Download Lab - Transformation of E. coli Bacteria with the pGLO

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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)?
4. What is agar?
5. What is ampicillin (amp)?
6. What is ara?
2009 HHMI Summer Workshop, Dept. of Molecular Biology, Princeton University
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 bacteria’s environment--the plate they are growing on--to best tell
if they are ampicillin resistant?
Very often an organism’s 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 bacteria’s 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.