The Lac Operon
The lac operon is a cluster of genes that function together to
import and metabolize the disaccharide lactose (lac) into
glucose and galactose. For most prokaryotes, glucose is the
preferred carbohydrate (sugar) because it can directly enter
glycolysis. If, for example, you drank a glass of milk, lactose
would be readily available to the E. coli living in your
intestines, but glucose would be in short supply. In such a
scenario, the lac operon would be switched ON to produce
the enzymes required for the E. coli to utilize the lactose.
The lac operon is, therefore, another example of gene
regulation in response to changes in environment conditions.
Regulation of the Lac Operon
The activity of the lac operon is controlled by two different
regulatory proteins.
The first is the lac repressor which is produced by the
regulatory gene called lacI. When active, the lac repressor
binds the lac operator and blocks transcription of lac operon
(Figure 1). Unlike the trp repressor, however, the lac
repressor is active by itself. A regulatory molecule called an
inducer (acts to induce transcription) is required to
inactivate the lac repressor. As lactose enters a cell, some is
converted into the inducer allolactose. Allolactose binds to
the lac repressor causing it to change shape which inactivates
the repressor (Figure 1). This form of regulation saves the
cell energy by only producing the enzymes required for
Name:___________________
breaking down lactose when lactose is present. The lac
operon is considered an inducible operon because its default
state is OFF and transcription is turned ON in the presence of
an inducer (allolactose).
The second regulatory protein is an activator (activates
transcription) called Catabolite Activator Protein (CAP).
When active, CAP attaches to its binding site next to the
RNA polymerase binding site and activates transcription by
recruiting RNA polymerase to the lac promoter (Figure 1).
As previously mentioned, glucose is the prefered energy
source for cells because the enzymes that metabolize glucose
(through glycolysis) are expressed continuously. When
glucose levels are low, the cell responds by producing a
molecule called cyclic AMP (cAMP). The cAMP binds to
and activates CAP. This enables the cell to metabolize
lactose as an alternative energy source.
The two different regulatory proteins, the lac repressor
and CAP, allow the lac operon to integrate two different
environmental signals, cellular lactose and glucose levels.
Further, the lac operon is only ON when those two
conditions have been met. The purpose of this activity is to
determine under what conditions the lac operon is ON (being
transcribed).
Figure 1 The Lac Operon
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Activity 2: Building the Lac Operon
In this activity you will be using a computational modeling and simulation software called Cell Collective to create a
computational model of the lac operon.
Part 1: Getting Started
a.
The first step in building a computational model of the lac operon is to determine what you want the model to do. The
goal of your model is to demonstrate the conditions required for the lac operon to be active. Specifically, the
activity of the operon in response to environmental glucose and lactose. The components of the lac operon, the cell,
and extracellular environment that you will need to include in your model (e.g., lac repressor) are listed in Table 1.
b.
Next, determine the positive and/or negative regulators for each of your components and complete Table 1 (See
“lactose metabolism” for an example).
c.
Sketch your OWN model in the box of Diagram 1, draw in the appropriate interactions using arrows (→) for positive
interactions and blunt-end lines (⊣) for negative interactions. Make sure to include all components from Table 1.
Diagram 1
Table 1
Component
List
Positive
Regulators
Negative
Regulators
enviro_glucose
enviro_lactose
Describe Relationship
external components
lactose
metabolism
lac enzymes
The lac enzymes are
responsible for breaking
down lactose
Lac enzymes
Lac mRNA
Lac operon
CAP
cAMP
environmental
glucose
NA
NA
external component
NA
NA
external component
Lac Repressor
allolactose
environmental
lactose
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Part 2: Access to Cell Collective
a.
Go to dev.cellcollective.org and log in using your username and password.
b.
Add a new model by clicking the “⊕ New Model” button on the top left of the Home page.
c.
Rename the New Model as “LacOperon_lastname” by clicking the name line on the top menu bar.
Part 3: Creating your Computational Model using Cell Collective
a.
Click “Model” on the top middle menu bar to enter the model construction workspace. In this workspace you will be able
to add components to your model and create relationships (positive and negative) between the components.
b.
Next, add all of the components from Table 1 to the “Internal Components” panel. Click the “⊕” symbol to add new
components to your list. Click the name space to change the name of the component.
c.
Click and drag the “environmental glucose” and “environmental lactose” components to the “External Components”
panel. These are the two environmental factors that you will be manipulating.
d.
Next, use the information from Table 1 and Diagram 1 to create the relationships between the components in your model:
To add a Positive Regulator:
i.
