MBIO 2020 / Lab 2: Lac operon

• What does it mean for a nucleic acid to be polycistronic? On that note, are both eukaryotes and prokaryotes polycistronic?

• What is an operon?

• What is an operator?

• What is a promoter?

• Polycistronic means that you would have more than one coding region or cistron. Eukaryotes are generally monocistronic, while bacteria are generally polycistronic.

• Operon: A cluster of genes for related proteins that will be transcribed into a polycistronic mRNA.

• Operator: Region of DNA at the beginning of the coding region, controlling transcription of operon and where repressor binds.

• Promoter: Region that binds to the RNA polymerase.

What’s an inducible operon?

An operon normally off but can be turned on in the presence of a specific molecule.

What does it mean for E. coli to have a lac operon?

The genes of the lac operon will only be transcribed when the proteins required to break down lactose are actually needed.

• What does lacI code for?

• What does lacZ code for?

• What does lacY code for?

• What does lacA code for?

• lacI: Repressor protein.

• lacZ: Beta-galactosidase.

• lacY: Lac permease.

• lacA: Transacetylase.

There are two control mechanisms for the expression of lac operon. What are the two control mechanisms? Explain them.

• The two control mechanisms work independently of each other, and both must be turned on before the lac operon will be expressed.

• Negative transcriptional control.

  • When there is no lactose in the environment, repressor is produced and is active. Repressor binds to operator, blocking the path of RNA pol, and thus preventing transcription.

  • When there is lactose in the environment, existing molecules of beta-galactosidase will convert some of the lactose into allolactose. The allolactose will act as the inducer to the repressor, which changes the repressor’s conformation, causing detachment from the operator, and unblocking the path of RNA pol, allowing transcription.

  • Negative control = repression = turning off.

• Positive transcriptional control.

  • Adenylate cyclase produces cAMP but is only activated by a protein that is involved in glucose transport into the cell. If this protein is busy with glucose transport, it won’t be able to turn adenylate cyclase on.

  • If glucose is depleted and lactose is available, this protein can turn adenylate cyclase on and produce cAMP. cAMP will then bind to a catabolite activator protein (CAP), which allows the complex to bind to the promoter. Th ebinding of the cAMP/CAP complex promotes efficient binding of RNA pol and activates transcription.

  • Positive control = activation = turning on.

What is the indication in this experiment that beta-galactosidase is present?

ONPG is cleaved by beta-galactosidase to form galactose and ortho-nitrophenol, which causes yellow colour and indicates production of beta-galactosidase by cell.

What are the questions that must be asked in this experiment?

• Does the presence of lactose in the environment affect production of beta-galactosidase?

• Does the presence of glucose affect production of beta-galactosidase?

• What happens when both lactose and glucose are present? Will beta-galactosidase be produced?

• And what if there are no sugars in the medium at all? (Just a non-preferential carbon source like glycerol.)

How do you add sugars to media aseptically?

• Tip.

• Milk dilution bottle: Remove cap. Bunsen. Get sample.

• Milk dilution bottle: Bunsen. Return bottle.

• Culture tube: Bunsen. Eject into tube.

• Culture tube: Bunsen. Return tube.

How do you inoculate the culture tubes?

1. Tip.

2. Starter: Bunsen. Get sample.

3. Starter: Bunsen. Return sample.

4. Culture: Bunsen. Eject sample.

5. Culture: Bunsen. Return sample.

6. Eject tip.

7. Tap the bottom of tube 5 times.

8. Set to 600 µL.

9. Tip.

10. Culture: Bunsen. Get sample.

11. Culture: Bunsen. Return sample.

12. Cuvette: Eject sample.

13. Eject tip.

How do you perform the beta-galactosidase assay?

1. Add 100µL of 1% SDS to cuvette.

2. Take a piece of parafilm and cover cuvette.

3. Invert cuvette 5 times to mix and permeabilize and kill cells.

4. Set to 200µL.

5. Add 200µL of ONPG to cuvette. Peel off a bit of the parafilm.

6. Invert cuvette 5 times to mix.

7. Place in bath for 10mins.

8. Add 0.2µL 1M Na₂CO₃ to stop the enzymatic reaction to make stable measurement. Peel off a bit of the parafilm.

9. Invert cuvette 5 times to mix. No bubbles.

How do you use the spectrophotometer for this experiment?

1. Use M9 + glycerol as blank for spectrophotometer, arrow facing light source.

2. Measure abs for the inoculated cuvettes.