Exp3: Pre-prac Blue White Screening

Question 1

What is the overall role/aim of Blue White Screening?

Answer 1

To identify which colonies have empty vectors (plasmid) or have vectors with the desired insert in them

Question 2

Which gene (and enzyme) does Blue White Screening take advantage of?

Answer 2

Beta-galactosidase gene (LacZ)
(B-gal)

Question 3

WHich plasmid is the B-gal fragment located on in our experiment of Blue White Screening?

Answer 3

pUC19

Question 4

What reagents are contained in the agar of a Blue White Screen?

Answer 4

Ampicillin
IPTG
X-gal

Question 5

What is the role of AMP in the agar of a Blue White Screen?

Answer 5

Amp kills off bacteria without the plasmid (because they don’t have the resistance gene located within the plasmid)

Question 6

What is the role of IPTG in the agar of a Blue White Screen?

Answer 6

Changed the shape of the repressor bound to the operator sequence of LacZ
=> dissociation of the repressor
=> increased expression LacZ
i.e. activator of lac operon and beta-gal expression

Question 7

What happens to X-gal when beta-gal is present?

Answer 7

glycosidic bonds in X-gal break
one product oxidises and dimerises
=>blue product

Question 8

What advantage is there to using X-gal in the indication of an insert being present or not in a vector plasmid?

Answer 8

It has a colorimetric change from colourless to blue (therefore easy to identify)
Also does not select against (kill) colonies that do not contain the gene insert if these are required

Question 9

Describe the very basic structure of beta-gal ?

Answer 9

4xidentical subunits
aka homotetrameric

Question 10

What is the outcome of Blue White Screen if the bacteria have no plasmid?

Answer 10

No colony formed because they do not have the Ampicillin resistance gene and therefore die before forming colonies

Question 11

What is the outcome of a Blue White Screen if bacteria contain pUC19 with no insert?

Answer 11

They survive and form beta-gal (which interacts with X-gal) forming blue colonies

Question 12

What is the outcome of a Blue White Screen if bacteria contain pUC19 and the insert at the MCS?

Answer 12

Disruption at the alpha peptide gene fragment (within the Lac operon)
causing no expression of the beta-gal
=> white colony

Question 13

In a Blue White Screening test, what result would be considered the most successful? why?

Answer 13

white colonies
indicates successful transformations with both plasmid and the desired insert

Question 14

What is the name of the gene that codes for beta-galactosidase (betra-gal)?

Answer 14

LacZ

Question 15

What does MCS stand for on a vector plasmid?

Answer 15

multiple cloning site