Lab VI: Simulating the Lac Operon in Excel
Objectives: Use Excel to represent the components of the lac operon system, their relationships and activities.
Lab Introduction: The key to understanding how the lac operon functions is to understand how its components
interact. One way to develop this understanding is by building a model that mimics the workings of the system.
Today you will use Excel to build such a model. The lac operon system can be characterized by its components,
which include substrates, cis-elements, genes, and proteins and by the states of these components. States
include the allelic states of genes (e.g. genes can be either wild-type or mutants), whether cis-elements have
proteins bound to them, etc.
I. Components of the lac operon system:
Substrates
Glucose
Lactose / Allolactose
Genes
lacI
lacZ
lacY
lacA
cis-elements
CAP site
operator
promoter
Gene Products
lacI product: The lac repressor
lacZ product:  galactosidase
lacY product: lac permease
lacA product: transacetylase
Other Proteins
Repressor
RNA polymerase
CAP
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II. States of the components in the lac operon system:
Substrate States
Present or Absent
Allelic States of Genes
+ wild type
- defective
S super (applies to a repressor that cannot bind inducer)
Allelic States of cis Elements
+ wild type
- defective (or absent)
C constitutive (applies to an operator that cannot bind repressor)
Functional States of cis Elements
bound Protein is bound to element
free Protein is not bound to element
Gene Product Synthesis (for lacI)
+ present (actively synthesized)
- absent (not actively synthesized)
Gene Product Synthesis (for lacZ, lacY and lacA)
none (there are no wild-type copies of this gene)
leaky (repressed)
low (not repressed, but not CAP activated)
high (not repressed and CAP activated)
Protein States (active requires that the protein is synthesized)
active (inducer is bound to repressor, or repressor is a superrepessor; cAMP is bound to CAP)
inactive (no inducer and repressor is not a superrepressor; cAMP, not bound to CAP)
III. Getting started
A. Download and open the spreadsheet
1. Download Lab 6.xls (Excel file) from the class website
(http://seahorse.louisiana.edu/biol224/)
2. Save in My Documents folder.
3. Navigate to your My Documents folder, and open lab 6.xls by double-clicking on it.
4. When the file opens, you should see this warning message near the top of the screen:
5. Click Enable Editing.
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6. Next you will see this warning:
7. Click Enable Content.
8. You will then see this message:
9. Click Yes. Now you are ready to begin.
IV. Overview of the spreadsheet for lab 6.xls.
It is essential that you begin by understanding how the spreadsheet is organized.
A. The labels for the lac operon components have been entered in the first column (A). You will not
change these. These are grouped into substrates, genes, cis elements, proteins, and gene products. There
are separate entries for genes and cis elements on the main chromosome versus those on the F’ factor
of a merozygote.
B. The second column (B) lists the possible states for the components.
C. The third column (C) is for the actual states of the components. Your goal is to fill in cells in column
C so that the spreadsheet will accurately represent how the components of the lac operon function in
response to the genotype and environment of the cell. Some of the states (in green boxes) you will
simply enter as givens, such as the presence or absence of glucose or lactose and the alleles of genes.
For others (in red boxes) you will enter Excel formulas that determine states based on other
components. A few of these formulas have already been filled-in for you (blue boxes).
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V. Start with a simplified model of a wild-type lac operon system, with the following characteristics:
A. There are only genomic copies of genes (no F' factor)
B. All of the genes and cis-elements have wild type alleles (+)
C. Levels of expression of lac operon genes are leaky, low, or high as follows:
Lactose
Present
+
+
Glucose
Present
+
+
-
lac Operon
leaky
leaky
low
high
D. Here is a walkthrough for setting up the simplified model of a normal, wild-type system.
1. Start by entering all of the “givens” that don’t require formulas. These are the cells within
green-bordered boxes. If you click on one of these cells, a little arrow appears to the right of the
cell. Click on this arrow to select one of the possible states from a pull-down list of choices.
a. Select absent for glucose (cell C5), and present for lactose (cell C6). You can change
these later to see how the cell responds to different conditions.
b. Enter + (wild-type) for the genomic copies of all the genes (C10 through C13). This
indicates the cell has wild-the genes on the main chromosome.
c. Enter – (not functional or absent) for the F' copies of all the genes (C16 through C19).