Click to select the component, whose
regulatory mechanism you want to
create, in the “Internal Components”
panel. When selected the name of that
component will appear in the
“Regulatory Mechanism” panel in the
middle of the screen (Red box, Figure
2).
ii.
Click, drag, and drop the positive
regulator to the green “Drop
Component” box in the “Regulatory
Mechanism” panel next to Positive
Regulators. For example, click and
drop “Lac enzymes” into the
“Positive Regulator” box for “lactose
metabolism” (Figure 2).
iii.
Before continuing, click “Save”.
Figure 2 Positive Regulatory Mechanism for “lactose metabolism”
When “lactose metabolism” is selected from the Internal Components panel you can view
and edit its Regulatory Mechanism. In this example, “Lac enzymes” are set as positive
regulators of “lactose metabolism.
To add a Negative Regulator:
i.
Click to select the component, whose regulatory mechanism you want to create, in the “Internal Components”
panel. When selected the name of that component will appear in the “Regulatory Mechanism” panel in the middle
of the screen.
ii.
Click, drag, and drop the negative
regulator to the red “Drop
Component” box into the “Negative
Regulator” box.
iii.
Next, set the “Dominance” of your
negative regulator if applicable (Red
box, Figure 3). This feature exists
because a specific “Negative
Regulators” may not be dominant
over all possible “Positive
Regulators” that activate a given
component. For example, click and
drop “Lac repressor” into the
“Negative Regulator” box for “Lac
Operon” then adjust the Dominance
Figure 3 Dominance and the Regulatory Mechanism for “Lac Operon”
The “Lac Repressor” is a negative regulator of the “Lac Operon” and its inhibitory effects
so that the “Lac repressor” is
are dominant over the positive effects of “CAP”.
dominant over “CAP” (Figure 3).
iv.
Before continuing, click “Save”.
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Part 4: Model Validation
In this section you will be validating the dynamics of your model using the “Simulation” feature of Cell Collective. Remember,
the goal of this particular model is to demonstrate the conditions required for the lac operon to be active. You will be treating
your model with four different combinations of glucose and lactose.
Simulation Setup:
a.
Go to the “Simulation” workspace and select the output species that represents the activity of the lac operon.
b.
Under Simulation Control, change the “Sliding Window Size” to 25.
c.
In the Internal Components panel, check the “
” icon box next to the “lac_operon”.
d.
Next, complete Experiments 1-4.
Experiment 1
i.
Adjust both the “enviro_glucose” slider and “enviro_lactose” slider to 0 (External Components).
ii.
Start the simulation by clicking on the play (▶) button under Simulation Control. Click the pause (॥) button
after ~75 cycles (shown on the x-axis).
iii.
Record the activity level (shown on the y-axis) of the lac operon in Table 2 under “test 1” and reset the
simulation.
Experiment 2
i.
Adjust the “enviro_glucose” slider to 100 but keep the “enviro_lactose” slider at 0.
ii.
Start the simulation then click “Pause” after ~75 cycles.
iii.
Record the activity level of the lac operon in Table 2 under “test 1” and reset the simulation.
Experiment 3
i.
Keep the “enviro_glucose” slider at 100 but adjust the “enviro_lactose” slider at 100.
ii.
Start the simulation then click “Pause” after ~75 cycles.
iii.
Record the activity level of the lac operon in Table 2 under “test 1” and reset the simulation.
Experiment 4
i.
Adjust the “enviro_glucose” slider to 0 but keep the “enviro_lactose” slider at 100.
ii.
Start the simulation then click “Pause” after ~75 cycles.
iii.
Record the activity level of the lac operon in Table 2 under “test 1” and reset the simulation.
d.
e.
When all four experiments have been completed, compare the observed activity of the lac operon to the expected. A
significant difference between the two suggests problems with your model.
Proceed to Part 5 to refine your model if it is NOT correct. If correct, proceed to Part 6.
Table 2 Validation Table.
lac_operon
observed
Experiment
enviro_glucose
enviro_lactose
lac_operon
expected
1
0
0
0
2
100
0
0
3
100
100
0
4
0
100
100
test 1
test 2
test 3
test 4
test 5
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Part 5: Model Refinement
In this section you will be refining your model and repeating the validation in Part 4. For each round of refinement, record
your modifications in detail in the tables below (additional revision tables are on the last page) and retest your model as in Part
4 (record your data as test 2). When your model has been validated, proceed to Part 6.
Model Revisions 1
Model Revisions 2
Part 6: Final Model Description
Draw the final (validated) version of your model in the box below.
enviro_glucose
enviro_lactose
external species
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