Name:
Cyan Fluorescent Protein (CFP) Expression Lab
Using E.Coli Cells As Protein Factories:
In today’s lab you will try to make E.Coli cells glow by putting a gene for Cyan
Fluorescent Protein (“CFP”) into them.
This process is called “ bacterial transformation”
and is done by heating the cells to a high temperature in order to make their
membranes leaky so that the CFP DNA can get inside.
If we are successful, the cells
should glow after a few days when we shine UV light on them.
This process is what
scientists use to get large amounts of a specific protein they want in a very short
amount of time.
They use the bacteria as little protein factories!!!
E.Coli Cell
transformation
CFP
DNA
E.Coli
DNA
42˚C
CFP
DNA
Antibiotics are Bacteria-Killers:
Ampicillin is an antibiotic, meaning it kills bacteria cells like E.Coli.
lots of different antibiotics that we use to kill different kinds of bacteria.
There are
People take
antibiotics to treat infections like pneumonia, but sometimes the antibiotic doesn’t work
and they have to try a different one.
resistant to the antibiotic.
This is because the bacteria has become
These resistant bacteria have a gene that makes a protein
which protects the cell from the antibiotic.
Bacteria can get these genes naturally from
sharing their DNA with each other, but scientists also can also introduce these genes to
create special strains of bacteria that will grow under conditions where others cannot.
Hunting Down the Ampicillin Resistance Gene:
In this lab you will work with bacteria that are resistant to Ampicillin.
is to hunt down the gene that makes these cells resistant.
DNA or the CFP DNA?
Your job
Is the gene on the E.Coli
In order to figure this out, you will attempt to grow cells on 2
agar plates, one containing only LB (cell food) and one containing LB and Ampicillin.
On
each plate, you will try to grow cells that have been transformed with the CFP DNA and
“control” cells without the CFP DNA. Your job is to figure out which samples will grow
on which plates.
Developed by Sharlene Denos for The Center for Physics of Living Cells Outreach Program
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Here’s What You Need To Do The Experiment:
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An ice bucket
1 tube of CFP DNA (on ice)
1 empty tube labeled “control” (on
ice)
1 tube of E. coli bacteria (on ice)
1 tube of LB broth
A micropipette with sterile tips
1 LB agar plate without Ampicillin
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1 LB agar plate with Ampicillin
1 box of micropipette tips
1 cell spreader with sterile tips
A Biohazard waste container
A shaking incubator at 37˚C
An incubator at 37˚C
UV lamp
Experimental Protocol
Before you begin, write your initials on the tops of the tubes labeled “CFP” and
“control” and on the top of each of your LB agar plates. Also, draw a line down the
center of each agar plate. Write “CFP” to the left of this line and “control” to the
right of it. IMPORTANT: USE A FRESH PIPET TIP EACH TIME YOU PIPET TO AVOID
CROSS-CONTAMINATION OF YOUR SAMPLES!!!!!
1. Pipet 20 microliters of E.Coli cells into the tube containing your CFP DNA.
2. Pipet 20 microliters of E. Coli cells into the tube labeled “control”.
3. Place both tubes back in the ice for 5 minutes.
4. Place both tubes in a 42˚C water bath for exactly 30 seconds and then immediately
put them back in the ice for 2 minutes.
5. Pipet 80 microliters of LB broth into each of your tubes and then place them in the
37˚C shaker for 5 minutes.
6. Pipet 50 microliters of the “CFP” sample onto the upper left corner of each plate and
50 microliters of the “control” sample onto the upper right corner of each plate.
7. Obtain a fresh tip for your cell spreader and use it to spell your first initial using
the “CFP” sample on each plate. Now use the spreader to spell your last initial with
the “control” sample on each plate. REMEMBER TO GET A FRESH TIP BEFORE YOU MOVE
ON TO THE NEXT SAMPLE ON EITHER PLATE!!!!
10. Allow the plates to soak up your cell solution for 20 minutes before putting them
upside down in a 37˚C incubator.
11. Your cells will need at least 12 hours to grow at 37˚C and another 12 hours to
make enough CFP protein so that you can see them glow.
Developed by Sharlene Denos for The Center for Physics of Living Cells Outreach Program
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Lab Questions
1. What is the reason for each step in the protocol above?
space under each step.
Write your answers in the
2. What variables are you testing for in this experiment? How are you testing for each
of these variables?
5. If the Ampicillin gene is on the E.Coli DNA, what do you expect your results to look
like? Which samples will grow on which plates? Indicate your answer using + for
growth and – for no growth on the diagram below.
CFP
Control
CFP
LB agar plate
Control
LB agar plate with Ampicillin
6. If the Ampicillin gene is on the CFP DNA, what do you expect your results to look
like? Which samples will grow on which plates? Indicate your answer using + for
growth and – for no growth on the diagram below.
CFP
Control
CFP
LB agar plate
Control
LB agar plate with Ampicillin
Developed by Sharlene Denos for The Center for Physics of Living Cells Outreach Program
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7. If the transformation reaction worked, what do you expect your results in this
experiment to look like? Indicate your predictions by writing “glow” and “no glow” in
the diagrams above.
8. Does your answer to question 7 change if the Ampicillin gene is on the E.Coli DNA or
on the CFP DNA?
Results
What do your plates look like after incubation at 37˚C for 2 days?
1. Was there cell growth? Indicate your answer using + for growth and – for no
growth on the diagram below.
CFP
Control
CFP
LB agar plate
Control
LB agar plate with Ampicillin
2. Compare your results to your predictions above. Based on these results, is the
Ampicillin resistance gene on the E.Coli DNA or the CFP DNA?
3. Now look at your plates under UV light. Which samples on which plates glow?
Indicate your answer using “glow” or “no glow” on the diagram above. Is this
result surprising? Do you still agree with your answer in number 2 above? If
not, where is the Ampicillin resistance gene and how do you know?
Developed by Sharlene Denos for The Center for Physics of Living Cells Outreach Program
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