The soluble form of the sensor was made by tethering together the two fluorescent proteins, ECFPenhanced cyan fluorescent protein, and Citrinea type of yellow fluorescent protein. This put the two proteins in proximity to one another so that the fluorescent emission of ECFPenhanced cyan fluorescent protein might be absorbed and re-emitted by Citrinea type of yellow fluorescent protein. GltIglutamate-binding protein was interposed between the two proteins. This protein from Escherichia coli binds glutamate and changes its shape when it has done so. The resulting protein emits yellow-green fluorescence to a degree dependent on the presence of glutamate, as described in the Introduction.
An artificial gene was made encoding these three proteins without interruption so that a single protein would be synthesized when the gene was expressed in the cell. An additional six amino acids (six histidines) were encoded at the beginning of the gene to facilitate purification of the protein in high amounts. This was accomplished by cloning into a special-purpose plasmid, pRSETB, used to express large amounts of protein in E. coli.