MBB 267 Week 6: Corrigan 4 Flashcards
What are fuse reporter genes used for?
They are genes that determine if a promoter region is switched on or off.
a. To test genes, can be targeted or randomly.
How are reporter genes measured?
Either;
a. Simple stranded enzyme assay (e.g. LacZ)
b. Other readout (fluorescence GFP)
What are the types of known reporter genes?
2 types;
a. Multiple-copy reporter on a plasmid
b. Single-copy reporter on the chromosome
What do unknown reporter genes use?
Using;
a. Modified transposons
b. Promoter traps
What is promoter trapping?
Promoter trapping is a method developed that uses the promoter regions as bait to trap proteins of interest and then purified using column chromatography.
How are transcriptional fused reporter genes used?
Transcriptional fusions; uses the promoter of the original gene but uses the translational elements (Ribosome binding site, Open reading frame and terminator sequence) of the reporter gene, this is to drive expression of the reporter.
How are translational fused reporter genes used?
Translational fusions; uses the promoter and the ribosome binding site of the original test gene, but uses the open reading frame and the terminator sequence of the reporter gene.
What is translational fused reporter genes used for?
Allows simple determination of gene expression in different environments
What is a chimeric protein fusion?
Fusion proteins or chimeric proteins are proteins created through the joining of two or more genes that originally coded for separate proteins
What are the commonly used reporter genes?
Include;
a. lacZ
b. cat
c. lux
d. gfp
What does the lacZ reporter gene do?
encodes β-galactosidase, stable therefore measures cumulative promoter activity, simple colorimetric (assay turns from clear to yellow colour)
What does the cat reporter gene do?
encodes chloramphenicol acetyltransferase, easily assayed and selectable – if expressed bacteria are resistant to chloramphenicol (easy screening)
What does the lux reporter gene do?
encodes luciferase, allows real-time measurement, needs oxygen to function (anaerobic)
What does the gfp reporter gene do?
encodes green fluorescent protein, cell imaging for protein expression and localization, needs oxygen to mature (anaerobic)
Transcriptional vs translational fusions?
Compare
a. Transcriptional fusion: the 5’ promoter region of a gene is fused with an upstream promoter-less gene, which retains the promoter-less’s ribosome binding site.
i. Produces the protein for the promoter-less gene and the N-terminal of the original fused gene
b. Translational fusion: the original 5’ promoter region is fused to the coding region of the next gene. The promoter and ribosome binding site are from the original gene. This produces a single hybrid protein
What does a single copy reporter fusion do?
Process;
a. Ligate your promoter of interest (POI) into a vector (e.g. pRS415) that contains a promoterless lacZYA operon (with translation initiation sequences) and a selectable marker (bla for ampicillin resistance).
b. Transform an E. coli lacZ mutant strain and select for ampicillin resistance in the presence of X-gal to detect β-galactosidase synthesis.
c. This is now a plasmid in the cytoplasm. Need to get it onto the c/some
d. infect transformants with bacteriophage RS45 – a modified lambda (l) phage.
e. It’s been modified to contain a truncated bla gene (bla’) and the lacZ cistron deleted for the promoter-proximal two-thirds (lacZSC), with wild-type versions of the lacY and lacA.
f. The lac-bla sequence of RS45 is homologous to sequences of your pRS415 derivative. This permits recombination (X) to generate a phage lysate containing bacteriophage genomes carrying the gene fusion.
g. The bacterial strain to be investigated is infected with the lysate carrying the gene fusion and grown on plates containing X-gal. Blue plaques are restreaked. Insertion of the prophage at the att is confirmed by PCR.
How do random single copy reporter fusions work?
Donor plasmid contains modified transposon (TN917-LTV3) and a temperature sensitive origin of replication.
How does GFP promoter trap method work?
Method;
a. Promoter library preparations; fragment chromosome
b. Transform DNA into bacteria
c. Use FACS (fluorescently actively cell sorter); clones that are active (fluorescent) are removed.
d. Inactive clones are inoculated into an infection model system (e.g. Mice)
e. Recover bacteria, separate using GACS and collect the active bacteria.
f. Bacteria are regrown again in culture?
g. Then rescreen the inactive bacteria agai
