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Life 23L > Lab C > Flashcards

Lab C Flashcards

1
Q

What does β galactosidase do?

A

it catalyzes the hydrolysis of lactose into galactose and glucose

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2
Q

How is β galactosidase transcription regulated?

A

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

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3
Q

What is an assay?

A

a method to assess how much of a given enzyme is produced per cell per unit time

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4
Q

What does a biochemical assay do?

A

measures enzyme activity

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5
Q

What is the natural substrate of B-gal?

A

lactose

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6
Q

What type of blank should be used for the B-gal enzyme activity assay?

A

“b-gal blank” made by your group

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7
Q

What is the product of the lac I gene?

A

the repressor

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8
Q

How does the lac repressor work?

A

it binds to the operator, blocking the RNA polymerase from transcribing the lac operon

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9
Q

How is the lac operon activated?

A

cAMP-CAP binds to the promoter

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10
Q

What is ONPG?

A

a colorless compound that produces a yellow compound, o-nitrophenol, when cleaved by β galactosidase

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11
Q

How is the amount of o-nitrophenol produced measured?

A

by determining the absorption of the sample

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12
Q

What do the two spectrophotometry readings measure?

A

OD₄₂₀ - proportional to the amount of o-nitrophenol produced

OD₆₀₀ - an estimate of cell density

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13
Q

What must happen before you measure optical density?

A

you have to measure an LB blank

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14
Q

When do you assay β galactosidase activity?

A
  • 20 minutes after E. coli added to sugar

- 70 minutes after E. coli added to sugar

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15
Q

What does luria broth do?

A

provides essential nutrients for bacterial growth

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16
Q

What does chloroform do?

A

partially disrupts the cell membrane, allowing cellular proteins to diffuse out of the cell

17
Q

What does the Z buffer do?

A

optimizes pH of the sample for β-galactosidase/ONPG reaction

18
Q

What does Na₂CO₃ do?

A

stops the β-galactosidase/ONPG reaction by increasing the pH

19
Q

What doe IPTG do?

A

mimicks allolactose to induce the cell

20
Q

What required special cleanup?

A

chloroform - hazardous waste

cell cultures - bleach bin

21
Q

Which reagent doesn’t need to be on ice?

A

Na₂CO₃

22
Q

Is the level of β galactosidase activity influenced by the carbon source in the media?

A

Yes, lactose must be present for there to be β-galactosidase activity. Glucose can deactivate β-galactosidase activity

23
Q

What explains the difference in β-galactosidase activity between the 20 minute and 70 minute samples?

A

β-gal production is time dependent

24
Q

Why must OD₆₀₀ be taken at both the 20 minute and 70 minute markers?

A

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

25
Q

According to the lab safety sheet in the B-gal assay lab, used gloves should be disposed of in the autoclave bag

A

true

26
Q

chemical/materials you will use in the B-gal lab include

A

Z buffer mix
E. coli cells
chloroform
ONPG

27
Q

What is the purpose of ONPG in this lab?

A

Substrate that gives a yellow color when cleaved

28
Q

Which of the following procedures should be done in the hood?

A

add chloroform into tubes for enzymatic assay

29
Q

According to Lab Safety Sheet provided for this week, when working with e. coli cells, you must wear

A

safety goggles
lab coat
gloves
standard lab attire

30
Q

What is the natural substrate of B-gal?

A

lactose

31
Q

What type of blank should be used for the B-gal enzyme activity assay?

A

“b-gal blank” made by your group

32
Q

According to the Lab Safety Sheet for the B-gal assay lab, it is okay to use chloroform at your bench work station as long as you use the dropper bottle and wear gloves.

A

false

33
Q

Microcentrifuge tubes with chloroform on the bottom must be

A

capped and placed in the labeled hazardous waste bin in the hood

34
Q

What type of blank should be used for cell density assay?

A

LB blank provided

Life 23L (11 decks)

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