BIOTECH Project, University of Arizona
Bacterial Transformation
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TEACHER GUIDE: Bacterial Transformation with Mystery DNA
This teacher guide is provided to give sample answers to questions. Most of the questions are open-ended,
so students may have correct answers that aren't included in this guide. Finally, although the experiment
is set up to yield one correct answer, there are variations in data between students. As long as students
examine their data carefully and can justify their answers based on their data, that's science! Data are
always right and there isn't necessarily a 'right answer'.
Some questions to get you thinking about today’s lab:
What can we use DNA for?
We can use DNA to code for proteins, to identify individuals (like when solving a crime), or to do genetic
engineering by inserting foreign DNA into an organism.
How can DNA be put into bacteria?
There are three strategies for getting DNA into bacteria, which you may or may not want to talk about
with students. Bacteria can insert DNA into each other by CONJUGATION; viruses can insert DNA into
bacteria by TRANSDUCTION; or we can insert DNA into bacteria using chemicals or electricity, which is
called TRANSFORMATION. During this lab, we will 'poke holes' in the bacteria using chemicals,
allowing the DNA to flow into the bacteria- this is called BACTERIAL TRANSFORMATION.
Why would we want to put DNA into bacteria?
We can use bacteria as little 'factories' to make more DNA, as they replicate, or to make protein, by
transforming them with genes for proteins we want to make (like insulin).
How can we tell DNA is in the bacteria once we put it there?
The DNA we insert is shaped in a little circle, called a plasmid. We can put one, two, or more genes in a
single plasmid. One of the genes in the plasmid codes for the ampicillin resistance protein, and thus will
allow bacteria with the plasmid DNA to grow in the presence of ampicillin.
What is a plasmid? What is ampicillin?
A plasmid is a small circle of DNA. Ampicillin is an antibiotic; antibiotics prevent bacteria from growing.
Ampicillin specifically prevents bacteria from making cell walls. Thus, ampicillin will not kill bacteria
(that already have a cell wall), but will prevent bacteria from reproducing (because they can't make new
cell walls).
Materials for each group (students should work in groups of 4):
Tube of mystery plasmid DNA (tubes numbered 1, 2, 3 or 4)
One tube of E. coli bacteria (on ice)
1 LB agar plate
1 LB agar plate with ampicillin for DNA (1 black stripes)
Q-tips or innoculation loops
1
1 tube of LB broth
micropipette
micropipette tips
two steriletransfer pipette
BIOTECH Project, University of Arizona
Bacterial Transformation
Materials to share:
Water bath at 42°C
ice
trash containers/biohazard waste bag
UV lights
Protocol:
1. Using the syringe pipettor and a sterile tip, pipette the DNA solution from your numbered DNA tube into your
E. coli bacteria tube and label the tube according to your DNA number (1, 2, 3, 4). Also mark your tube so that
you will recognize it compared the other groups.
Be sure the students number the top of the tube with which DNA they added to the bacteria.
2. Put tubes on ice for 5 minutes. Why do you think we put the tubes on ice?
To get the DNA into the bacteria, we have to poke holes in them with the chemical calcium chloride
(CaCl2). CaCl2 will dissociate into Ca2+ and 2 Cl-, and the positive charge of the Ca2+ cancels the
negative charge of the DNA, allowing it to cross the cell wall and cell membrane. The holes poked to allow
the DNA in leaves the bacteria leaky. If we don't keep them on ice, they'll 'bleed' to death.
3. In the meantime, each group should get one LB agar plate and one LB agar + ampicillin plate. You will be
plating bacteria with DNA on an LB agar plate and on an LB agar +ampicillian pate. Mark these two plates with
the DNA number on your tube, your group members initials and class period. Also label which plat contains
Ampicillian. Where is the best place to label your plates? What is the control you are conducting
Always label plates on the bottom. The lids can get mixed up accidentally, so if the bottoms are labeled,
the label will stay with the bacteria (which are growing in the bottom). The LB agar plate will look for the
existence of viable bacteria cells. If nothing grows on the LB agar plate then your bacteria are dead and
you cannot expect transformation or growth on the LB agar + ampicillin
4. Put tubes directly from ice into 42°C water bath for 50 seconds. What do you think heating the tubes does?
Heating the bacteria helps open the holes. It's called heat shock. Back to the ice which acts to close the
holes in the cell and keep the DNA inside.
5. Put your tube directly from the water bath onto ice for 2 minutes.
Back to the ice which acts to close the holes in the cell and keep the DNA inside
6. With a sterile transfer pipet, add the LB broth into your tube. Incubate at room temperature or in the warmth
of your hand for 10 minutes. What is the LB broth for? Why can you now allow the cells to warm up to the
temperature of your hand?
The LB (Luria-Bertani) broth is both food and water for the bacteria. It will help make the bacteria
healthy after poking holes in them, shoving DNA into them. Now the bacteria can become metabolically
active and begin making proteins, specifically the amp resistance gene, which they will need this protein
before being placed into the environment of Ampilcillian.
7. With the other transfer pipet, pipet the half the content of your tube onto your LB agar plate and the other half
onto your LB agar + ampicillin plate.
8. Spread the solutions on the plates. Be careful not to stab the agar.
The same Q-tip can be used for both plates as long as it is kept sterile (don't touch it to anything!).
9. Put your plates in a 37°C incubator for 24 hours. Why 37°C?
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BIOTECH Project, University of Arizona
Bacterial Transformation
These bacteria are E. coli, which grow in human intestine. Because they grow in humans, they will grow
best at human body temperature (37°C). You could ask students to calculate what 37°C is in Fahrenheit,
or what 98.6°F is in Celsius.
Challenge for Day 2: What is ampicillin and why do you think we used it in some of the plates?
