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methods > Final 2 > Flashcards

Final 2 Flashcards

1
Q

What are the components of an expression vector?

A

Promoter
Operator
Tag site
Cleavage site
MCS
Cleavage site
Tag site
Stop codon
Terminator

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2
Q

What is the promoter and where does it come from in the Pet29b vector?

A

T7 promoter, Origin from bacteriophage

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3
Q

What is the operator in the Pet29b vector?

A

Operator
- Concept comes from lac operon

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4
Q

What is the Lac I gene in the Pet29b vector? What does it have?

A

Lac I gene: Makes the repressor to bind to the operator so the protein is not expressed
Promoter for Lac I gene

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5
Q

What is used for expression? What is the one we use call?

A

Expression host ; BL21De3

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6
Q

Define an Operon

A

Operon: multiple genes (more than 1 gene) under the same promoter, controlling a biochemical pathway. All gene products are responsible for a single biochemical reaction.

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7
Q

What are the three Lac Operon genes, name and state the function. What do tehy have flanking tm

A

3 genes (Z, Y, A)
All under the same promoter
Goal of the genes is to produce beta galactosidase
Gene Z: Beta galactosidase
Gene Y: Lac permease
Gene A: Transacetylase
All the genes are responsible to break down lactose

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8
Q

What type of expression is the lac operon?

A

Inducible

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9
Q

Is the operator closed or open? Why? What is it produced by?

A
  • Always closed due to the binding of a repressor protein.
  • The repressor protein binds to the operator
  • The repressor protein is produced by a gene
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10
Q

How is the repressor protein produced by? How? What type of expression is it?

A
  • Produces the repressor protein
  • The repressor protein binds to the operator
  • The Lac I has its own promoter
  • It is constitutive
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11
Q

What happens to the lac operon when induced?

A

When there is no glucose but lactose
The repressor protein will unbind from the operator through lactose.
- Lactose coverts to allolactose and binds to the repressor protein changing its confirmation and it falls off.
The operon will transcribed, (Z, Y, and A).
A mRNA will be produced
Then translation will happen and produce the three proteins from the three genes.

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12
Q

What happens if in a gel electropheresis water is used? Why?

A

A solution containing few ions (i.e distilled water or benzene) would carry very little current.

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13
Q

What is mobility? What is its function?

A

how easily ions move in an electrical field.
the rate of migration traveled within a voltage.

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14
Q

What is electrophoresis?

A

Electrophoresis is the process by which charged molecules are separated in an electrical field due to their differential mobilities.

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15
Q

What are factors affecting mobility?

A

: charge of the molecule, voltage of the gradient of the electrical field, and the frictional resistance of the supporting medium impeding their movement.

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16
Q

define mobility of a protein

A

the distance a protein migrates to that of the ladder

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17
Q

Why do proteins migrate in a electrophoresis?

A

Proteins have a net charge at any pH other than their isoelectric point, pI, and thus when placed in an electric field will migrate towards the electrode of the opposite charge.

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18
Q

What is the PI of most proteins?

A

For most proteins, the pI is in the range of 3-10 with a majority of protein of less than 8.

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19
Q

What is the ideal support medium for a electropheresis?

A

An ideal support medium should be strong, hydrophilic to prevent hydrophobic interactions between the proteins in the sample and the support medium, and stable over a range of temperature, pH, and osmolarity, and have a carefully controlled and adjustable porosity.

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20
Q

Why is the pore size of the medium important?

A

The pore size of the supporting medium is important because it is a major contributor to the frictional coefficient f.

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21
Q

What two things contribute to the separation process?

A

Both the sieving effect of the support medium and the charge/mass ratio of the molecules contribute to the separation process.

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22
Q

What does SDS PAGE stand for> What is the primary means of fractination?

A

In sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) the sieving effect is the principal means of fractionation.

23
Q

How does SDS page simplify seperation of proteins? What type of proteins will migrate easier?

