BI 302

Fall 2014

Lab 4 Catabolite Repression

Introduction

In this lab experiment we will study Catabolite Repression, as demonstrated by Nobel Prize
winning scientists Jacob and Monod. This experiment builds on your previous knowledge
of the regulation of gene expression in E.coli as demonstrated through the lac operon.

There is a direct relationship between the amounts of sugar available (metabolized) and
the rate of growth of a microorganism. Jacob and Monod demonstrated that E.coli in a
medium with two types of sugar, Glucose (directly metabolized) and Lactose (further
processing required), the organism would use glucose first and then the other sugar.

The reason being that glucose is a monosaccharide and thus a simple sugar, thereby
requiring less energy and resource to metabolize. Lactose, on the other hand, is a
disaccharide and must therefore be broken down to its constituents glucose and galactose
before energy, in the form of ATP, can be attained from its metabolism. The breakdown of
lactose in E.coli is mediated by -galactosidase, encoded by the lacZ gene on the lac operon.

The presence of preferred carbon sources (Glu) prevents the expression, and often also the
activity, of catabolic systems that enable the use of secondary substrates (Lac); this is called
Catabolite Repression. Catabolite Repression therefore explains the state whereby in the
presence of Glu, the Lac Operon is turned OFF.

Catabolite Repression regulation is mediated by a regulatory protein called CAP (Catabolite
Activator Protein) and cyclic AMP (cAMP). cAMP is produced from the breakdown of ATP.
Glucose inhibits the activity of adenylate cyclase, the enzyme that catalyzes the production
of cAMP from ATP. Therefore an inverse relationship exists between Glu & cAMP. When
Glucose is HIGH, cAMP is LOW and, when Glucose is LOW, cAMP is HIGH.

CAP requires cAMP to bind to the lac operon. The lack of Glu in the medium allows
adenylate cyclase to function, whereby it converts ATP into cAMP. High concentrations of
Glucose reduces the amount of cAMP, hence less CAP-cAMP complexes are available in the
cell to recruit RNA Polymerase to the lac promoter to initiate transcription of the lac
operon. Once the amount of CAP-cAMP complex increases in the cell, RNA polymerase can
be recruited to the lac promoter to initiate transcription of the lac operon, thereby enabling
the production of enzymes required for the transport and breakdown of Lactose in the cell
(Figure 2).

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Fall 2014

Figure 1 The Lac Operon and its control elements. The repressor must be removed before transcription of
the operon can occur. Release of the repressor is mediated by Allolactose, which is converted from Lactose
by -galactosidase. When [Glu] is HIGH, [cAMP] is LOW in the cell. CAP remains unbound by cAMP. Once
[Glu] decreases, [cAMP] increases. The CAP-cAMP complex recruits RNA Pol to the Lac Promoter to initiate
expression of the Lac Operon, in presence of lactose. When Lac is converted to Glu the levels cAMP levels
decrease.
Note: CAP is also known as CRP (cAMP Receptor Protein).

Figure 2: Structures of Lactose and IPTG

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Fall 2014

When bacteria are grown on lactose, the lactose in the medium is eventually used up and
the level of intracellular -galactosidase drops. There are several lactose analogs or "look-
alikes" that also cause the production of -galactosidae, but unlike lactose they are not
broken down by the enzyme. One such molecule is isopropyl--d -thiogalactoside (IPTG).
Its structure and that of lactose are shown above. Note the regions of similarity.

-galactosidase can be assayed by measuring hydrolysis of the chromogenic substrate, o-
nitrophenyl--D-galactoside (ONPG) as shown below (Miller, 1972).

The amount of o-nitrophenol formed can be measured by determining the absorbance at
420 nm. If excess ONPG is added, the amount of o-nitrophenol produced is proportional to
the amount of -galactosidase and the time of the reaction. The reaction is stopped by
adding Na2CO3 which shifts the reaction mixture to pH 11. At this pH most of the o-
nitrophenol is converted to the yellow colored anionic form and -galactosidase is
inactivated.
The reaction can be run using whole cells that have been made permeable to allow ONPG to
enter the cytoplasm. However, since whole cells are present, the absorbance at 420 nm is
the sum of the absorbance due to o-nitrophenol and light scattering due to the cells. The
contribution of light scattering can be determined by measuring the absorbance at 550 nm
where o-nitrophenol doesn't absorb. The light scattering at 420 nm is 1.75x the light
scattering at 550 nm, so the absorbance of o-nitrophenol is determined by subtracting 1.75
x OD550. The corrected absorbance is then used to calculate the activity of -galactosidase.

!
!
!
!