This corresponds to a cell without an F' plasmid.
d. Enter + (wild-type) for all of the genomic cis-elements (C23 and C24).
2. Now start to fill-in the cells that require formulas to calculate states of components based on
the states of other components (the cells within red-bordered boxes in column C). Begin by
entering a formula to determine the synthesis status of the genomic copy of the lac repressor
protein (C46).
a. The synthesis status can be either + or -. A + means the gene product (protein) is being
made (translated), a – indicates it is not being made.
b. The repressor will be synthesized only if there is a wild-type allele (+) for the genomic
copy (C10) of the lacI gene
c. The following formula in C46 accomplishes this:
=IF(C10="+", "+", "-")
d. This formula says that if there is a wild type allele for the lacI gene on the main
chromosome (indicated by a + in cell C10), then the lacI gene product will be made (+),
otherwise it will not be made (-).
e. If you entered this formula correctly, the cell (C46) should display +.
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3. Next, you need a formula to determine if there is an active repressor (C40 is active) or an
inactive repressor (C40 is inactive)
a. There are two requirements for the wild-type repressor to be active:
i. There must be a wild-type repressor protein (C60 is +), and
ii. There must not be an inducer (C6 is absent) that would bind to the repressor.
b. If these requirements are not met, the wild-type repressor is inactive.
c. Remember, IF formulas have the following structure:
IF (logical_test, value_if_true, value_if_false)
In this case, the logical test is whether a wild-type protein is present and the inducer is
absent, so an AND formula is also needed. AND formulas have this structure:
AND (logical1, logical2)
…and are true only if both logical statements are true
d. The following IF formula in C40 accomplishes this:
=IF(AND(C60="+", C6="absent"),"active", "inactive")
e. If you entered this formula correctly, cell C40 should now display inactive.
4. The next component to consider is CAP. CAP is active (C41 is active) when glucose is absent
(C5 is absent).
a. The formula for C41 is:
=IF(C5="absent","active","inactive")
b. After you enter this formula, cell C41 should display active.
5. Now move to the state of a cis element, the operator (C33 for the genomic copy). The
operator can either have a repressor protein bound to it (bound) or not (free).
a. Repressor is bound to the operator if there is either an active wild-type lac repressor
protein (C40 is active) or a superrepressor protein (C42 is active). The formula is:
=IF(OR(C40="active", C42="active"), "bound", "free")
When this formula is entered into cell C33 it should display free.
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6. For the CAP site cis element (C32), CAP protein is bound if the protein (C41) is active:
a. The formula is:
=IF(C41="active", "bound", "free")
b. When entered, cell C32 should now display bound.
7. Finally, enter the formulas for the levels of expression of the three structural genes of the lac
operon (C48 to C50). Start with the lacZ gene (C48). First, expression is none if there is no
wild-type allele (C11 is -). Otherwise, expression is: leaky if the operon is repressed, low if the
operon is depressed but not CAP-activated, and high if the operon is derepressed and CAPactivated. Try this formula yourself; here are some hints for lacZ (C48):
a. You will need three nested IF statements that follow this pattern:
IF (A is true, B, IF (C is true, D, IF (E is true, F, G)))
b. Expression is none if the genomic copy is defective (C11 = "-");
c. The operon is leaky (repressed) if the operator (C33) is bound (has repressor).
d. If not leaky, expression is high if the CAP site (C32) is bound, otherwise expression is
low.
e. Now fill-in the formulas for the lacY and lacA genes (C49 and C50). These are almost
the same as the formula in C48, except for part b. above.
8. Congratulations! You now hopefully have a functional model of a wild-type lac operon.
VI. Interactive graphics
A. There are two bar graphs on the spreadsheet. One displays levels of glucose and lactose in the
environment, and the other displays levels of expression for the three genes of the lac operon.
1. Experiment with changing the presence of substrates (C5 and C6) and the allelic states of the
genomic copies of genes (C10 through C13). As you change these, watch how the graphs
change.
2. Complete Part I of the lab report, checking your answers based on your understanding of
the lac operon against the results shown in the interactive graphic. Resolve any discrepancies by
either changing the model, or rethinking your answer.
Present 10
High
10
9
8
7
6
Low
5
4
3
2
Absent
Leaky
None
0
Glucose
Lactose
1
0
B galactosidase
Permease
Transacetylase
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VII. Stress test your model of the lac operon
A. Built into this spreadsheet is a program to test your model of the lac operon, with each test
corresponding to a question on your lab report.
