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Lab - Transformation of E. coli Bacteria with the


pGLO plasmid
I. INTRODUCTION:
Green Fluorescent Protein is used extensively in biology research and
work. It is one among several fluorescent proteins that allow DNA to be
sequenced automatically by lasers. It is used quite frequently to label cells
for studying how organisms develop as embryos and it is a key part of
ongoing stem cell research. The protein itself does not glow but it can be
made to fluoresce with a green light when exposed to ultraviolet (UV) light.
Bacteria grow well with the food source Luria Broth (LB). You can grow
them either in liquid LB or on the surface of a petri dish with a mixture of LB
and agar that solidifies into a thick gel. Antibiotics are sometimes also added
to the agar to help control the growth of bacteria that are unwanted at the
end of the experiment. Most antibiotics either kill the bacteria or keep them
from reproducing. We will be using an antibiotic called ampicillin (amp).
Lab groups will prepare bacterial cells using calcium chloride to help the
small plasmid circles of DNA get through the membrane of the cell. Then,
you will get a plasmid into half of these cells by weakening the membrane
further with heat shocks (cold, hot cold). A
small percentage of the millions of bacteria in
your sample should successfully take in
plasmids.
Each plasmid contains a gene coding for
green fluorescent protein (GFP), a gene
coding for an enzyme (beta-lactamase) that
breaks down the antibiotic ampicillin and a
region that is like a lock or on/off switch for
the green fluorescent protein gene. The
on/off switch or lock is called an inducible
operon that is usually in the off or locked
position. The sugar arabinose (ara) is the key that opens the lock and turns
on the GFP region of the plasmid.
You will put your transformation mixes (and your control sample) onto
petri dishes to compare what happens with ones that have the plasmid to
ones that have not received the plasmid. After the bacteria have had a
chance to grow we will look at them under normal and ultraviolet light. It will
take at least a day to show results.

Pre-lab Questions
1. What is a plasmid?
2. What is a gene?
3. What is luria broth (LB)?
2009HHMISummerWorkshop,Dept.ofMolecularBiology,PrincetonUniversity

4. What is agar?
5. What is ampicillin (amp)?
6. What is ara?

2009HHMISummerWorkshop,Dept.ofMolecularBiology,PrincetonUniversity

Hypothesis:
What do you think will be the results of your experiment? Will the four plates
turn out the same? If any bacteria grow what do you think they will look like; the
original colonies on the starter plate, something different? Make a prediction of
what will happen for each of your four plates.
LB DNA (no plasmid)
LB/amp DNA (no plasmid)
LB/amp +DNA (plasmid)
LB/amp/ara +DNA (plasmid)

Data
Observe the results you obtained from the transformation lab under normal
room lighting. Then turn out the lights and hold the ultraviolet light over the
plates.
Observe and draw what you see on each of the four plates carefully. Draw then
as they appear under UV light in an appropriate color. Record your data to allow
you to compare observations of the + DNA cells with those you record for the
non-transformed E. coli. Write down the following observations for each plate.

Control Plates

-DNA LB/amp

-DNA LB

Transformation Plates
Plate

Drawing

Amount of
Growth

Color Under UV

# of colonies

+DNA LB/amp

+DNA
LB/amp/ara

Analysis
1. Do you have any colonies on your LB/amp plate(s)? If you do not take a look
at the plates from several other groups, most of them will probably have
some colonies on at least one of those plates. Given the fact that ampicillin
kills normal bacteria (or at least keeps them from forming visible colonies),
what does this evidence show about the success of transformation of the
bacteria on the LB/amp plate(s)?
2. If the genetically transformed cells have acquired the ability to live and
reproduce in the presence of the antibiotic ampicillin, then what might be
inferred (implied) about the other genes on the plasmid that you used in your
transformation procedure? Did they get those genes as well? Why or why
not?
3. Antibiotic resistance is often used as a way to separate successfully
transformed bacteria from those that are not. Why might this technique be
particularly useful when trying to get bacteria to make a hormone such as
human insulin instead of a colored protein?
If a fluorescent green color is observed in the E. coli colonies when exposed to
UV light then a new question arises. What are the possible sources of
fluorescence within the colonies when exposed to UV light?
4. Recall what you observed when you shined the UV-light source onto a
sample of original plasmid DNA, or do so now, and describe your
observations.
5. Which of the possible sources of the fluorescence can now be eliminated?
6. So what is the source of the fluorescence and what does that have to do with
the plasmid?

7. Look again at your four plates. Do you observe some E. coli bacteria growing
on a plain LB plate?
8. From your results, can you tell if these bacteria are ampicillin resistant by
looking at them on the LB plate? Explain your answer.
9. How would you change the bacterias environment--the plate they are
growing on--to best tell if they are ampicillin resistant?
Very often an organisms traits are caused by a combination of its genes and its
environment. Think about the green color you saw in the genetically
transformed bacteria:
10. What two factors must be present in the bacterias environment (NOT
inside the bacteria) for you to see the green color? (Hint: One factor is in the
plate and the other factor is in what you use when you look at the bacteria).
11. What do you think each of the two environmental factors you listed above
is doing to cause the genetically transformed bacteria to turn green?
Conclusion and Explanation On a separate sheet of paper TYPE up your
conclusion and explanation to this lab, submit it to turnitin.com and
as a printed copy to the in box.
1. Conclusion - Discuss whether your data supported or did not support your
hypothesis. Explain any experimental sources of error. Mistakes you made.
Problems with equipment or materials, etcetera. What would you do
differently next time to better assure you had success/had success again?
2. Explanation - Explain the process of transformation including the steps
needed to make it occur, what happens at each step, and what you learned
about how genetics influences appearance/traits. This is NOT the place to
discuss your individual results, think of a group that succeeded in
transformation. Also discuss how this lab has informed you about gene
expression and protein synthesis.