PHAR1422: Enzyme Induction in Bacterial Cells .

PHAR1422 Biochemistry/Biotechnology II
Laboratory 5: Enzyme Induction in Bacterial Cells

Introduction

The enzyme β-galactosidase of E. coli strain K12 is an inducible enzyme - that is,
its synthesis is stimulated by the presence of a metabolite (the inducer) in the growth
medium. β-galactosidase hydrolyzes β-galactosides, e.g. the milk constituent sugar
lactose. When E. coli is grown in the absence of lactose or other β-galactosides, it
produces very small amount of β-galactosidase (only a few enzyme molecules per cell).
On the other hand, when the same strain is grown in the presence of a β-galactoside
(e.g., with lactose as the major carbon source), the rate of β-galactosidase synthesis is
greatly increased. Therefore, the quantity of β-galactosidase in such induced cells is
much higher than that in cells grown in the absence of the inducer. The inducer needs
not to be a substrate for β-galactosidase. In the following experiment a gratuitous
inducer, isopropylthio-β-galactoside (IPTG), which is not a substrate for β-
galactosidase, is used to induce synthesis of the enzyme.

The lactose operon is a gene cluster, which coordinates the regulation of the three
enzymes needed for the metabolism of lactose. It consists of three structural genes
encoding β-galactosidase, permease and transacetylase and a control region that contains
a promoter and an operator site. RNA polymerase binds to the promoter site and, the
binding is enhanced by a cyclic AMP receptor protein (CAP), which also binds in this
region. Immediately adjacent to this area is the operator, which is the binding site for a
repressor coded by lactose regulatory gene. In the absence of an inducer, the repressor
binds to the operator site and blocks RNA polymerase so that the structural genes cannot
be expressed. However, the inducer binds the repressor protein and converts it to an
inactive form that can no longer recognize the operator site, so that the operon can be
transcribed.

The synthesis of β-galactosidase in E. coli K12 cells is regulated not only by the
presence of inducers, but also by the availability of alternate carbon sources. Certain
preferred carbon sources, such as glucose, have the capacity to repress the synthesis of
β-galactosidase even in the presence of an inducer. This phenomenon is called
catabolite repression. Other carbon sources, such as succinate, acetate, and glycerol,
are poorer catabolite repressors.

The compound o-nitrophenyl-β-galactoside (ONPG) is the substrate for assays of

β-galactosidase activity in this experiment. The enzyme hydrolyses the colourless
compound to o-nitrophenol (ONP), which instantly ionizes in alkali to the o-
nitrophenolate anion, a yellow-coloured compound that absorbs at 420 nm.
PHAR1422: Enzyme Induction in Bacterial Cells .

Materials

i) Growth of cells: Take an inoculum of E. coli K12S and grow it in a lactose-free

medium consisting of 1% tryptone, 0.5% NaCl and 1% glucose. Grow cells until
it reaches an optical density of 0.3 - 0.6 at 660 nm (1 cm cuvette).

ii) Reagents: 0.08 M phosphate buffer, pH 7.7; 1.0 M Na2CO3; 0.02 M IPTG (freshly
prepared); 2.5 mM ONPG (freshly prepared); toluene.

iii) Apparatuses: Mirco-centrifuge tubes, pipets for measuring volumes of 5, 0.5 and
0.05 ml, cuvettes, test tube racks, parafilm; wash bottle, ice bath, and 37°C water
bath; spectrophotometer; droppers.

Experimental procedures

* Pay attention to biological safety regarding the

handling and disposal of bacterial (E.coli) cells. *

1. Label 10 tubes containing 0.5 ml of lactose-free medium as tube 1 to 10.

2. Add 0.05 ml of distilled water to tubes 1 to 3, and 0.05 ml of 0.02M IPTG to tubes
4 to10. Incubate all tubes in a 37°C shaking water bath for 5 min.

3. After incubation, quickly add 0.5 ml of E. coli K12S cells to each of the 10 tubes
while keeping the tubes in the 37°C water bath. Immediately withdraw tube 4
(with IPTG) and tube 1 (no IPTG), and add a drop of toluene to the tubes. Shake
gently for 10 sec and incubate the tubes on ice. (What is the function of toluene in
this step?)

