Experiment 3: The LAC operon
Questions
1. What is the purpose of using chloroform when you assess β-galactosidase
activity with ONPG?
Chloroform will make the bacterial membranes more permeable to small molecules;
this will be needed to get ONPG into the cell, where it can be cleaved. Also, β-
galactosidase can cleave lactose.
2. What induces cAMP production?
When the glucose levels are 0, and glucose is consumed. Because of the complete
consumption of glucose, IIAGIc is phosphorylated and will stimulate the enzyme
Adenylyl cyclase, which produces cAMP from ATP.
3. Which enzyme makes cAMP?
The enzyme Adenylyl cyclase makes cAMP.
4. Does IPTG respress or induce the LAC operon?
IPTG functions as an inducer of the LAC operon.
5. What will happen if a bacterium is mutated in the LacY gene (lactose
permease)?
The rate of growth will be low because the mutated cells of the bacterium can only
metabolize lactose that diffuses in the cells.
6.
Substrate cAMP present? Is CAP inducing LacO Can lactose be
present LAC operon? respressed by metabolized?
LacI?
glucose+lactose no yes no no
only lactose yes no no yes
only glucose no yes yes no
no substrate yes no yes no
The aim of the experiment
The aim of this experiment is to examine what will happen when the LAC operon in
E. coli is induced and what will happen when the LAC operon in E. coli is repressed.
In this experiment, we will study the activity of the enzyme β-galactosidase in
different E. coli strains. β-galactosidase is encoded by the lacZ gene. Also, I will be
examining the effect of blocking translation with the antibiotic chloramphenicol on β-
galactosidase activity. For the mutants of E. coli, strain TG1 and strain CSH7, I will
be checking whether it is disturbed in the β-galactosidase or in the lactose
permease. Lastly, a negative control to make sure that cells without β-galactosidase
do not show a yellow colour and a positive control to make sure that cells with β-
galactosidase do show a yellow colour will be included in this experiment.
Questions
1. What is the purpose of using chloroform when you assess β-galactosidase
activity with ONPG?
Chloroform will make the bacterial membranes more permeable to small molecules;
this will be needed to get ONPG into the cell, where it can be cleaved. Also, β-
galactosidase can cleave lactose.
2. What induces cAMP production?
When the glucose levels are 0, and glucose is consumed. Because of the complete
consumption of glucose, IIAGIc is phosphorylated and will stimulate the enzyme
Adenylyl cyclase, which produces cAMP from ATP.
3. Which enzyme makes cAMP?
The enzyme Adenylyl cyclase makes cAMP.
4. Does IPTG respress or induce the LAC operon?
IPTG functions as an inducer of the LAC operon.
5. What will happen if a bacterium is mutated in the LacY gene (lactose
permease)?
The rate of growth will be low because the mutated cells of the bacterium can only
metabolize lactose that diffuses in the cells.
6.
Substrate cAMP present? Is CAP inducing LacO Can lactose be
present LAC operon? respressed by metabolized?
LacI?
glucose+lactose no yes no no
only lactose yes no no yes
only glucose no yes yes no
no substrate yes no yes no
The aim of the experiment
The aim of this experiment is to examine what will happen when the LAC operon in
E. coli is induced and what will happen when the LAC operon in E. coli is repressed.
In this experiment, we will study the activity of the enzyme β-galactosidase in
different E. coli strains. β-galactosidase is encoded by the lacZ gene. Also, I will be
examining the effect of blocking translation with the antibiotic chloramphenicol on β-
galactosidase activity. For the mutants of E. coli, strain TG1 and strain CSH7, I will
be checking whether it is disturbed in the β-galactosidase or in the lactose
permease. Lastly, a negative control to make sure that cells without β-galactosidase
do not show a yellow colour and a positive control to make sure that cells with β-
galactosidase do show a yellow colour will be included in this experiment.