OD420 and OD550 are read from the reaction mixture,

OD600 reflects the cell density just BEFORE assay
t = time of the reaction in minutes
dil = dilution of culture used in the assay

REFERENCE
Miller, J. 1972. Experiments in Molecular Genetics, p. 352-355. Cold Spring Harbor
Laboratory, NY.
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Fall 2014

Students will be working in Groups of 4:

SUPPLIES:

Culture see below

Experimental Flasks see below

Hotplates (2) and beakers (2) per group

Thermometer 2 per group

Timer 1 per group

Ice bucket and ice

Spectrophotometer (Spec 20) 2 per group

Cuvettes 4 per group

Rinse bottle

Beaker to collect waste

PopCulture Reagent and rLysozyme solution

Sterile microfuge tubes for the distribution of Lysis Master Mix per bench

4 mg/ml ONPG (prepared fresh and stored in a brown bottle) 20 ml per group

Minimal glycerol broth (for Blank and if needed to dilute for OD readings) 50 ml

Z buffer Stock Solution 250 ml per section (40 ml per group)

-mercaptoethanol (fumehood)

500 ml bottle to make Z buffer with -mercaptoethanol

6 x 50 ml bottles/flasks for the distribution of Z buffer

1M Na2CO3 solution 20 ml per group

Micropipettes P10, P100, P1000

Sterile micropipette tips

Sterile slide cap tubes 19 per group

Vortex
CULTURE: E. coli ATCC # 11303 with (i+ z+ y+) or any (i+ z+ y+ E. coli).
The genetic symbols used in this lab instructions are as follows: lac+(-), ability (inability) to
ferment lactose; z, structural gene for galactosidase; y, structural gene for galactoside
permease; a, structural gene for thiogalactoside transacetylase.
Set up the following FLASKS each with 45 ml of minimal 0.2% glycerol medium:
1. Control (0.2% glycerol medium)
2. Glucose (1ml of 10% solution)
3. Lactose (1ml of 10% solution)
4. Glucose and lactose (1ml of 10% solution each)
5. Glucose and cAMP (1ml of 10% solution glucose and 0.1 M cAMP)
6. Glucose, Lactose and cAMP (1ml of 10% solution each: glucose and lactose and 0.1M
cAMP)
7. IPTG (1ml of 10% solution each: glucose and IPTG)
8. Glucose and IPTG (1ml of 10% solution each: glucose and IPTG)
9. Glucose, IPTG and cAMP (1ml of 10% solution each: glucose and IPTG and 0.1 M
cAMP)

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Fall 2014

BEFORE CLASS (Lab Technologist):

Grow 200 ml of E. coli ATCC # 11303 with (i+ z+ y+) in 0.2% minimal glycerol broth
supplemented with 0.1% glucose overnight at 37C

Inoculate each flask, except the Control flask, with 5 ml of the O/N culture (approx.
109) and incubate the 8 flasks for 1 hour at 37C in the water bath shaker (before the
beginning of the class)

Provide 5 ml of inoculum for each class for OD600 measurement

TEN MINUTES BEFORE CLASS (Instructor):

Tuesday 8:30 am Instructor: Prepare Lysis Master mix solution by adding 17 l of

rLysozyme mixture to 30 ml Popculture. Mix well by gentle vortexing. Keep the
tubes on ice at all times. After distributing for your class, keep the rest in the fridge.

Wednesday 8:30 am Instructor: Add 17 l of rLysozyme to 30 ml of Popculture.

Mix well by gentle vortexing. Keep the tubes on ice at all times. After distributing for
your class, keep the rest in the fridge.

Thursday 3:30 pm Instructor: Add 5.5 l of rLysozyme mixture to 10 ml of

Popculture. Mix well. Keep the tubes on ice at all times.

All instructors: Distribute 1.5 ml of rLysozyme-Popculture Lysis Master Mix to 6

microfuge tubes (1 tube per group). Keep the tubes on ice at all times.

Add 680 l of -mercaptoethanol to 250 ml of Z buffer (in the fumehood) and mix well
(40 ml per group x 6 groups per class) Distribute 40 ml each into 6 bottles (1 per group)

PROCEDURE (per Group of 4 Students):

1. Setup two 37C water baths at your bench (using beakers and hotplates) monitor and
adjust the temperature using a thermometer
2. Label 10 sterile test tubes SET A: A1, A2, A3, A4, A5, A6, A7, A8, A9 and Blank
3. Label 9 sterile test tubes SET B: B1, B2, B3, B4, B5, B6, B7, B8 and B9

