January 11th, 2011

Induction of the lac operon in Escherichia coli

Aim

To determine the conditions needed to switch on the lac operon in E.coli.

Introduction

Gene transcription can be switched on and off by gene regulation proteins. The example of
regulation of proteins biosynthesis is the lac operon in E. coli.
The lac operon of E. coli encodes proteins which catalyze the transport and degradation of the sugar
lactose. The cell can use lactose as an energy source by producing the enzyme β-galactosidase to
digest that lactose into glucose and galactose.
E. coli live in intestines and metabolize lactose to glucose as energy source. However, it makes
proteins for lactose metabolism only when certain conditions are met: glucose level is low and
lactose is present. This adaptation allows saving energy as only necessary proteins are produced
when needed.

Figure 1. The effect of an inducer on the synthesis of the proteins of the lac operon
(http://www.biochem.wisc.edu/courses/biochem651/labs/ing.aspx ).
Hypothesis: Lactose activates β-galactosidase gene expression. The enzyme is not present, or is
present in only very low concentration in the bacterial cells until lactose is added to the solution.
Prediction:
Tube 1 Tube 2 Tube 3 Tube 4 Tube 5 Tube 6
Colour yellow colourless yellow colourless dark yellow colourless
No ONPG No ONPG control control

Method

The apparatus, chemicals and samples were collected.

The bench was wiped down with disinfectant and the test tubes were labelled 1 to 6.

5 cm3 sodium phosphate solution was add to each test tube. Then five drops of methylbenzene to
speed up the formation of any yellow colour was added to each test tube.

Using aseptic technique 1 cm3 of the E.coli in nutrient broth was transferred to test tubes 1 and 2.

Then using aseptic technique 1 cm3 of the E. coli in nutrient broth with lactose was transferred to
test tubes 3 & 4.

1 cm3 of the enzyme ß-galactosidase was added to test tubes 5 & 6.

Finally 1 cm3 of the ONPG solution was added to test tubes 1, 3 and 5 and 0.05 g of lactose powder
to test tubes 1 and 2.

Then test tubes were covered with a stopper and shaken to help distribute the ONPG.

All test tubes were put in a water bath set at 35 °C for 50 minutes.

Results

Tube 1 Tube 2 Tube 3 Tube 4 Tube 5 Tube 6

sodium 5 cm3 5 cm3 5 cm3 5 cm3 5 cm3 5 cm3
phosphate
methyl 5 drops 5 drops 5 drops 5 drops 5 drops 5 drops
benzene
E.coli in 1 cm3 1 cm3
nutrient broth

E.coli in 1 cm3 1 cm3

nutrient broth
with lactose
lactase 1 cm3 1 cm3
ONPG 1 cm3 1 cm3 1 cm3
lactose 0.05g 0.05g
powder
Colour pale yellow pale ivory yellow pale ivory dark colourless
yellow
Figure 1 Test tubes 1-5 before water bath set at 35 °C for 50 minutes

Figure 2 Test tubes 1-5 after water bath set at 35 °C for 50 minutes

Analysis

The test tube 5 confirm that when enzyme is present ONPG brakes down into glucose and yellow
dye. From the results it can be concluded that the best conditions were in the test tube 3, which
confirms the hypothesis. On the beginning there was a low concentration of enzyme due to lactase
in nutrient broth. Straight after addition there was no colour change which meant that the
concentrations of ß-galactosidase remained the same. After incubation in water bath the mixture in
test tube 3 changed colour which proved that the lactose present in ONPG activated β-galactosidase
gene expression. There was minor change in colour in the test tube 1. The nutrient broth in the test
tube 1 did not contain lactose so there was no enzyme on the beginning. It can be concluded that
the gene expression was triggered but was slower. This was because bacteria in the test tube 1 were
not having optimal condition to multiply.

References:

1. http:/ /www.biochem.wisc.edu/courses/biochem651/labs/ing.aspx