PRACTICAL 6

Control of protein synthesis in

prokaryotes
PRACTICAL 2

Control of protein synthesis in prokaryotes – the induction of β-galactosidase in E.coli

AIM
To investigate the mechanism of enzyme induction by galactoside.
To measure the activity of β-galactosidase in a culture of E. coli that has been exposed to lactose
(high concentration of glucose).
To compare the β-galactosidase activity with cultures that has not been exposed and with specific
inhibitors.

INTRODUCTION
Gene regulation is an essential system for life on Earth. Prokaryotes of the same species can be
significantly varied in terms of which genes are turned on at a specific point of time. Therefore,
with a complex and adjustable system of gene regulations, organisms are deemed to be robust
and they are able to survive in diverse environments.

The most established models for gene regulation are the lac operon in E. coli. The lac operon
contains three genes (lac Z, lac Y and lac A) which code for proteins vital to the metabolism of
lactose. The lac operon regulates transcriptional process as a repressor and an activator. An
operon is a collection of linked genes under common, coordinated control. Lac operon is
regulated by several factors including the availability of glucose and lactose.

Figure 1: Lac operon – a genetic regulatory mechanism in prokaryotes.

The first control mechanism is the regulatory response to lactose, which uses an intracellular
regulatory protein called the lactose repressor to hinder production of β-galactosidase in the
absence of lactose. If lactose is missing from the growth medium, the repressor binds very tightly
to a short DNA sequence just downstream of the promoter near the beginning of lacZ called the
lac operator. When cells are grown in the presence of lactose, however, a lactose metabolite
called allolactose, which is a combination of glucose and galactose, binds to the repressor,
causing a change in its shape.

This experiment focuses on lac Z gene which encodes for the enzyme β-galactosidase. This
enzyme catalyses the hydrolysis of lactose to galactose and glucose. β-galactosidase is produced
in a very amount when glucose is absent. However, this is reversed when lactose or related
compounds are present in the mixture. Isopropyl β-thiogalactoside (IPTG) is a powerful inducer
of β-galactosidase. It is not a substrate thus could not be used as a carbon source. In this practical,
E. coli cells will be grown in the presence or absence of IPTG. The cells will be disrupted and the
amount of enzyme present will be determined. The induction or increase in enzyme activity could
be achieved by:

 Increased synthesis of β-galactosidase mRNA leading to increase enzyme synthesis

 Increase enzyme synthesis without increased mRNA synthesis
 Increase stability of enzymes
 Activation of an inactive enzyme precursor

β-Galactosidase activity could be measured easily by using o-nitrophenylβ-galactoside (ONPG) as

a substrate:

The ONPG is a colourless compound but with the presence of a substrate (β-galactosidase) it will be
converted to galactose and o-nitrophenol (ONP). The ONP is yellow and can be measured by its absorption
at 420 nm. The amount of ONP produced is proportional to the amount of enzyme present and to the
time the enzyme reacts with the OPNG. This reaction is stopped with the presences of Na2CO3 which shifts
the pH to 11 where the enzyme is inactive.
REAGENTS AND EQUIPMENT

One set of each of the following reagents is provided per group of students.

 A log phase culture of E. coli  Isopropyl β-thiogalactoside (IPTG)

 37 °C water bath  Streptomycin
 Ice bath  Rifampicin
 Falcon tubes  Glucose
 Centrifuge  Lactose
 Timer  3.3 mM o-nitrophenylgalactoside
 Vortex (ONPG)
 CTAB  1 M Na2CO3
 Autoclaved distilled water  Spectrophotometer

METHOD

Your demonstrator will inform you which experiments you should perform. You will be given
the appropriate number of flasks.

Label each flask clearly with your initials.

*Ensure that the flasks are in the 37 °C water bath throughout the experiment.
1. Label seven falcon tubes as 0, 6, 12, 18, 24, 30 and 36 minutes. Place all tubes on ice.

2. Add 1 mL of CTAB into each falcon tube.

TAKE NOTE:

 You will now perform a time-module experiment.

 You will make additions to each flask at zero time and six minutes.
 Sample will be removed from the flask at six minutes interval.
 At ZERO time
3. Take 2 mL of E. coli sample from the flask and place it into the tube labeled 0 minutes, store
on ice, until further analysis.

4. There are 9 flasks filled with E. coli incubated in 37°C water bath.

5. Immediately add the following reagents into each flask as described in the table 6.1 below:
Time Content Flask number
(minutes) (in mL) 1 2 3 4 5 6 7 8 9

H2O 1
0
IPTG 1 1 1 1 1

Mix contents in the flask

1. Incubate mixture in 37°C water bath for 6 minutes.

At 6 minutes
7. Take 2 mL of E. coli sample from each flask and place it into the tube labeled 6 minutes, store
on ice, until further analysis.

8. Immediately add the following reagents into each flask as described in the table 6.2 below:
Time Content Flask number
(minutes) (in mL) 1 2 3 4 5 6 7 8 9

Streptomycin 1

Rifampicin 1
6
Glucose 1 1 1

Lactose 1 1 1

Mix contents in the flask

9. Incubate mixture in 37°C water bath for 6 minutes.

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 At 12 minutes

*No additions should be made to the flask

7. Take 2 mL of E. coli sample from each flask and place it into the tube labeled 12 minutes, store
on ice, until further analysis.

