Induction of -galactosidase in Escherichia coli
Pre-Class Assignment: Please view the following video prior to class. There will be a fun team
competition (prizes!) to assess your understanding before we begin the exercise.
http://www.dnaftb.org/33/animation.html
Principle:
We are going to test the hypothesis that specific environmental signals cause certain genes within
a cell to become active. In Part I of the experiment transcription of the lac operon within E. coli
cells was induced by addition of an inducer, thiomethyl--D-galactoside (TMG). TMG is a nonmetabolizable (it is not broken down) analog of lactose. It binds to the lac repressor allowing
transcription of the lac operon. In part II, because it is technically difficult to measure increased
synthesis of the lac operon mRNAs, induction was indirectly assessed by measuring the
enzymatic activity of one of the proteins that will be produced from the mRNAs synthesized
following induction of the lac operon. Specifically, the activity of -galactosidase was measured.
This enzyme normally hydrolyzes lactose (milk sugar) to glucose and galactose:
lactose + H2O
-galactosidase > glucose + galactose
Following induction, a colorimetric assay was used to determine -galactosidase activity. Instead
of using lactose as the substrate, o-NPG (o-nitrophenylgalactoside) was used. o-NPG is a
colorless substrate analog and is useful because it turns yellow when it is hydrolyzed. This
provides us with a simple spectrophotometric assay of -galactosidase activity. It is important to
note that TMG acts as an inducer of the lac operon, but it is not a substrate for  galactosidase.
On the other hand, o-NPG does not function as an inducer of the operon, but can serve as a
substrate for  galactosidase. Data from the experiment is shown below.
Time in min
0
10
20
40
60
No TMG
1.5625E-11
2.5E-11
1.77083E-11
1.14583E-11
2.8125E-11
Enzyme Activity
TMG
6.35417E-11
7.08333E-11
1.60417E-10
5.58333E-10
7.42708E-10
TMG + GLC
2.70833E-11
1.875E-11
1.45833E-11
1.35417E-11
2.08333E-11
Lab Report:
Each team will turn in one lab report that will consist of a computer-generated graph that
accurately portrays the data above, and that answers the questions below.
Generating a graph
Use Microsoft Excel (or your favorite graphing program) to generate a scatter graph that uses
“time after addition of TMG (min)” on the x-axis and “units of enzyme activity per cell
(micromoles of o-nitrophenol produced per cell per minute)” on the Y-axis. On the same graph,
plot the results for the uninduced cultures (No TMG), the induced (TMG) and induced in the
presence of glucose (TMG+Glu).
Questions
1. What is the natural substrate and inducer of the lac operon?
2. Why is it preferable to use TMG rather than lactose as the inducer?
3. Why is o-NPG a better substrate than the natural substrate?
4. What are the 2 transcription factors that regulate the lac operon? Which one is the
repressor and which one is the activator?
5. What happens over time in the culture that was induced with TMG?
6. Whap happens to gene expression from the lac operon in the presence of TMG and
glucose?