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methods > Final 8 > Flashcards

Final 8 Flashcards

1
Q

What assay is used to characterize the enzyme?

A

Activity Assay/ Enzyme Activity Assay

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2
Q

What is the enzyme in the experiment called? What reaction does it carry our? What is another name for the reaction?

A

Enzyme is carbonyl reductase
* The enzyme carries out a oxidation reduction reaction
* Also called redox reaction

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3
Q

What does the enzyme activity graph look like? What are the axes? Why does the change occur?

A

The rate increases then decreases or plateaus due to the taking up of the substrate

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4
Q

What are the Enzyme Activity Assay Components?

A
  • Substrate
  • Buffer
  • Cofactor - Phosphate buffer
  • Present experiment: NAD+
  • Enzyme
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5
Q

What are the 5 things assay performance depends on?

A
  • The enzyme concentration
  • Ionic strength of the buffer
  • PH
  • Temperature
  • Purity of the enzyme preparation
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6
Q

What are the 4 principles of the CR assay?

A
  • Continuous assay - We will monitor it throughout
  • NAD+ - No absorbance at 340nm, absorbance is 0
  • NAD is the reduced form- It absorbs maximally at 340nm (lambda max)
  • Use these to make the assay possible
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7
Q

What is the activity assay specific to?

A

The enzyme

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8
Q

Define denature and degrade

A

Denature: the enzyme is losing its 3 dimensional structure (tertiary structure goes to primary structure)
Degraded: The primary structure of the enzyme is broken.

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9
Q

How is the substrate determined? What are we using?

A

Some enzymes can digest multiple substrates, others may be specific to one.
* For example, CR is specific to steroid. We will be using cholic acid

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10
Q

What buffer are we using?

A

Buffer
* We are using sodium pyrophosphate (pH 8.9)

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11
Q

What is the cofactor/coenzyme?

A
  • We are using NAD+
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12
Q

What is the absorbance we are using? Is there color?

A

Spectrophotometer - UV 340nm
* No color product

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13
Q

What is the general formula of the reaction? What is the formula for this experiment?

A

The substrate + NAD + Enzyme - product + NADHTH + Enzyme
Present experiment: Colic acid + NAD+ + Enzyme -> CR = product + NAD H +
* The enzyme carries out a reaction but stays the same

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14
Q

What is R on the graph? What does it represent? What is the highest point? Why does it plateaus? What is the S?

A

R is the rate over time
* the rate the substrate is being reacted by the enzyme
* Reaches a maximum velocity
* Plateau because the substrate finishes or the enzyme is no longer active.
* S is substrate

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15
Q

What are the steps of the experiment?

A

First in a cuvette place the NAD, Buffer, and Substrate.
* Max 1mL cuvette. Can be used within the UV range.
* Used as a starting point to see activity with and without the enzyme.
Read the cuvette at 340nm
* Use spectrophotometer to measure OD at 340nm.
* To get an initial reading. Should be 0 because there is no enzyme, therefore no absorbance.
* However, sometimes there is background noise and therefore a 0 is not achieved.
* Will read as a zigzag.
Add the enzyme after the first measurement has plateaued.
* Once the enzyme is added, the absorbance increases then plateaus after the substrate is consumed.

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16
Q

What is the obtained graph from the spectrophotometer? What are the axes? What does it look like?

A

Absorbance over time. The Y axes is Absorbance, the X is time. It first pleateus, then accelerates to a peak, then plateus.

17
Q

How is the absorbance calculate? What is proportional to?

A

A= e (absorbtivity constant/ extinction coefficient/molar absorbtivity/molar absorption coefficient) * c (concentration) * l (path length of cuvette (usually 1 cm))
e: How strongly the chemical absorbs light at the specific wavelength.
A=ecl
* l is always 1
Absorbance is proportional to concentration

18
Q

What does The Beer-Lambert law state?

A

that there is a linear relationship between the concentration and the absorbance of the solution, which enables the concentration of a solution to be calculated by measuring its absorbance.

19
Q

How is delta absorbance calculated?

A

delta A = (change in OD)/ (change in time)

20
Q

How is enzyme activity calculated?

A

Enzyme activity = (deltaA/min) x (Total volume in ml) x (Dilution (if there was any dilution))/ (e )x (Volume of enzyme used (ml))

21
Q

How is the enzyme activity expressed.

A
  • in units. The amount of substrate (in a unit)/ time (in a unit)
  • Ex: in our experiment the substrate is in mMol (micromol) and we are using time as a measurement. Ours would be mMol/ minute. Translates to x micromol of the substrate per minute
22
Q

What is the definition of a restriction enzyme unit?

A

Restriction enzyme units: ug (microgram) of DNA at 37 deg, at a specific pH.

23
Q

What is total volume?

A
  • Total volume is always 1mL.
24
Q

Why is dilution done? How is the dilution factor caluculated?

A

sometimes the purified product is so pure the enzyme activity will go too fast.
* Ex: If you take 1mL of enzyme and 9mL of dH20, it is a 10X dilution

25
Q

What is e?

A
  • e (Epsilon): The molar extinction coefficient/ molar absorptivity of the cofactor at the absorbance.
26
Q

How many replicates does each sample need? Can 2 readings be the same? What should they be and what should be done with them?

A

Each sample needs 3 replicates - triplicate
* 2/3 readings should be close. Should take the average of the closest samples.
2 readings cannot be the same.

27
Q

Do these 5 equations.
1. How many ul (microliter) of 20% sugar solution should be used to make 2mL of 5% sucrose.
* C1V1 = C2V2
2. How to prepare 1L of 1X Tris Borate EDTA Buffer from 10X TBE solution. (Dilution)
3. Prepare 100mL of 40% (w/v) polyethylene glycol. (need molecular weight)
4. How to prepare 200mL of 95% (v/v) solution of ethanol. (just multiply)
5. How would you prepare 50mL of 20mm NaOH2. (39.997 g/mol)

A
28
Q

What are the two issues with leaky expression?

A

Can be a waste of energy and create a metabolic burden because there may not be enough protein.
If the protein is toxic, the leaky expression can create proteins that can kill the bacteria prior to induction

29
Q

How can leaky expression be controlled? How is it produced?
What does it do?

A

Can control the leaky expression through a lysozyme.
The lysozyme will be produced by another gene and will bind to the RNA polymerase and repress it, controlling the leaky expression.

30
Q

What can be used in the host to get the genes to control leaky expression? How are there expression? What host can be used and what does it have?

A

Can use alternative plasma with lyse genes
Lyse genes are constitutively expressed
Can also use BL21DE3 PLYSS/E host
* Has the lysozyme plasmid in it

31
Q

When will leaky expression only occur?

A

Leaky expression will only occur when induction does not occur because there is an excess of RNA polymerase.

methods (14 decks)

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