74
INDUCTION OF ß-GALACTOSIDASE IN E.COLI
The machinery of RNA and protein synthesis needs a great amount of
energy. The strictly controlled energy metabolism of living cells demands
very selective and careful control of these processes. Enzyme induction
and repression serve both in procaryotes and in eucaryotes as a main
pathway of regulation to fulfil these demands.
One of the best known and most thoroughly studied enzyme induction
systems is the lactose operon of E. coli.
The structure genes of the lac operon encode the information of three
proteins (ß-galactosidase, transacetylase and lactose permease)
necessary to be synthesized by the cells utilizing lactose as energy
source. ß-galactosidase catalyzes the hydrolysis of lactose into glucose
and galactose. Besides the structure genes the lac operon contains
control sites (operator and promoter regions). From the regulator gene
that is not part of the operon, a repressor protein is synthesized
continuously. This repressor protein binds to the operator gene to prevent
the binding of polymerase and transcription. Lactose, isopropylthiogalactoside and other inducers can bind to the repressor protein
removing it from the operator, so RNA polymerase can move through the
operator to transcribe the lac operon. The mRNAs are translated and the
elevated protein production is continued while lactose is present in the
medium.
Induction of ß-galactosidase
Besides its natural substrate, lactose, ß-galactosidase also hydrolyzes the
synthetic substrate ortho-nitrophenyl-ß-D-galactoside (ONPG). The
liberated ortho-nitro-phenol is colored, so therefore its amount can be
determined spectrophotometrically. Instead of lactose, isopropylthiogalactoside, a compound that is not hydrolyzed by the enzyme, is
used as an inducer; therefore its concentration is not changed during the
experiment.
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Separation of the process of transcription and translation by the use of
specific inhibitors
The process of enzyme induction, as described earlier, can be divided
into subsequent steps:
1.) inactivation of the repressor by the inducer.
2.) mRNA synthesis
3.) translation of mRNAs into proteins
The sequence of events can be studied by specific inhibitors, inhibiting
either the transcription (rifampicin) or the translation (chloramphenicol). If
rifampicin or chloramphenicol is added at 0 minute of induction, there is no
ß-galactosidase production. When rifampicin is added at the 15th minute of
enzyme induction, there is some further enzyme production with the help of
the mRNA made during the 15 minute incubation. When chloramphenicol is
added at 15th min. of enzyme induction there is no further enzyme
production.
Solutions:
1.) Esherichia coli suspension
2.) isopropy-ß l-thiogalactoside (IPTG) 0.5 mg/ml
3.) toluene
4.) ortho-nitrophenyl-ß-D-galactoside (ONPG) 1 mg/ml
5.) 0.02 M phosphate buffer, pH 7.5
6.) 0,5 M Na2CO3
7.) 0.5 mg/ml chloramphenicol
8.) 2 mg/ml rifampicin
76
Set up the following experimental points:
materials ( ml)
1
2
3
4
5
6
7
8
9
suspension of E.
coli
1
1
1
1
1
1
1
1
1
H2O
0.1
0.1
0.1
-
-
0.2
chloramphenicol
at 0 min
-
-
-
0.1
-
-
-
-
-
rifampicin
at 0 min
-
-
-
-
-
-
0.1
-
0.1
IPTG inducer
0.1
0.1
0.1
0.1
0.1
-
0.1
0.1
-
chloramphenicol
at 15 min
-
-
-
-
0.1
-
-
-
-
rifampicin
at 15 min
-
-
-
-
-
-
-
0.1
-
incubation
at 37 oC
0'
15'
30'
30'
30'
30'
30'
30'
30'
0.1
Add one drop (50 µl) of toluene into each tube immediately after the
incubation to stop the induction and to permeabilize the bacterial cell wall.
Keep the samples together on ice until all the incubations are done.
Incubate each sample for 15 minutes at 37oC; shake the samples in the
waterbath.
Estimation of the ß-galactosidase activities of the samples
Pipet into test tubes the following ingredients:
0.1 ml of permeabilized bacterial suspension,
0.3 ml of phosphate buffer,
0.1 ml ONPG solution.
Incubate the samples for 15 minutes at 37oC.
Add 1.0 ml of Na2CO3 solution of each tube.
Read the optical activities of the tubes 1-5 using the 6. as blank at 420
nm. Since the rifampicin itself is colorful, the 9th tube is the blank for
tubes 7-8.
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Results:
Samples:
Extinction:
1
2
3
4
5
6
--
7
8
9
--