Final 4

Question 1

In a good design, what makes a good expression vector?

Answer 1

• Strong promoter: Greater expression, more production of mRNA, more protein
• Controllable Promoter - and inducible promoter
• done by adding an operator.
• Ex: Lac I in the lac operon, producing an repressor protein binding to the promoter to keep it off while IPTG changes the conformation of the repressor protein to turn it on

Question 2

In a good design, what kind of plasmid copy number do we desire? What else is important about the plasmid? What about the host cell?

Answer 2

• Plasmid copy number
• Desire a high copy number
• Plasmid stability
• The plasmid is considered a foreign DNA to the host and it could possibly be attacked
• Host-cell physiology.
• Our host cell is BL21DE3
• Codon bias: preference to use a specific codon to code for a specific amino acid.

Question 3

What does the Lac I gene give?What does it have an affinity for? How is it expressed?

Answer 3

Lac I gene gives a repressor protein
* The repressor protein has a high affinity for the operator
* The Lac I gene is constitutively expressed

Question 4

What is the shape of the E.coli genome? What has been added to the genome in out experiment? Is there an operator? What king of promoter? What does the gene in the genome produce? What is it controlled by and done by?

Answer 4

• It is a circular genome
• The genome in the host cell as been engineered in such a way that it has an added T7 gene 1
• There is also an operator
• There is also a lac promoter
• T7 gene 1 produces T7 RNA polymerase
• It is a lysogen, a virus that does not cause destruction to the cell.
• E.coli has E.coli RNA polymerase naturally
• The gene is controlled by the lac promoter, an inducible promoter.
• Must be induced
• Done through IPTG

Question 5

Why the the CR gene need a host? How is the promoter activated and what activates it?

Answer 5

• Does not have a gene to produce T7 polymerase
• Also has an inducible promoter
• Must be induced
• Done through IPTG

Question 6

What two things it the PET system does IPTG induce? How does it work?

Answer 6

• Induces both the Lac promoter (e.coli) and the T7 promoter (pet29bcr)
• IPTG binds to the repressor protein, changing the confirmation of the repressor protein, creating a repressor IPTG complex. The protein can no longer bind to the operator.

Question 7

What does the repressor IPTG complex result in and allow? What is produced? Is it produced before or after transcription?

Answer 7

• Since the protein cannot bind to the operator, the operator site is free for RNA polymerase to move forward and transcribe.
• T7 RNA polymerase is produced from the E.coli gene
• Occurs before the transcription of pet29bCR

Question 8

What and where does T7 RNA polymerase bind to?

Answer 8

• Binds to the T7 promoter on the pet29bCR and transcription occur.

Question 9

Why does a leaky promoter occur? Why is it common and preferable in bacteria?

Answer 9

• Dependent on how strong the binding between the promoter and the gene
• The repressor and operator binding is not 100% tight
• Is common and preferable in bacteria.
• Assists with having resources for the biochemical reaction, prior to induction.

Question 10

What are the 3 experiments in Immunoblotting?

Answer 10

o SDS page
o Western Blotting
o Immuno activity assay

Question 11

What is done after expression of the protein?

Answer 11

Purification

Question 12

When does purification start?

Answer 12

• Starts when you lyse the cell

Question 13

What are the 3 ways you can lyse a cell?

Answer 13

Physical pressure through french press
Ultrasonication
Enzymatic lysis

Question 14

Describe the french press method.

Answer 14

• If there are a lot of cells, you can apply a french press method where physical pressure is applied and the cell breaks

Question 15

Define ultrasonication.

Answer 15

Use sound waves to break the cells

Question 16

Define ad state the 4 features of enzymatic lysis.

Answer 16

• Enzymatic lysis: Break down the cells using various enzymes and buffers
• A good buffer is one maintaining the solution at a specific pH well.
• For small samples
• Centrifuge
• Take supernatant

Question 17

What does SDS PAGE stand for?

Answer 17

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis

Question 18

What is Polyacrylamide used?

Answer 18

• Polyacrylamide has smaller pores allowing molecules to migrate

Question 19

Why is SDS used?

Answer 19

• Proteins, unlike DNA which is negatively charged naturally, have differing charges due to the functional group.
• Therefore, we need to use SDS to unwind the protein and make the charge of the protein negative so it will migrate through gel.

Question 20

What type of gel is in SDS PAGE and what does it do?

Answer 20

• The gel is a denaturing and breaks the non covalent bonds to unwind the protein

Question 21

What is electrophoresis and how do molecules migrate?

