Bacterial Transformation: the PGLO system

Question 1

Bacterial Transformation: the PGLO system illustrates what?

Answer 1

• Bacterial transformation,
• use of an antibiotic-selective medium to identify transformed cells.
• the operon as a mechanism of microbial genetics regulation

Question 2

What is bacterial transformation?

Answer 2

the process by which component bacterial cells pick up DNA from the environment and make use of genes it carries

Question 3

What is used as a transforming solution in the PGLO system?

Answer 3

CaCl2

Question 4

What did we use to make our Escherichia coli cells competent?

Answer 4

a CaCl2 transforming solution and heat shock

Question 5

What are operons?

Answer 5

structural and functional genetic units of prokaryotes

Question 6

What are plasmids?

Answer 6

small, naturally occurring circular DNA molecules that possess only a few genes.

Question 7

What does the antibiotic resistance gene (bla) produces?

Answer 7

Produces an enzyme called B lactamase

Question 8

In what strain of bacteria was bacterial transformation first demonstrated?

Answer 8

Pneumococcus

Question 9

The promoter in the arabinose operon can be abbreviated as…?

Answer 9

Pbad

Question 10

The three structural genes on the arabinose operon are abbreviated as…?

Answer 10

araB, araA, and araD

Question 11

What part of the pGLO plasmid is necessary for DNA replication?

Answer 11

ori (the origin of replication)

Question 12

Which section of the pGLO plasmid produces Beta-lactamase, which allows it to hydrolyze/become resistant to certain antibiotics including penicillin and ampicillin?

Answer 12

bla

Question 13

Which section of the pGLO plasmid is an indicator of gene transcription and cell transformation?

Answer 13

GFP

Question 14

In this procedure, we transferred the +DNA and -DNA tubes from ice, to hot water, to ice again. What was the purpose of this?

Answer 14

To make the cells permeable to the uptake of DNA

Question 15

Why don’t the two -pGLO plates fluoresce?

Answer 15

Because they do not contain the pGLO plasmid vector, which carries the GFP gene.

Question 16

Why does the LB/amp/ara +pGLO plate fluoresce under U.V. light, while the LB/amp +pGLO plate does not?

Answer 16

because it contains the DNA binding protein araC, while the LB/amp +pGLO plate does not. Therefore, the LB/amp/ara +pGLO plate was able to take up the GFP gene due to the fact that it contains the DNA binding protein.

Question 17

The culture on the left contains what gene carried by what vector?

Answer 17

the Green Fluorescent Protein (GFP) gene carried by the pGLO plasmid

Question 18

What is the regulatory molecule for the arabinose promoter? In the presence of arabinose, this molecule has a shape that allows RNA polymerase to bind to the promoter. Without arabinose, this molecule’s shape prevents RNA polymerase from binding to the promoter.

Answer 18

DNA binding protein (araC)

Question 19

What is the attachment site for RNA polymerase during transcription of the gene encoding GFP on the pGLO plasmid?

Answer 19

the arabinose promoter (Pbad)