8. Repeat this sampling procedure at t = 18, 24, 30 and 36 minutes.

9. Proceed with the β-galactosidase activity assay after completing the 36 minutes sampling
time.

Assay of β-galactosidase

1. Place ALL the falcon tubes into a 37 °C water bath for five minutes.

2. Add 0.5 mL 3.3 mM o-nitrophenylgalactoside (ONPG) to ALL the tubes. Mix the tubes by
inverting. *Note the start time

3. Continue to incubate ALL the tubes at 37 °C.

4. When a definite yellow colour appears in the tube, add 1.3 mL Na2CO3 to stop the reaction.
Note the exact end time at which Na2CO3 is added to the tube.

*Incubation time is the time between additions of ONPG and Na2CO3

5. After 20 minutes of incubation, if there are any tubes which do not have yellow colour
development, add 1.3 mL Na2CO3 to stop the reaction.

6. Mix the contents of each tube and centrifuge at 4000 rpm, 37 °C for 10 minutes.

7. Measure the absorbance of the supernatant at 420 nm using water blank.

8. Record all the data (simulated data provided).

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 Prepare a SHORT REPORT - METHODS are not required.

Your short report should contain:

Pre-Laboratory Questions

1. Name THREE (3) regulatory portion of the lac operon. [3 marks]

2. Name THREE (3) structural genes of the lac operon. [3 marks]

3. When are these genes expressed? [2 marks]

4. What is the regulatory gene of the lac operon? [1 mark]

5. Where is the binding site of the repressor protein? [1 mark]

6. What is isopropyl β-thiogalactoside (IPTG)? What is the effect of IPTG on lac operon?
[3 marks]

7. What is the function of CTAB being added to the cell culture before performing the
enzyme induction experiment? [3 marks]

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RESULTS - SIMULATED DATA

CONTENT A​420​ at time

Flask
Number H​2​0 IPTG Strep Rifampicin Glucose Lactose 0 min 6 min 12 min 18 min 24 min 30 min 36 min
1.571 abs 1.591 abs 1.599 abs 1.549 abs 1.503 abs 1.534 abs 1.566 abs
1 ✓ (17 min) (17 min) (17 min) (17 min) (17 min) (17 min) (17 min)
1.806 abs 2.289 abs 1.938 abs 1.827 abs 1.843 abs 1.868 abs 1.804 abs
2 ✓ ✓ (16 min) (16 min) (16 min) (16 min) (16 min) (16 min) (16 min)
1.953 abs 1.914 abs 1.865 abs 1.856 abs 2.500 abs 1.934 abs 1.744 abs
3 ✓ ✓ (12 min) (12 min) (12 min) (12 min) (12 min) (12 min) (12 min)
1.505 abs 1.543 abs 1.712 abs 1.770 abs 1.803 abs 1.544 abs 1.744 abs
4 ✓ ✓ (15 min) (15 min) (15 min) (15 min) (15 min) (15 min) (15 min)
1.607 abs 1.538 abs 0.103 abs 1.630 abs 1.669 abs 1.457 abs 1.457 abs
5 ✓ ✓ (10 min) (10 min) (10 min) (10 min) (10 min) (10 min) (10 min)
1.547 abs 1.628 abs 1.580 abs 1.731 abs 1.805 abs 2.096 abs 1.784 abs
6 ✓ (13 min) (13 min) (13 min) (13 min) (13 min) (13 min) (13 min)
1.957 abs 1.676 abs 1.659 abs 1.519 abs 1.902 abs 1.777 abs 1.686 abs
7 ✓ ✓ (10 min) (10 min) (10 min) (10 min) (10 min) (10 min) (10 min)
1.852 abs 2.031 abs 2.186 abs 2.225 abs 2.471 abs 2.453 abs 2.500 abs
8 ✓ (9 min) (9 min) (9 min) (9 min) (9 min) (9 min) (9 min)
2.500 abs 2.216 abs 2.500 abs 2.500 abs 2.500 abs 2.422 abs 2.389 abs
9 ✓ (4 min) (4 min) (4 min) (4 min) (4 min) (4 min) (4 min)
Results:
Flask CONTENT β-galactosidase activity (U/mL)
number
H2O IPTG Strep Rifampicin Glucose Lactose 0 min 6 min 12min 18 min 24 min 30 min 36 min

1 √

2 √ √

3 √ √

4 √ √

5 √ √

6 √

7 √ √

8 √

9 √

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Table 1: Induction of ß-galactosidase production in ​E. coli​ under different conditions

Flask IPTG Other molecules β​-galactosidase activity

present present (No change / increased / decreased)

1 No No

2 Yes Streptomycin

3 Yes Rifampicin

4 Yes Glucose

5 Yes Lactose

6 Yes No

7 No Glucose &
Lactose

8 No Glucose

9 No Lactose
RESULTS

1. Calculate the β-galactosidase activity (U/mL) using the following formula:

𝑈𝑛𝑖𝑡𝑠 (𝐴420𝑇𝑒𝑠𝑡 − 𝐴420 𝐵𝑙𝑎𝑛𝑘)(𝑡𝑜𝑡𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑎𝑠𝑠𝑎𝑦)

=
𝑚𝐿 (𝑡𝑖𝑚𝑒 𝑖𝑛 𝑚𝑖𝑛𝑢𝑡𝑒)(4.6)(𝑒𝑛𝑧𝑦𝑚𝑒 𝑣𝑜𝑙𝑢𝑚𝑒)

Total volume of assay = 4.8

Millimolar extinction coefficient of o-nitrophenol at 420nm = 4.6

Enzyme volume = 2

Show an example of your calculation. (2 marks)

Prepare a table showing all of the calculated values. (2 marks)

2. Plot your results in one graph (Enzyme activity vs time) (5 marks)

3. Based on your graph, describe the changes of β-galactosidase activity in different

conditions. Discuss your observation for every conditions. (40 marks)

4. Conclusion (5 marks)

5. References (5 marks)

Marks obtained: / 75 marks

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