Reporter gene fusions

Question 1

What reporter gene fusions are done on known genes?

Answer 1

-mulit-copy reporter on plasmid (not v. accurate)
-single-copy reporter integrated into chromosome (better)

Question 2

What reporter gene fusions are done on known genes?

Answer 2

-modified transposons (incl reporter gene)
-promotor traps

Question 3

What are transcriptional fusions?

Answer 3

Fusion of gene of interest and reporter gene where the promotor comes from the gene and the ribosome binding site for the expression of the reporter comes from the reporter gene
-Monitors transcription of gene

Question 4

What are translational fusions?

Answer 4

Fusion of gene of interest and reporter gene where both the promotor and ribosome binding site comes from the gene of interest for the expression of the reporter gene
-Monitors transcription and translation

Question 5

Name some commonly used reporter genes

Answer 5

-lacZ
-cat
-lux
-gfp

Question 6

How is lacZ used as a reporter gene?

Answer 6

-encodes β-galactosidase (v stable)
-can measure cumulative promotor activity (all of β-galactosidase produced) in colorimetric assay

Question 7

How is cat used as a reporter gene?

Answer 7

-encodes chloromphenicol acetyltransferase (antibiotic resistance)
-screen for antibiotic resistance on plates

Question 8

How is lux used as a reporter gene?

Answer 8

-encodes luciferase
-luciferase is unstable so allows real-time measurement

N/B: Need oxygen (aerobic bact)

Question 9

How is gfp used as a reporter gene?

Answer 9

-encodes green fluorescent protein
-imaging of fluorescence

N/B: Oxygen needed for protein to mature