Answer 21

Migration of the ions through a matrix in an electric field
- Charged molecules migrate in an electric field with a velocity proportional to its overall density, size, and shape.

Question 22

What 2 things are molecular separation based on?

Answer 22

Sieving effect: the amount of space in the gel
- Proportional to the percentage of acrylamide percentage. Similar to agarose gel, the greater the percentage the smaller the pores.
Electrophoretic Mobility: depends on the charge.
- The larger the protein, the longer it takes to migrate, and need to make sure there is a uniform charge so it moves based on the charge.

Question 23

What are the charges in SDS? What does it impart? What is the proportion of SDS to protein?

Answer 23

Sodium is negative and sulfate is positive
The sds imparts a negative charge to the protein
1.4 g SDS/g of Protein

Question 24

What is the solubility of dodecyl? What occurs even after treatment with it? What is the protein coated with and why?

Answer 24

Dodecyl is hydrophobic
- Unwraps the protein by disrupting the 3d structure (tertiary structure) of the protein
The protein we will use is globular and has a tertiary structure and therefore needs to be unwrapped
- Even after treatment of dodecyl, due to disulfide bonds from the amino acid cysteine there are still bonds remaining
- The entire protein is coated in sulfate from the dodecyl treatment.

Question 25

How are disulfide bonds broken?

Answer 25

• To break the bonds use: DTT (Dithiothreitol or B – Mercaptoethanol and apply heat).
• Breaks disulfide bond

Question 26

What is done after dentauring the protein? What is the machine similar to? What is the arrangement of the wells? How many bands are ideal? Why can’t you get the ideal? What do you obtain?

Answer 26

• After denaturing the protein, run the SDS gel
• Similar to gel electrophoresis machine
• In the first well, place a protein ladder
• In other wells, run protein sample
• Ideally get one band but proteins are not 100% purified.
  In the gel, can obtain smears

Question 27

What are the 4 levels of protein structure? Which ones do all proteins have? What is required for the highest structure?

Answer 27

Proteins have 4 levels of structure: primary, secondary, tertiary, and quaternary.
- All proteins have the first 3, for quaternary there needs to be 2 polypeptide subunits

Question 28

What is done after the gel is done? Define the second step.

Answer 28

• Cut the gel
• Place ½ the gel in the coomassie blue (stain)
• Place ½ gel for blotting
• blotting: transfer the protein from the gel to a PVDF membrane (Western Blotting).

Question 29

In the immuoassay what is done? Why can it be difficult?

Answer 29

• Wash/ treat the membrane with an antibody solution
• Uses an immunoglobulin (antibody) specific to the protein
• The antibody binds to the antigen in the protein.
• However, difficult to find an antibody specific to the protein

Question 30

In the present experiment, what antibody do we use for the gene and what will it bind to? What is thething it binds to called and what are the two things it is used for?

Answer 30

*In the present experiment, use an antibody specific for the CR gene. The antibody will bind to the his-tag region of the CR protein.
his tag is used for identification and purification
* Can use the antibody for the his tag for identification (primary antibody)
* The mechanisms is there in a protein (CR protein)
* The his tags of the CR protein bind to the his tag antibody to create a Antigen - Antibody complex
* Need to identify the complex
* Add a secondary antibody (enzyme)
* The his tags of the CR protein bind to the his tag antibody to create a Antigen - Antibody complex

Question 31

What are the components of the immunoblotting assay in the present experiment? What are you trying to identify? What is the other antibody called and what does it allow for? What doyou observe if everything is a success? Where is everything done?

Answer 31

• Need to identify the complex
• Add a secondary antibody (enzyme)
• Overall the component contains the CR Enzyme, secondary antibody (enzyme), and primary antibody.
• Since there is a secondary antibody, can use a substrate with a color releasing agent so when it reacts with the secondary enzyme a color is released.
• A band will be present.
• Everything is done on the PVDF membrane.

Question 32

Define epitope? What is the eptiope in the present experiment and what happens to it?

Answer 32

• In the present experiment, the his-tag is the epitope and we use the antibody to it.

Question 33

What does the expressed protein in the present experiment have? What binds there and what does it have? What are the two parts of the protein and what is it called? What does the tag have and what does it allow to make easier?

Answer 33

Currently the expressed proteins will have a hist tag(6 histidine)
* the antibody binds there
* The antibody has fluorescence
The protein is a recombinant protein, the CR and the aspects of the pet29b vector (his tag)
The tag allows for easier protein detection and purification.