A

SDS page simplifies the separation of proteins by relying on only the size of the polypeptide chains. Small proteins will migrate in the gel with minimal impediment whereas migration of larger proteins will be slower.

24
Q

What is the percentage of the gel determined by in a polyacrylamide gel?

A

percentage of acrylamide

25
Q

How are proteins affected in an SDS PAGE

A

Proteins are denatured by detergent sodium dodecyl sulfate.
Dodecyl sulfate binds to the proteins and causes the folded proteins to become denatured into extended rods coated with dodecyl sulfate.
Coated chains will have a much more negative charge than uncoated
Charge to mass/ratio for proteins will be almost identical and proteins will only be separated by size.

26
Q

What does a stacking gel do?

A

Use a stacking gel causing the protein to be concentrated at a narrow zone at the top of the resolving gel, permitting effective and reproducible separation of the SDS coated proteins by size.
The stacking gel is polymerized with a small percentage of acrylamide to insure high porosity and is buffered with Tris-HCL buffer at a highest pH.

27
Q

What is added to eliminate the disulfide links?

A

b-mercaptoethanol

28
Q

What is used to visualize bands in SDS PAGE

A

Coomassie Brilliant Blue is dissolved in dilute acid and methanol.
To visualize the protein bands, the usual practice is to stain the gel until it is uniformly blue and then remove the stain not adhering to the protein using dilute acid and methanol.
Large bands stain deeper than smaller.
In SDS page, the bands corresponding to the protein of interest should become more prominent relative to those of the other proteins.

29
Q

How is silver used for visualization?

A

The gel is soaked in a dilute solution of methanol to fix the proteins to the gel and is then incubated with an acidic solution of silver nitrate. The silver nitrate reacts with the proteins.

30
Q

How are radioactive isotopes used to label proteins?

A

Radio labeled protein bands may be detected by placing the dried gel against a piece of X ray film in the dark.
Decay of the radioactive compound will expose the X ray film, revealing the presence of the protein bands.

31
Q

How can imunobloting be used?

A

If an antibody to the protein of interest is available, the protein may be detected in the gel by immunoblotting.
Can use a camera to take a photograph of the gel directly for a lab notebook or publication.
Can also dry the gel on a piece of white filter paper, gel dryer, or on cellophane at ambient temperature.

32
Q

What is SDS PAGE good for?

A

estimation of the purity and quantity of purified proteins.
SDS PAGE is frequently used to estimate the molecular mass of the polypeptide chains.

33
Q

How is the resolution of the protein affected?

A

The resolution of the bands will be affected by the amount of protein loaded as well as the current applied.

34
Q

What is immunobloting also known as?

A

Western blotting

35
Q

Define immunobloting

A

The technique is used to detect and quantify a protein reacting with a certain antibody.

36
Q

Describe the procedure of immunoblotting.

A

-The SDS gel is removed from the glass plates and placed flat onto a nitrocellulose membrane.
-Absorbent pads are used to support the gel and membrane as a sandwich, and the assembly is held together by a supporting clamp.
-An electrical field transeverses to the sandwich and forces the proteins to migrate out of the gel onto the membrane.
-The membrane has free sites remaining, and coated with a mixture of non specific proteins to block the free sites.
-Often milk containing a high concentration of casein is used.
-The membrane is the soaked in a solution containing an antibody to the protein of interest (protein antibody).
-Since all the protein binding sites on the membrane are blocked, the antibody can only adhere to the membraned if it interacts with its specific antigen.
-The detection of the presence of the antibody can be done through a secondary antibody. The secondary antibody will react with any antibody from the same biological source as the primary antibody.
-The secondary antibody can be assayed by imaging the membrane in substrate of a coupled enzyme, whose reaction creates a chromogenic reaction (release of color).

37
Q

What was done to create the recombinant DNA molecule in cloning?