4. Repeat step 3 to the rest of the tubes according to the schedule listed in Table 1.
While waiting for the other samples, proceed to step 5.

5. Prepare eleven test tubes each containing 1 ml of 0.08 M phosphate buffer (pH 7.7)
and 0.2 ml 2.5 mM ONPG. Label the tubes as 1 to 10 and “Blank” (see Table 2).

6. Quickly add 0.2 ml of the toluene-treated cells obtained in steps 3 & 4 into the
corresponding test tubes (complete this step within one minute). For “Blank”, add
0.2 ml of water instead of bacterial cells. Incubate all samples at 37°C for 15 min.

7. After incubation, terminate the reaction by adding 0.2 ml of 1 M Na2CO3 to all 11

tubes. Record the absorbance at 420 nm using the “Blank” for zero setting.
PHAR1422: Enzyme Induction in Bacterial Cells .

Table 1: Induction of β-galactosidase in E. coli K12

Relevant procedures Samples

A. Label tube 1 2 3 4 5 6 7 8 9 10
B. Add lactose-free medium
0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
(ml)
C. Add distilled H2O (ml) 0.05 0.05 0.05 - - - - - - -
D. Add 0.02 M IPTG
- - - 0.05 0.05 0.05 0.05 0.05 0.05 0.05
(ml)
E. Shake at 37°C (5 min.)
F. Quickly add (ml) E. coli
0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
K12
0 min  - -  - - - - - -
5 min - - - -  - - - - -
Keep shaking at
10 min - - - - -  - - - -
37°C
15 min -  - - - -  - - -
20 min - - - - - - -  - -
25 min - - - - - - - -  -
30 min - -  - - - - - - 

 Withdraw the tubes indicated followed by adding one drop of toluene. Shake gently for 10 sec and
incubate the cell lysates on ice until the whole series of experiment is completed.

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

Table 2: β-galactosidase Activity Assay

Samples
Relevant procedures
no IPTG with IPTG
Sample no. 1 2 3 4 5 6 7 8 9 10
Equivalent time Blank
0 15 30 0 5 10 15 20 25 30
interval (min)
A. Add 0.08 M PO4
1 1 1 1 1 1 1 1 1 1 1
Buffer (pH7.7) (ml)
B. Add 2.5 mM ONPG
0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2
(ml)
C. Add the cell lysates
0.2
from the corresponding 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2
tubes 1 to 10 above (ml) dH2O

D. Incubate at 37°C for 15 min

↓
E. Add 0.2 ml 1 M Na2CO3 to terminate the reaction
↓
F. Read absorbance at 420 nm
PHAR1422: Enzyme Induction in Bacterial Cells .

There is no Write-up Form for this practical. Please answer the following questions
and the required calculations in the format of a Laboratory Report.

The completed Laboratory Report (stapled with the Computer Grade Form) should
be submitted to Ms Kaman Lee at BMSB Rm 410 by 17 March 2011, 5 pm.

Results and Calculations

1. For quantitative measurement of the β-galactosidase activity, one unit of enzyme

is arbitrary defined as the activity of the enzyme that hydrolyzes ONPG to generate
an increase in optical density of 0.01 at the wavelength of 420 nm in 1 cm light
path, in 30 min. Calculate the units of the enzyme per ml of the bacterial culture
for the samples taken at different times following the addition of the inducer.

2. Plot a graph showing the time course of β-galactosidase induction. How does the
specific activity of the enzyme change during the incubation period?

Questions for Discussion

1. In this experiment the growth of E. coli (or the increase in bacterial cell number or
cell concentration) was monitored by measuring the absorbance of the bacterial
culture at 660 nm. Why is it possible to use absorbance to measure the bacterial
growth? Can you suggest another method for estimating the cell concentration or
the cell mass of a bacterial culture?

2. What is the experimental advantage of using IPTG instead of lactose as an inducer

of the lac operon?

3. What is the purpose of adding toluene to the cell suspension prior to the assay of
β-galactosidase activity? Why the mixing of the bacterial suspension with toluene
should be done gently?

4. How would chloramphenicol and cycloheximide affect the induction of β-

galactosidase in E. coli.

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