Biosensors are devices leveraging biological modules for the detection of molecules of interest, including biomarkers of inflammation and cancer. Using elements capable of detecting small molecules, such as transcription factors, aptamers, and antibodies, they specifically and sensitively interact with a target analyte, producing a detectable response. By applying traditional and synthetic biology approaches, these modules can be engineered to detect different molecules. A whole-cell biosensor (WCB) is a subtype of biosensor in which a whole living organism with an active metabolism is used to detect a compound. WCBs based on synthetic transcription factors are adaptable and accessible sensing tools for the detection of small soluble molecules. In this chapter, we describe a pipeline for the construction of a prokaryotic biosensor strain bearing a genetic circuit that integrates a custom-made transcription factor. As an example, we use the strain Escherichia coli MG1655 (pCKT-Sphnx, pHC_DYO-LacI-R), which has been engineered to detect neuraminic acid, an oral cancer biomarker present in saliva.