Reporter Gene Technology Flashcards
What is a reporter gene?
A reporter gene is a gene that researchers attach to a gene of interest to monitor its expression or repression.
What are the prerequisites for a reporter gene?
- non toxic
- not present in cell
- sensitive
- easily quantifiable
- rapid activity
What is transcriptional fusion? What does it show us?
Reporter gene is attached to a promoter of interest.
Shows where/when promoter of interest is turned on.
What is translational fusion?
Reporter gene is fused to gene of interest.
When gene of interest is transcribed and translated, so is reporter gene.
Protein will express reporter gene product.
How can reporter genes be used to identify if gene of interest has been taken up during transformation?
gene of interest fused to reporter protein.
linked to same promoter.
if reporter gene product showing in bacteria, gene of interest is also present.
How does the gene reporter dye Beta-galactosidase work?
LacZ gene is attached to promoter of interest.
When promoter is activated, LacZ produces Beta-galactosidase.
Beta-galactosidase cleaves X gal to produce blue precipitate.
What is luminescence?
Luminescence is the emission of light by a substance that has not been heated.
What is bioluminescence?
Emission of light resulting from a biochemical reaction
What is fluorescence?
Type of luminescence caused by absorbance of light at one wavelength followed by emission at a longer wavelength.
Which enzyme/protein do fireflies use to luminesce?
Firefly luciferase protein
What is the reaction that firefly luciferase mediates?
Luciferase oxidises luciferin to create oxyluciferin and causes emission of light.
Which two molecules does the luciferase reaction require?
ATP and magnesium
What is the structure of GFP?
beta barrel structure
What region is at the centre of the beta barrel structure of GFP?
chromophore region
What makes up the chromophore region of GFP?
three amino acids in a specific sequence
Which two reactions occur in the unique environment of the beta barrel structure? (to form mature GFP)
cyclisation and dehydration
What is a cyclisation reaction?
where the amino acids form a ring structure through new bonding
What is the length of the emission released by mature GFP?
511nm
How is the brighter EGFP produced?
by replacing Serine with Threonine
How is EBFP produced?
by replacing Tyrosine with Histidine
How is ECFP produced?
by replacing Tyrosine with Tryptophan
How is EYFP produced?
by adding a second Tyrosine ring
Which three amino acids make up GFP?
Serine, Tyrosine and Glycine
Which amino acid of GFP can never be changed?
glycine as it allows the orientation of the triad to form the fluorophore
Which animal did scientist find a second fluorescent protein?
DsRed was found in a mushroom reef coral
