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Induction and repression of lacZ expression
Introduction.
Synthesis of the enzyme beta-galactosidase is induced in wild type E. coli strains in response to
the presence of lactose, the enzyme's natural substrate, or IPTG (isopropyl-beta-Dthiogalactoside), a non-metabolizable (i.e., "gratuitous") inducer. Synthesis of the enzyme is also
subject to catabolite repression, meaning that beta-galactosidase is not synthesized rapidly in
the presence of lactose when a better carbon and energy source, such as glucose, is present.
When given both sugars, E.coli will not synthesize beta-galactosidase until all of the glucose is
first exhausted from the medium, then will induce beta-galactosidase synthesis to use the
remaining lactose.
This experiment is designed to demonstrate both catabolite repression and induction of betagalactosidase synthesis during growth of E. coli.
Procedure:
PERIOD ONE
1. Prepare to harvest samples for the beta-galactosidase assays: Label 10 microfuge tubes so
that your group can identify them in the freezer. Number the tubes 1-10. Store the tubes on
ice.
2. Each group will be provided with 6 x 100 ml of LB medium in a 500-ml flask. You will add
either IPTG, glucose, both, or neither to your LB medium before inoculation (see Table 3.1
below):
Table 3.1.
Group: A
IPTG* no
Glucose
no
**
B
yes
C
no
D
yes
no
yes
yes
E
yes
at 0.3
OD***
F
at 0.3 OD***
yes
*Add 1 ml of IPTG stock solution (100 mM); final IPTG concentration = 1 mM.
**Add 5 ml of 10% glucose stock solution; final glucose concentration = 0.5%
***Add one compound first, then inoculate culture, then add the other compound when the
culture reaches an OD600 of approximately 0.3.
3.A fresh overnight culture of E. coli in LB medium will be provided . Determine the OD600 of
the overnight culture, then inoculate your 100 ml of LB with sufficient overnight culture to
obtain a starting OD600 of approximately 0.1. Remove a 5-ml sample from the culture, then
place the flask in the 37°C shaking water bath at 200 rpm. Record the time and the initial optical
density.
4. The 5-ml sample from the culture will be treated as follows:
 a. 4 ml for OD600 reading. Place 4 ml of culture in a cuvette and read the OD600.
(Dilute sample appropriately if the OD600 reading is >0.4).
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
b. 1 ml for beta-galactosidase assay: Add 1 ml of culture to a eppendorf tube.
Centrifuge for 1 minute, discard the supernatant, and place the tube containing the cell
pellet on ice.
5. At 15-min. intervals (record times!), remove 5-ml samples from your culture and treat them as
described in Step 4. At the end of the lab period, all eppendorf tubes will be placed in racks
provided in the -20°C freezer in the prep room.
PERIOD TWO
6. Assay cells for beta-galactosidase:
The principle of the assay is based on the fact that when ONPG (o-nitrophenyl-beta-Dgalactopyranoside) is cleaved, the chromogen o-nitrophenol (ONP) is produced. At alkaline pH,
the chromogen absorbs light efficiently
at 420 nm
a.Remove the tubes containing your samples from the -20C freezer and place at room
temperature.
b.Resuspend each pellet completely in 1.0 ml of Z buffer and transfer to a clean 13-mm test tube.
Z buffer:
0,1 M sodium phospate buffer pH 7,0
0,001 M MgSO4
0,0002 M MnSO4
0,1 M 2-mercaptoethanol
c.Add 2 drops of chloroform to each tube. Vortex each tube at maximum speed for 10 seconds.
d. Add 0.6 ml of 4mg/ml ONPG to each, mix and record the time.
e. Incubate the tubes until the yellow color appears.
f. Add 1.3 ml of 1 M Na2CO3 Stop Solution and mix. Take note of the total assay time for each
sample.
g. Read the absorbance of each assay tube at 420 nm and the OD at 550 nm.
Use glass cuvettes and use as a blanco in both falls the following mixture:
1,0 ml Z buffer + 2 drops of chloroform + 0,6 ml of 4 mg/ml ONPG + 1,3 ml 1M Na2CO3
7. Calculate "Miller Units" (named in honor of Jeffrey Miller) of beta-galactosidase activity
using the equation:
Miller Units = A420 - 1,75 (OD550)
t . v . OD600
A420 and OD550 are read from the reaction mixture,
OD600 is the culture optical density at the time the sample was taken for assay
t is the reaction time, in minutes
v is the volume of culture assayed, in ml.
8.Plot all of the data; OD600 vs. time, Miller U. vs. time.
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