Life Sciences 1a Laboratory – Fall 2006
Lab 7: Got milk?
(AKA Investigating the Lac Operon)
Goals
1.
2.
3.
of the lab:
To observe how environment can influence gene expression
To understand how expression of the lac operon is controlled
To use the scientific method to determine bacterial genotypes
Introduction:
As you learned in lecture, the lac operon is a classic example of gene control,
where the expression of different genes varies depending upon the
environmental situation. Specifically, the gene that encodes β-galactosidase,
lacZ, is normally only expressed when the bacteria is in the presence of lactose,
and in the absence of any glucose. Many different genes and gene products are
involved in this regulation, and in this lab you will learn how they work together
to coordinate the expression of lacZ.
The lac operon
Three enzymes are involved in lactose metabolism in E. coli: β-galactosidase –
an enzyme that cleaves lactose into glucose and galactose; galactosidase
permease – an enzyme that promotes the uptake of β-galactosides, like lactose,
from the extracellular environment; and β-galactosidase transacetylase – an
enzyme that catalyzes the acetylation of β-galactosides as a detoxification
reaction. The genes that encode these three enzymes are named lacZ, lacY and
lacA respectively.
When grown in the absence of lactose, only traces of β-galactosidase or
permease are synthesized by the cell. Thus the cell does not waste any energy
making enzymes it doesn’t currently need. However if lactose is present as the
only carbon source, the expression of these genes increases approximately a
thousand fold. This activation is controlled by the regulator gene lacI. This gene
encodes the repressor protein that prevents the transcription of the lac operon,
and thus the production of β-galactosidase and permease. When the repressor
binds to allolactose, a metabolite of lactose, the repressor is stabilized in a nonDNA binding form and is thus no longer able to prevent the transcription of the
lac operon.
As Dan mentioned in lecture, Jacob and Monod and many other scientists
deduced how the lac operon is regulated through the use of special chemicals
and various lac operon mutants. In this lab, you will recreate some of their
experiments and deduce the identities of four different strains of bacteria based
on their responses to growth in different media. More specifically, each lab pair
will study the regulation of β-galactosidase synthesis in either wildtype, lacI-,
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Life Sciences 1a Laboratory – Fall 2006
lacZ-, or lacY- E. coli. You will measure β-galactosidase activity by following the
hydrolysis of the indicator ONPG which will turn yellow when cleaved by the
enzyme.
Protocol:
Reagents
Medium
A
B
C
D
Color Code
Red
Green
Yellow
Blue
Ingredients
Glycerol (2 x 10-3 M)
Lactose (2 x 10-2 M)
Glycerol (2 x 10-3 M); TMG (2 x 10-3 M)
Lactose (2 x 10-2 M); glucose (4 x 10-2 M)
Each lab pair will examine 1 of 4 bacteria strains. They will then record their
results with the rest of the lab to determine the identities of each strain.
1.
2.
3.
4.
5.
Collect a set of 4 tubes containing 4 different media
Label each tube with your initials and the bacteria strain
To each tube, add 5 drops of bacteria
Place your tubes in the shaking incubator, and shake at 37° C for 1 hr
After 1 hr, take your tubes to the hood and add 1 drop of toluene to each
tube. Return the tubes to the incubator and shake for another 10
minutes maximum.
a. The toluene makes the bacterial cell wall permeable to ONPG
6. After 10 minutes, to each tube add 2 drops of ONPG
7. Return the tubes to the incubator and continue shaking as the color
develops
8. Check your tubes periodically and record the time and the observed color
changes (if any)
9. After your reaction is complete (or your TF tells you it is), tally your
results on the board in the front of the classroom.
10. Record the results from all of your lab mates, both in your section and the
other sections in the same time slot.
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Life Sciences 1a Laboratory – Fall 2006
Questions:
1. Attach a table with your entire labs (all sections belonging in a given time
slot) results, and explain them.
a. What strain did your lab pair test?
b. What were the results for your strain?
c. Were the results of every other lab pair testing the same strain
identical? Why or why not?
d. Were the results for the other strains consistent between different
groups?
e. What is the identity of each strain? Explain.
2. In this lab the bacteria in the dropper bottles had been prepared in media
containing glycerol as the carbon source. Why did we have to incubate
the bacteria for an hour in the new media?
3. You have observed that different bacteria strains grown on different
media exhibit different gene expression patterns. Based on your
observations, and your knowledge of the lac operon, write a hypothesis
explaining how lacZ expression is regulated.
4. Bacteria grown solely on lactose will grow, as will bacteria grown only on
ONPG. However cells grown solely on TMG will not grow, and will instead
eventually die. Based on the structures provided below, why are cells
capable of growing on ONPG and lactose, but not TMG alone?
HO
O
+O
S
HO
O
O
O
HO
HO
OH
OH
thiomethyl-beta-D-galactopyranoside
HO
O
N
O
HO
HO
O
OHO
OH
OH
OH
OH
OH
Ortho-nitrophenyl-ß-galactoside
OH
lactose
5. Would you have been able to conclusively determine the identities of all 4
strains if media C were not used in the testing of any of the 4 strains?
Explain.
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Life Sciences 1a Laboratory – Fall 2006
6. In this lab, 3 of the 4 media contained either an inducer or repressor of
lac operon expression. Which compounds are inducers and which are
repressors? Additionally, based on the results of media A, what is the
default state of lac operon expression in wildtype cells (induced or
repressed). Explain.
7. As Dan mentioned in lecture, the lac repressor is able to work in trans.
Explain what this means, and how it differs from a repressor that works in
cis.
8. Based on your understanding of the lac operon, what do you expect
would have been the results of this experiment had cells from the
different strains been fused together? Explain your reasoning.
Media A
Glycerol
Media B Lactose
Media C
Glycerol, TMG
Media D
Lactose, Glucose
wt/lacIwt/lacZwt/lacYlacI-/lacZlacI-/lacYlacZ-/lacY-
9. Gene control is essential in embryonic development. It is known that skin
cells and nerve cells develop from the same group of embryonic cells. A
gene called noggin is essential in regulating the fate of these cells. The
genes expressed in cells exposed to noggin protein are vastly different
from those not exposed to the protein. If noggin is over-expressed in an
embryo, excess amounts of nerve cells are made and less skin is
produced. However if noggin is deleted (removed from the genome) the
nervous system is underdeveloped and excess skin is formed. Based on
this information, do you believe that noggin induces a nerve cell fate by
activating a nervous cell specific pathway? Briefly explain your logic.
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