DATE: Oct 23 2022

Experiment 3: the LAC operon

Aim of the experiment: to study the effects of repressing and inducing the LAC operon in
Escherichia Coli by blocking translation with chloramphenicol on the LacZ gene. Additionally, E
coli mutants (CSH7 strain and TG1 strain) will be experimented on to see if lactose permease or
beta galactosidase is affected. There will also be a negative and positive control to make sure
that the effects on the LAC operon are due to the independent variables tested.

Hypothesis: The solution with no beta galactosidase expression will not be yellow, the solution
with the ONTP and beta galactosidase will be yellow because of the ONP metabolite. The
negative control will not show a yellow colour and the positive control does show a colour.
CSH7 has a lacY mutation, meaning it does not produce the permease required to bring ONPG
into the cell, even though IPTG does not need LacY permease to induce the LAC operon. Since
tube 4 has the CSH7 but also chloroform, the LacY mutation doesn’t matter because te cell
membrane is made permeable anyway. Therefore CSH7 will alsoh ave ONPG enter the cell and
break into ONP metabolite, making the tube yellow.

Materials
5 glass tubes
• LB medium
• The E. coli strains (CSH7, TG1,
CSH7)
• IPTG (5 mg/ml)
• ONPG (4 mg/ml, in 60mM
Na2HPO4/40mM NaH2PO4 buffer)
• Shaker at 37 degrees
• Vortex (machine to mix liquids in a
tube)
• Chloramphenicol (20 mg/ml
ethanol)
• Chloroform

Method (taken from BMW – Genetics – Manual 2022):

Day 1:
- The assistant sets up precultures of E.coli CL4B, the lac mutant TG1, and the CSH7 in
bottles with medium. These cultures are grown overnight in the 37C shaker
Day 2
- Fill and inoculate glass test tubes according to table 5.
- Please note: 2.5mL LB + 0.5mL cells is a total of 3mL, therefore dilute the cell 6 times.
- Do not use plastic tubes as they will be affected by chloroform
- Label tubes correctly
 -
- Put the caps on the glass tubes and incubate all tubes in a shaker 37C for 30 mins
- Add 45 microlitres of IPTG (5mg/ml) to tubes 2 and 5. Add 3 microlitres of
chloramphenicol to tube 3 as well. Incubate for at least 30 minutes
- Add 100 microlitres of chloroform to tubes 1 through 5 to make cells permeable.
- Add 0.5 ml of ONPG to all tubes
- Mix on a vortex, asses the colour of the various tubes after some minutes. Complete the
result list:

Results:

Slightly yellow

Yellow

Not yellow

Yellow

Not yellow

Conclusion
With IPTG as an artificial inducer, the lac operon was able to be transcribed by RNA polymerase
and the LacZ gene was able to produce beta galactosidase. This then broke up the ONPG into
ONP, which gave the solution its’ yellow colour. With no IPTG, the repressor blocked RNA
polymerase from transcribing for beta galactosidase so ONPG remained intact, and there was
only slight background colour . CSH7 had a lacY mutation but the effects of this were not seen
as chloroform broke down the cell membrane instead of permease transporting ONPG into the
cell. With Cm, transcription is stopped because it is an antibiotic; by killing e.coli.

Questions:
What is the purpose of using chloroform when you asses beta galactosidase with ONPG: ONPG
is an extracellular molecule while beta galactosidase is an intracellular enzyme, so chloroform
makes the cell membrane permeable. This allows for ONPG to enter the cell and be
metabolized into ONP by the beta galactosidase
What induces cAMP production? When there is low glucose/ all glucose is used, the IIAGc is
phosphorylated, so adenylyl cyclase levels increase, and these produce cAMP
Which enzyme makes cAMP? Adenylyl cyclases
Does IPTG repress or induce the LAC operon: IPTG is an artificial inducer of the LAC operon
What will happen if a bacterium is mutated in the LacY gene (lactose permease). Lactose cannot
enter the cell without the permease as it is a transporter. Without the permease (or mutated
LacY gene), lactose cannot enter the cell to be metabolised by beta galactosidase

No Yes No Yes

Yes Yes No Yes

No No Yes
No
no yes
No no