamp over the
plates and observe. Sketch what you observe on each plate and explain in the space beside the
diagram why this happened. Describe the distribution of colonies on the plate, the color of the
colonies in room light and under UV light, the size of the colonies, and the number of bacterial
colonies (1 spot = 1 colony) on the plate as you did for the starter plate on day 1.
-pGLO
LB
-pGLO
LB+AMP
+pGLO
LB+AMP
+pGLO
LB+AMP+ARA
The applications of this technology are far-reaching. Would you be in favor of using bacteria to
produce proteins that could be used for medicine? How about genetically modified crops, to
produce better-tasting or disease-resistant foods? What about genetically modified animals, to
grow organs that wouldn’t be rejected by the human body, or pork with less fat? Would you be in
favor of using technology that enabled us to cure diseases in babies before they are born? What
about altering other characteristics, such as height, weight, or math ability?
*Adapted from the pGLO™ Bacterial Transformation Kit, 166-0003EDU, Biotechnology Explorer (TM)
instruction manual, Rev. E. copyright 2005 by Bio-Rad Laboratories, Life Science Education. 1-800-4BIORAD (800-424-6723), www.explorer.bio-rad.com
pGLO Bacterial Transformation
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Extension:
Advanced Analysis of Results: Transformation Efficiency
In many experiments, it is important to genetically transform as many cells as possible. For
example, in some types of gene therapy, cells are collected from the patient, transformed in the
laboratory, and then put back into the patient. The more cells that are transformed to produce the
needed protein, the more likely the therapy will work.
The transformation efficiency tells a researcher how effective the procedure is at getting DNA
molecules into the bacterial cells. In this lab, the transformation efficiency represents the total
number of bacterial cells that express the green protein, compared to the amount of DNA used in
the experiment. You can calculate your group’s transformation efficiency in this lab by using the
following formula:
Transformation efficiency =
Total number of cells growing on the LB/AMP/ARA plate
Amount of plasmid DNA (in bacterial cells) spread on the agar plate
Use the space below to calculate the transformation efficiency for your group. The units for
transformation efficiency are transformants/g, and the numbers are usually large, so scientists
frequently use scientific notation when describing these numbers. You will also need the
following information about the plasmid DNA we used:
1. 10 l of pGLO DNA at a concentration of 0.08 g/l was added to the bacterial cells
2. 100 l of cells containing the pGLO were spread on the petri dish, from a total volume of
510 l of bacterial cells that received the pGLO DNA
Number of colonies growing on LB/amp/ara plate: ______________
Amount of plasmid DNA in the bacterial cells spread on the plate: _____________
Using the above formula, our group’s transformation efficiency is: ______________
1. Biotechnologists are in general agreement that the transformation protocol that you have just
completed generally has a transformation efficiency of between 8.0 x 102 and 7.0 x 103
transformants per microgram of DNA. How does your transformation efficiency compare with
these numbers?
In the table below, report the transformation efficiency of the other teams in the class.
Team
Team
Efficiency (tranformants/g)
Efficiency (tranformants/g)
2. What might cause different groups to have different transformation efficiencies?
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Teacher Notes: pGLO Bacterial Transformation
Time for completion: 2 x 45 minute periods separated by overnight incubation
Target Grade Level: Advanced high school biology; AP Biology lab #6A
Objectives
Students will be able to describe, perform, and analyze the results of the genetic engineering
technique of bacterial transformation and relate this knowledge to applications and impacts in
today’s world.
Major Concepts:
Students should know what a gene is and should understand the relationship between genes and
proteins. Students would also benefit from knowledge of the idea and practices of sterile
technique, both for the success of the lab and for the safety of the students. The host organism for
this lab is E. coli HB101, a nonpathogenic bacteria strain. Components of the lab that have been
in contact with bacteria should be collected for sterilization. The lab requires lead time on the
part of SIM to prepare the agar plates and an overnight incubation of starter plates before the day
of the lab. The lab itself consists of a very full 42 minute period for day 1, an overnight
incubation, and recording and analysis of results on day 2.