A

Used Genomic DNA and PCR out the double stranded DNA (CR ORF)
Ligated in the PGEMT easy vector to create recombinant DNA molecule with the CR insert and the vector.

38
Q

Define recombinant DNA and state how it can be constructed.

A

joining two different fragments of DNA
To construct a recombinant DNA molecule need: DNA from two or more sources, restriction sites, and DNA ligase.
Need PCR to add appropriate overhangs for ligation.

39
Q

What is the goal of subcloning and what overall goal is it linked to? What does it depend on?

A

Goal: Overexpression
Protein expression
Depends on the quality of the design

40
Q

What are the design components of a sublconing experiment? What do you need to make sure of about the insert? Name what should be present when sequencing in the present insert.

A

Expression vector
present class: pet29b as it is designed for a high level of expression
Has a MCS/ polylinker
Need to make sure the CR insert does not have any mutations and have the components we desire
present study: CR insert and histag

41
Q

What is the operator in the expression vector based on? Name and define it.

A

The lac operator is the lac operon from e.coli
a group of genes under one promoter for one biochemical reaction is an operon. When it is a lac operon, the goal is to break down lactose. Only expressed when only lactose is present.

42
Q

What is the origin of the lac promoter? T7 promoter?

A

Bacteria; Virus (bacteriophage)

43
Q

How big is the lac promoter? T7 promoter?

A

40bp; 23bp

44
Q

What are the Bases necessary for promoter activity, lac promoter? T7 promoter?

A

-10 region (5’TATAAT3’)
Also called pribnow box
-35 region: (5’TTGACA3’)
Conserved sequence
;
Bases necessary for promoter activity: -7 to -11 region
Conserved sequence

45
Q

What mutations affect promoter activity lac promoter? T7 promoter?

A

Mutations at -10 or -35 affects promoter activity;
Mutation in any bases in -7 and -11 affects promoter activity.

46
Q

What polymerases bind to lac promoter? T7 promoter?

A

E.coli: RNA polymerase binds to the lac promoter
(interactions are very specific)
T7 RNA polymerase binds to the T7 promoter
(interactions are very specific)

47
Q

Answer the following about RNA polymerase?
How many subunits
Name the subunits
What kind of structure and why?
MW?

A

RNA polymerase – multi subunit enzyme
2 alpha polypeptide
1 beta
1 b’ polypeptide
1 omega polypeptide
1 sigma polypetptide
- Quaternary structure (more than two subunits)
MW = 480kDa

48
Q

Answer the following about T7 RNA polymerase?
How many subunits
What kind of structure?
MW?

A

T7 RNA polymerase – single subunit
(Tertiary structure)
MW = 90 kDa

49
Q

What type of promoter is lac promoter? T7 promoter? Name and define the types? Why is the lac promter like it is?

A

Weak promoter/ leaky promoter
- Weak promoter: The rate of transcription is slow. The amount of mRNA produced per unit of time is low
- Due to size
Leaky promoter: transcription occurs independent of induction.
;
T7 promoter is a strong promoter.
- Strong promoter: The rate of transcription is fast. The amount of mRNA produced per unit of time is high.

50
Q

What is integral to a proteins function? Which do not have functions?

A

A proteins structure is integral to its function. Primary and secondary have no functions.

51
Q

Name the cloning and the expression host.

A

BL21DE3 is the expression host
JM109 was a cloning host

52
Q

What species and strain are the host cells? What virus? What doe the cells produce and how?

A

Species: E.coli
Strain: BL21DE3
lambda DE3 prophage (virus)
The cells produce T7 RNA polymerase through the promoter LacUV5
produced through induction by IPTG.

53
Q

What is produced by the host cells and what happens?

A

The T7 RNA polymerase will be produced and binds to the T7 promoter of the recombinant DNA molecule
when exposed to a stressor the recombinant molecules will be replicated

54
Q
A

methods (14 decks)

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