What do you expect to grow on each of the plates?
LB agar
bacteria + DNA
Would expect to see
bacterial lawns
LB agar + ampicillin
(1 black stripe)
Would expect see colonies,
could count the number of
colonies
Do you expect to see any
difference in bacterial
growth on the two plates?
Yes
What do you think would have grown on these plates if no DNA had been added to these bacteria?
bacteria
(without DNA)
LB agar
LB agar + ampicillin
(1 black stripe)
Would see
bacterial lawns
Would see nothing.
Do you expect to see any
difference in bacterial
growth on the two plates?
Yes
Day 2:
What do you see on your plates?
Students should look at their plates to see what they look like. They may see any of three things:
- little dots, called colonies (each colony starts as a single bacterium because they reproduce
asexually, so each colony is like a house with a family of related bacteria)
- a big smear, called a 'bacterial lawn' (this is like a city of bacterial houses, where there are so many
colonies that we can't tell them apart any more)
- nothing, where no bacteria are growing (ampicillin may kill all of the bacteria or the students may not
have spread their bacteria around the plate correctly, e.g. they may have put it on the liddon't laugh! it happens!)
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BIOTECH Project, University of Arizona
Bacterial Transformation
E. coli are normally white to off white, do any of the bacteria have a different color??
Now look at your plates with UV light. What do you see?
Fill in the table with your data and the class datawhat does each group (#1, 2, 3, 4) see on each type of plate?
Mystery DNA
(number)
LB agar
#1
students should see
bacterial lawns
#2
students should see
bacterial lawns
#3
students should see
bacterial lawns
#4
students should see
bacterial lawns
LB agar + ampicillin
(3 black stripes)
Based on the phenotype,
what is the DNA?
students should see colonies,
they should count the number
of colonies they see and
record this number in their
data table
the bacterial phenotype
is GROWING and
GLOWING GREEN, so
the DNA must be both
the ampicillin resistance
gene and the GFP gene
students should see colonies,
the bacterial phenotype
they should count the number
is GROWING and
of colonies they see and
BLUE, so the DNA must
record this number in their
be the ampicillin
data table
resistance gene and BFP
students should see colonies,
the bacterial phenotype
they should count the number
is GROWING and
of colonies they see and
GLOWING PURPLE, so
record this number in their
the DNA must be both
data table
the ampicillin resistance
gene and the PFP gene
students should see colonies,
the bacterial phenotype
they should count the number is GROWING and RED,
of colonies they see and
so the DNA must be the
record this number in their
ampicillin resistance
data table
gene and the RFP gene
What does each DNA type (1, 2, 3, 4) allow the bacteria to do?
#1 allows the bacteria to GROW and GLOW GREEN
#2 allows the bacteria to GROW and be BLUE
#3 allows the bacteria to GROW and GLOW PURPLE
#4 allows the bacteria to GROW and be RED
Possible follow up questions:
What would the bacteria do on each type of plate (LB agar and LB agar + ampicillin) if you added no DNA?
The bacteria would form a lawn on the LB agar plate. On the plate with ampicillin, the bacteria wouldn't
grow or glow (you would see nothing) because they wouldn't have either the ampicillin resistance gene,
the Green Fluorescent Protein (GFP) gene, etc.
After the students fill in their data tables, I usually talk about the results with them in this order.
I ask the question and help them brainstorm the answers.
After this discussion, students should be able to tell what each DNA type allows the bacteria to do.
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BIOTECH Project, University of Arizona
Bacterial Transformation
1. Where do the bacteria grow best? On the LB agar plates, because it provides them with food, water, and
shelter. Also, the plates were stored at 37°C, which is their favorite temperature.
2. If the bacteria can grow on LB agar so well, why didn't they grow on LB agar with ampicillin? This is an
opportunity to talk about what ampicillin is. The bacteria aren't growing on LB agar with ampicillin because
ampicillin is an antibiotic.
3. If ampicillin is an antibiotic, why doesn't it completely stop the bacteria from growing? Because of the
DNA we added, the bacteria are now resistant. In fact, the DNA we added is called the 'ampicillin resistance
gene'.
4. Do both DNA types have the ampicillin resistance gene? They both should grow, thus they both have the
ampicillin resistance gene.
5. What would happen if no DNA is added? The bacteria could not grow in the presence of ampicillin if they
do not contain the ampicillin resistance gene.
6. #1 DNA contains a gene that codes for Green Fluorescent Protein (GFP) , which is why #1 bacteria
GLOW. What are #1 bacteria able to do? GLOW AND GROW. What are #2 bacteria able to do? GROW.
7. Why do you want to do this kind of GENETIC ENGINEERING experiment? Say you know someone
who is diabetic. They have to take the protein insulin to be healthy. We can put the insulin gene into a plasmid
and then insert that plasmid into bacteria. These bacteria will make insulin for diabetics to use. Before genetic
engineering was invented, we used to have to kill pigs to get their insulin. Now we can use bacteria to make
human insulin (instead of using pig insulin, especially important if someone is allergic to pigs) and we don't
have to kill any animals to do it.
8. Why do some of the bacteria fluoresce only under a UV light? (This should be a review of previous
materials). Different light reactive chemical groups have different excitation wavelengths- some fall in the
range of the light/energy emitted by the UV light. The energy provided by the UV light (in samples 1 and 3)
allow excitation of a light reactive chemical group. These specific excitation wavelengths provide a specific
energy to get the electrons in the light reactive chemical group to a higher energy state. Eventually the electrons
will return to their normal (ground) state and in doing so, emit a photon that carries a different amount of energy
than the original amount absorbed. Samples 2 and 4 only require energy found in white (or visible) light to excite
the electrons to a higher energy state.
Difference between fluorescence and phosphorescence…
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