Lab C Flashcards
What does β galactosidase do?
it catalyzes the hydrolysis of lactose into galactose and glucose
How is β galactosidase transcription regulated?
by the presence or absence of lactose in the environment; allolactose binds to the repressor and causes a conformational change, making it fall from the operator
What is an assay?
a method to assess how much of a given enzyme is produced per cell per unit time
What does a biochemical assay do?
measures enzyme activity
What is the natural substrate of B-gal?
lactose
What type of blank should be used for the B-gal enzyme activity assay?
“b-gal blank” made by your group
What is the product of the lac I gene?
the repressor
How does the lac repressor work?
it binds to the operator, blocking the RNA polymerase from transcribing the lac operon
How is the lac operon activated?
cAMP-CAP binds to the promoter
What is ONPG?
a colorless compound that produces a yellow compound, o-nitrophenol, when cleaved by β galactosidase
How is the amount of o-nitrophenol produced measured?
by determining the absorption of the sample
What do the two spectrophotometry readings measure?
OD₄₂₀ - proportional to the amount of o-nitrophenol produced
OD₆₀₀ - an estimate of cell density
What must happen before you measure optical density?
you have to measure an LB blank
When do you assay β galactosidase activity?
- 20 minutes after E. coli added to sugar
- 70 minutes after E. coli added to sugar
What does luria broth do?
provides essential nutrients for bacterial growth
What does chloroform do?
partially disrupts the cell membrane, allowing cellular proteins to diffuse out of the cell
What does the Z buffer do?
optimizes pH of the sample for β-galactosidase/ONPG reaction
What does Na₂CO₃ do?
stops the β-galactosidase/ONPG reaction by increasing the pH
What doe IPTG do?
mimicks allolactose to induce the cell
What required special cleanup?
chloroform - hazardous waste
cell cultures - bleach bin
Which reagent doesn’t need to be on ice?
Na₂CO₃
Is the level of β galactosidase activity influenced by the carbon source in the media?
Yes, lactose must be present for there to be β-galactosidase activity. Glucose can deactivate β-galactosidase activity
What explains the difference in β-galactosidase activity between the 20 minute and 70 minute samples?
β-gal production is time dependent
Why must OD₆₀₀ be taken at both the 20 minute and 70 minute markers?
you are trying to measure o-nitrophenol produced per minute PER BACTERIUM, and since E. Coli continues to grow between 20 and 70 minutes, you want to know that the difference is not due to the proliferation of E. Coli over time
