A technique used to locate and isolate Fur sites via cloning. The genome of a target organism is digested with restriction enzymes, and ligated into high copy plasmids to create a plasmid library, which is then cloned into a cell line with a fur-repressed reporter. In cells where the introduced high copy plasmid contains a fur site, fur will be titrated away from it's binding site on the reporter (and throughout the genome), marking the colony. The cell can then be isolated, the plasmid extracted and sequenced for further study.
Quantitative PCR (or quantitative real time polymerase chain reaction) is a modification of the conventional PCR reaction used to amplify and simultaneously quantify a targeted DNA molecule. In its more standard incarnation, a inactive fluorescent dye is added to the PCR mix. This dye is activated upon binding double-stranded DNA; as the PCR iterates, there is more and more dsDNA and therefore more fluorescence intensity.
Regulated genes for each binding site are displayed below. Gene regulation diagrams
show binding sites,
both positively and negatively regulated
genes, genes with unspecified type of regulation.
For each indvidual site, experimental techniques used to determine the site are also given.