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Fall 2014

4. Take 4 ml from each flask (back bench) and place it in the appropriate test tube in SET
A. For Blank, add 2 ml of Minimal glycerol broth. Go back to your bench and hold SET
A tubes on ICE.
5. Transfer 2 ml of each to its respective test tube in SET B (hold SET B tubes at room
temperature).
6. Add 150 l of lysis solution (containing Popculture and rLysozyme) to each test tube
in SET B (ONLY). Gently vortex each tube to completely mix the contents.
7. Let SET B tubes stand at room temperature for 15 minutes (set your timer) to ensure
complete lysis.
8. Add 150 l of sterile water to each tube in SET A + Blank to maintain the same volume.
9. Add 2 ml of complete Z buffer to each tube in SET A + Blank and SET B. Mix well.
10. Add 1 ml of ONPG (4 mg/ml) to each test tube (SET A, Blank and SET B tubes),
gently vortex to completely mix the contents. START TIMER.
11. Incubate all tubes at 37C until yellow colour develops in ONE OF THE TUBES in
SET B. The tubes should develop yellow colour within 10-30 minutes. STOP THE
REACTION IMMEDIATELY by adding 1 ml of 1M Na2CO3 to each tube (SET A,
Blank and SET B). STOP TIMER and Record the time it took to develop colour.
12. Qualitatively rate the intensity of the yellow colour in all SET A and SET B tubes: 1
(LEAST INTENSE) " 9 (MOST INTENSE)
13. Measure the OD600 for the original cultures (measurement of bacterial growth i.e. cell
density) Monitors will perform this step and record the absorbance on the white
board.
14. Quantitatively measure the intensity of the yellow colour for the SET B (measure first)
and SET A tubes at both 420nm and at 550 nm using the Blank. Record the absorbance
on the Table 1 (pg 8) and Table 2 (pg 9).
15. Record your results and CALCULATE the units of galactosidase produced by
E.coli under each substrate /combinations using the equation on pg.7.

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Fall 2014

!
!
!
!

OD420 and OD550 are read from the reaction mixture,

OD600 reflects the cell density just BEFORE assay
t = time of the reaction in minutes
dil = dilution of culture used in the assay

The units are proportional to the increase in o-nitrophenol per minute per bacterium. They are
convenient because a fully induced culture grown on glucose has approximately 1000 units, and
an uninduced culture has approximately 1 unit.
Alternatively, instead of correcting for the cell debris interference it is also possible to spin
down the debris in a small centrifuge, and eliminate the 550 nm reading.

SAMPLE CALCULATION:
IPTG induced culture with OD600 (cell density) of 0.60. Add 0.2 ml of culture to 1.8 ml of Z
buffer with lysis buffer and ONPG (This is the same as a 1:10 ratio so we use 0.1 ml as the
volume in the formula). The reaction is stopped after 15 minutes.





OD420 = 0.90
OD550 = 0.050 (1.75 X 0.050 = 0.088)






Units = 902 units of -galactosidase

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Fall 2014

Data Table
Section:
Group:
Members:

Table 1. Measurement of galactosidase production by visual ranking in whole E.coli (11303)

cells when grown on different substrates

Substrate Type

Tube #

Visual
Ranking
(1"9)

Absorbance

OD420 nm
Glycerol

A1

Glucose

A2

Lactose

A3

Glu + Lac

A4

Glu + cAMP

A5

Glu + Lac +
cAMP

A6

IPTG

A7

Glu + IPTG

A8

Glu + IPTG +
cAMP

A9

Calculated gal units

OD550 nm

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Fall 2014

Data Table
Section:
Group:
Members:

Table 2. Measurement of galactosidase production in E.coli (11303), released by cell lysis,

when grown on different substrates by OD measurements

Substrate Type

Tube #

Visual
Ranking
(1"9)

Absorbance

OD420 nm
Glycerol

B1

Glucose

B2

Lactose

B3

Glu + Lac

B4

Glu + cAMP

B5

Glu + Lac +
cAMP

B6

IPTG

B7

Glu + IPTG

B8

Glu + IPTG +
cAMP

B9

Calculated gal units

OD550 nm

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Fall 2014

Reagents
Recipe for Z Buffer Stock Solution(1 liter):
16.1 g of Na2HPO4-7H2O (if using Na2HPO4 add 8.54 g)
5.5 g of NaH2PO4-H2O
0.75 g of KCl
0.246 g of MgSO4-7H2O
Sterile H2O to 1 liter.
Need 1.8 L for all sections (40 ml per group x 45 groups)
DO NOT AUTOCLAVE
Store at room temperature
For complete Z-buffer -- Prior to class use mix:
250 ml Z-buffer
680 l -mercaptoethanol
ONPG (4 mg/ml) (make fresh)
80 mg o-nitrophenyl--D-galactoside
20 ml dH2O
Store in a brown bottle away from light
1M Na2CO3 (store in refrigerator)
5.3 g Na2CO3
50 ml sterile dH2O
Need 900 ml (20 ml per group x 45 groups)

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