Preparation:
Nutrient agar plates have been prepared for this lab. These plates should be refrigerated in the
plastic sleeves if several days precede the actual lab. Starter plates must be prepared 24-36 hours
before the transformation laboratory. Using a sterile pipet, rehydrate the lyophilized E. coli
HB101 by adding 250 μl of transformation solution directly to the vial. Recap the vial and allow
the cell suspension to stand at room temperature for 5 minutes. Shake to mix before streaking on
LB starter plates. Each lab team will need their own starter plate as a source of cells for the
transformation. The plates should be streaked for single colonies as shown below:
Place the plates upside down inside the incubator overnight at 37° C. Use for transformation
within 24-26 hours. Do Not refrigerate before use.
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On the day of the lab prepare the pGLO plasmid by adding 250 μl of transformation solution
into the vial of lyophilized pGLO plasmid DNA with a sterile pipet. If possible store the hydrated
in the cold.
Before the laboratory aliquot 1 ml of transformation solution (CaCl2) and 1 ml LB nutrient
broth into separate color-coded 2 ml microtubes. Label the tubes.
Each student work station should contain 1 starter plate, 4 poured agar plates (1 LB, 2 LB/amp,
1 LB/amp/ara), tube of transformation solution, tube of LB nutrient broth, inoculation loops,
micropipettor, tips, foam rack, containers of ice, and a sharpie. The shared materials include the
rehydrated plasmid DNA, the heat block set to 42° C, and the incubator set to 37° C.
Typical results:
–pGLO, LB plates: An even lawn of bacteria is present on this plate. The lawn appears offwhite. (this is the same plate condition and bacteria as the starter plate)
–pGLO, LB/amp plates: No bacterial growth present on this plate. (no plasmid; no antibiotic
resistence)
+pGLO, LB/amp: Many transformed colonies of bacteria (optimally ~75). Colonies appear
white. (Plasmid was taken up by these bacteria rendering them resistant to ampicillin and thus
able to grow)
+pGLO, LB/amp/ara: Many transformed colonies of bacteria (optimally ~75). Colonies appear
white in the room light but appear bright fluorescent green when exposed to UV light. (Plasmid
was taken up by these bacteria rendering them resistant to ampicillin and thus able to grow.
Colonies glow because the presence of arabinose allows for the expression of the green
fluorescent protein.)
Sample calculation:
Transformation efficiency
1. Determining the number of green fluorescent colonies: Count the colonies on the plate. (for
numerator)
2. Determine the amount of pGLO plasmid DNA in the bacterial cells spread on the plates:
a. Determine total amount of DNA
DNA (μg) = (concentration of DNA ( μg/ μl) x (volume of DNA in μl)
You used 10 μl of plasmid at a concentration of 0.08 μg/ μl.
Total DNA = 0.08 μg/ μl x 10 μl = 0.8 μg total DNA
b. Determine the fraction of this total DNA spread on each plate.
Fraction of DNA used = volume spread on plate (100 μl) / total volume in tube (510 μl)
100 μl/510 μl = 0.2
c. Determine how many μg of DNA was spread on the plates:
pGLO DNA spread (μg) = Total DNA used (0.8 μg) x fraction of DNA (0.2)
= 0.16 μg plasmid DNA/plate (for denominator)
3. Determine the transformation efficiency
Transformation efficiency =
Total number of cells growing on the LB/AMP/ARA plate
Amount of plasmid DNA (in bacterial cells) spread on the agar plate
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Answers to questions:
1. Examine the E. coli bacteria growing on the stock culture plate. Describe:
distribution of colonies on the plate
color of the colonies in room light and under UV light
size of the colonies
number of bacterial colonies (1 spot = 1 colony) on the plate.
2. Why do we heat shock the bacteria in the transformation solution?
To increase the permeability of the cell membranes to permit the entry of the plasmid DNA
3. Why do we add nutrient broth to the cells after heat shocking them?
To revive the cells that survive the heat shock process by incubating with nutrient broth to
allow the bacteria to express the new genes
4. Which plates serve as the control? What purpose does a control serve?
A control plate is a guide that is used to help you interpret the experimental results. Both
–pGLO plates are control plates. The LB only –pGLO plate shows that bacteria were
transferred and grew under the incubation conditions used. The LB/amp control –pGLO
plate showing no growth in the presence of ampicillin is compared to the LB/amp +pGLO
plate. This comparison shows that transformation produces colonies that can grow on
ampicillin plates as the plasmid contains the ampicillin resistance gene. Without this control
one would not know if the colonies on the LB/amp +pGLO plate were really transformants.
Extension:
The transformation efficiency section of the lab is optional and is appropriate for the AP lab.
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