A reporter gene fusion allows the studying of transcriptional output. There are a several different commonly used reporter genes:
- lacZ codes for beta-galactosidase. A simple colorimetric assay can then be used to quantify transcriptional levels. As beta-galactosidase is highly stable, this provides cumulative transcriptional activity.
- lux codes for luciferase. This is a fluorescent protein that does not require external illumination, but is only functional in aerobic bacteria, as it requires oxygen to function. Luciferase is unstable, and so provides real-time measurements and not cumulative.
- cat codes for chloramphenicol acetyltransferase. This is a protein providing resistance to chloramphenicol (an antibiotic). This can be used to screen bacteria on agar containing chloramphenicol, allowing simple screening.
- GFP is a widely used fluorescent protein, which can be used to identify protein synthesis sites. GFP does require illumination by UV light to fluoresce.
To fuse a reporter gene to the gene of interest, it should be inserted within the same operon. This allows for the expression of the reporter gene at the same time as the gene of interest (GOI). Where the reporter is inserted under the same promoter as the GOI, it will be expressed as a single protein product with the GOI and reporter in the same molecule. This is due to the lack of splicing or other post-transcriptional modification, as well as the ability of bacteria to produce polycistronic transcripts.
Using a technique like lambda red to insert the reporter gene into the desired location would be a potential cloning strategy that could be used. This would allow the highly specific targeting of homologous recombination, enabling the reporter gene to be inserted under the same promoter for a translational fusion.
This process, of fusing a reporter gene to a gene of interest, is beneficial in determining promoters active in vivo, allowing vaccine candidate screening in model animals. (Using fluorescence aggregated cell sorting).