1. When you are satisfied that your model is working, and have
answered the Part I questions on the lab report, click on the button
that is on top of cell M3 that looks like this:
C. The program will rapidly change the states in column C, and check to see if the operon model is
responding correctly. The results of the tests will appear in column M. At this point, just worry about the
first 7 tests, which correspond to the 7 rows in Part I of your lab report.
VIII. Mutant lac operons
A. Now that you have the basic wild-type model working, add some mutants! Some of the ground work
has already been laid: 1) the state of the operator (C33) already takes into consideration the presence of
superrepressors; 2) synthesis of lac proteins already checks for mutant structural genes; and 3) cells C60
through C64, which look at the net effects of protein synthesis from both genomic and F' plasmid copies
of genes, have are already filled-in.
B. Complete entries for superrepressor
1. Cell C47: is the genomic copy of lacI synthesizing a superrepressor? If the allele (C10) is S,
then +, otherwise -.
2. Cell C42: what is the superrepressor state? It’s active if there is a superrepressor being
synthesized, i.e. C61 is +.
3. Test your new modification. Try switching the genomic copy of the lacI gene to the S allele
and observe the effects on the interactive graph.
C. Constitutive Operator
1. At this stage, the state of the genomic copy of the lac operator (C33) doesn’t take into account
the possibility of a mutant constitutive operator (allelic state is C).
2. Modify the formula for C33 so that if the allele is C, then the operator is free. This will
require another IF statement that encloses the formula that is presently in this cell.
3. Test you new modification.
D. Complete Part II of the lab report.
E. Run the stress test again. Tests 8 – 11 correspond to part II of the lab report.
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IX. Merozygotes
A. The last thing to add to your model is the F' plasmid to your model.
1. Start by switching the alleles for the F' plasmid lac structural genes (C17 through C19) from –
to +. Also make sure the cis elements (C27 and C28) are set to wild type (+).
2. Enter a formula for the state of the CAP site on the F' plasmid (C36). This should be exactly
the same formula as in cell C32 (be careful if you cut and paste, the cell references will change).
3. Enter a formula for the state of the operator on the F' plasmid (C37). This should be similar to
the formula in cell C33, but not exactly. It should test for a C allele at the F' plasmid copy of the
operator (C27) rather than the genomic copy (C23).
4. Enter formulas for synthesis of F' plasmid copies of the repressor (C53 and C54) and
structural genes (C55 through C57). These will be similar to the formulas for the genomic copies
(C46 through C50), except they refer will refer to F' plasmid copies of alleles and cis-elements.
B. Complete Part III of the lab report.
C. Stress test your model of the lac operon, Tests 12-15 correspond to Part III of the lab report.
X. Have your TA rate your model
A. When your model passes all of the tests (or you run out of time), ask your TA to come over and run
the stress test. This, along with your lab report, will be the basis of your grade for this lab.
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Name _________________________
Section _____________________
Lab V: Simulating the Lac Operon in Excel – Lab Report
Part I. Fill in the table below indicating the level of synthesis for each protein as none, leaky, low or high.
Glucose
absent
absent
present
present
absent
absent
absent
Lactose
absent
present
present
absent
present
present
present
lacZ
allele
lacY
allele
lacA
allele
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
Synthesis of
galactosidase
Synthesis of
permease
Synthesis of
transacetylase
Part II. Assume the alleles for the genomic copies of the three lac operon structural genes are wild-type.
Glucose Lactose lacI
absent
absent
absent
present
absent
absent
absent
absent
+
S
+
S
operator
Synthesis of 
galactosidase
+
+
C
C
Part III. Indicate the level of synthesis for each protein as none, leaky, low or high.
Substrates
Genomic Copies
F' Copies
Glucose Lactose
absent
absent
present
absent
absent
absent
present
present
lacI
operator
lacZ
+
+
+
+
+
C
+
C
+
+
+
+
lacY
lacA
lacI
+
-
+
-
+
+
S
S
Protein Synthesis
operator
lacZ
lacY
lacA
+
+
+
+
+
-
+
+
+
+
+
+
+
+
B galactosidase
permease
transacetylase
Part IV. Stress test your model, ask your TA to see that your model passes all of the tests.
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