Lab Final

Question 1

What is Microplusin?

Answer 1

Antimicrobial Peptide

Question 2

What are antimicrobial peptides?

Answer 2

Peptides that help attack bacteria

Question 3

Is the antimicrobial peptide that we are working with anionic or cationic?

Answer 3

Catatonic

Question 4

What organism is Microplusin derived from?

Answer 4

Isolated from the cell-free hemolymph and egg of the tick Boophilus microplus

Question 5

What is PRSETA?

Answer 5

It is a vector used for high level protein expression

Question 6

PRESTA has an antibiotic resistance gene to what antibiotic?

Answer 6

Ampicillin

Question 7

What are competent cells?

Answer 7

Cells that can uptake foreign DNA

Question 8

Define transformation.

Answer 8

Foreign DNA introduced into a cell

Question 9

What type of E. coli were used the transformation?

Answer 9

BL21

Question 10

What media was used to grow the transformed cells?

Answer 10

LB Media

Question 11

What antibiotic was used?

Answer 11

Ampicillin

Question 12

Describe the Lac Operon

Answer 12

Cluster of genes used to regulate lactose

Question 13

What is heat shock and why is it performed?

Answer 13

To form pores in the cell for DNA uptake

Question 14

What is IPTG? How does it work?

Answer 14

IPTG is an inducer that mimics lactose and is used to induce protein expression

Question 15

For how many hours did we incubate the induced protein after induction with IPTG?

Answer 15

4 hours

Question 16

True or False. In order to express a gene that is controlled by the T7 promoter, you need to add IPTG to block the function of the lac repressor.

Answer 16

True

Question 17

Name the method for colorimetric detection and total quantification of protein.

Answer 17

BCA assay

Question 18

How does the BCA assay work? What does BCA stand for?

Answer 18

bicinchoninic acid assay; works by measuring the level of amino acid composition of the protein.

Question 19

BCA is what type of reaction? Explain.

Answer 19

Colometric measure the absorption

Question 20

True or False. The total quantification of the protein was measured in absorbance

Answer 20

True

Question 21

What does SDS-PAGE stand for? How does it work?

Answer 21

Polyacrylamide gel electro

Question 22

What is SDS? What does it do to proteins?

Answer 22

Sodium deodecyl Sulfate gives a negative charge to proteins

Question 23

What are the metals used for greatest affinity for histidine?

Answer 23

Nickel

Question 24

How many histidine does PRSETA have?

Answer 24

6

Question 25

What does IMAC stand for?

Answer 25

Immobilized metal affinity chromatography

Question 26

How does the nickel resin column work? What does the histidine tag bind to?

Answer 26

His-tag binds tightly to the immobilized metal ions because the side chain of Histidine.

Question 27

What does imidazole bind to? What does imidazole compete with when loaded into the nickel resin column?

Answer 27

imidazole, has a specific binding affinity to metal ions (in this case, nickel II). As a result, the desired protein is binded tightly to the beads while other proteins flow through the column easily.

Question 28

What is bromo-phenol blue?

Answer 28

A pH indicator, a color marker to monitor the progress of polyacrylamide gel electrophoresis.