Microbial-based biosensors

A hormone biosensor comprising:

• a) a microbial allosteric transcription factor polypeptide conjugated to one or more first fluorescent molecules of a Fluorescence Resonance Energy Transfer (FRET) pair, the allosteric transcription factor comprising one or more ligand binding domains (LBDs) and one or more DNA binding domains (DBDs),
• b) a nucleic acid probe conjugated to one or more second fluorescent molecules of the FRET pair, the nucleic acid probe having a sequence comprising one or more transcription factor binding domains (TFBDs) that is specific to the DBD of the transcription factor,
• wherein in the absence of a hormone of interest, the DBD of the microbial allosteric transcription factor is bound to the TFBD of the nucleic acid probe, and the first fluorescent molecule and the second fluorescent molecule of the FRET pair emits a fluorescent signal, and in the presence of the hormone of interest, the hormone binds to the ligand binding domain (LBD) resulting in a conformational change that decreases the affinity of the DBD for the TFBD such that the DBD dissociates from the TFBD, resulting in the first fluorescent molecule and the second fluorescent molecule of the FRET pair no longer emitting a fluorescent signal (OFF biosensor);
• or
• wherein in the absence of the hormone of interest, the DBA of the microbial allosteric transcription factor is not bound to the TFBD of the nucleic acid probe, and the first fluorescent molecule and the second fluorescent molecule of the FRET pair does not emit a fluorescent signal, and in the presence of the hormone of interest, the hormone binds to the ligand binding domain (LBD) resulting in a conformational change that increases the affinity of the DBD for the TFBD such that the DBA binds to the TFBD, resulting in the first fluorescent molecule and the second fluorescent molecule of the FRET pair emitting a fluorescent signal (ON biosensor),
• wherein the first fluorescent molecule or second fluorescent molecule, or both is a quantum dot (QD).

A hormone biosensor comprising:

• a) a microbial allosteric transcription factor polypeptide conjugated to one or more electroactive molecules, the allosteric transcription factor comprising one or more ligand binding domains (LBDs) and one or more DNA binding domains (DBDs),
• b) a nucleic acid probe attached to a conducting surface, the nucleic acid probe having a sequence comprising one or more transcription factor binding domains (TFBDs) that is specific to the DBD of the microbial allosteric transcription factor polypeptide
• wherein in the absence of a hormone of interest, the DBD of the microbial allosteric transcription factor is bound to the TFBD of the nucleic acid probe, resulting in an increase in flow of electrons from the electroactive molecule to the conductive surface which is detected by the presence of, or an increase in a current across the surface, and in the presence of the hormone of interest, the hormone binds to the ligand binding domain (LBD) resulting in a conformational change in the microbial allosteric transcription factor that decreases the affinity of the DBD for the TFBD such that the DBD dissociates from the TFBD, resulting a decrease in the flow of electrons from the electroconductive molecule to the conductive surface which is detected by a decrease in current across the surface (OFF biosensor);
• or
• wherein in the absence of a hormone of interest, the DBA of the microbial allosteric transcription factor is not bound to the TFBD of the nucleic acid probe, and no flow of electrons from the electroactive molecule to the conductive surface which is detected by absence of a current across the surface, and in the presence of the hormone of interest, the hormone binds to the ligand binding domain (LBD) resulting in a conformational change in the microbial allosteric transcription factor that increases the affinity of the DBD for the TFBD such that the DBA binds to the TFBD, resulting in an increase in flow of electrons from the electroactive molecule to the conductive surface which is detected by the presence of, or increase in a current across the surface (ON biosensor).

A system for detecting an analyte of interest in a sample;

• a) contacting the sample with a biosensor comprising:
• i. allosteric transcription factor polypeptide conjugated to one or more first fluorescent molecules of a Fluorescence Resonance Energy Transfer (FRET) pair, the allosteric transcription factor comprising one or more ligand binding domains (LBDs) and one or more DNA binding domains (DBDs), and
• ii. a nucleic acid probe conjugated to one or more second fluorescent molecules of the FRET pair, the nucleic acid probe having a sequence comprising one or more transcription factor binding domains (TFBDs) that is specific to the DBD of the transcription factor,
• wherein the biosensor is an OFF biosensor such that in the absence of an analyte of interest, the DBD of the transcription factor is bound to the TFBD of the nucleic acid probe, and the first fluorescent molecule and the second fluorescent molecule of the FRET pair emits a fluorescent signal, and in the presence of the analyte of interest, the analyte binds to the ligand binding domain (LBD) resulting in a conformational change in the allosteric transcription factor polypeptide that decreases the affinity of the DBD for the TFBD such that the DBD dissociates from the TFBD, resulting in the first fluorescent molecule and the second fluorescent molecule of the FRET pair no longer emitting a fluorescent signal (OFF biosensor);
• or
• wherein the biosensor is an ON biosensor such that in the absence of an analyte of interest, the DBA of the transcription factor is not bound to the TFBD of the nucleic acid probe, and the first fluorescent molecule and the second fluorescent molecule of the FRET pair does not emit a fluorescent signal, and in the presence of the analyte of interest, the analyte binds to the ligand binding domain (LBD) resulting in a conformational change in the allosteric transcription factor polypeptide that increases the affinity of the DBD for the TFBD such that the DBA binds to the TFBD, resulting in the first fluorescent molecule and the second fluorescent molecule of the FRET pair emitting a fluorescent signal (ON biosensor), and
• wherein the first fluorescent molecule or second fluorescent molecule, or both is a quantum dot (QD);
• b) measuring the fluorescence in the sample, and identifying the presence of the analyte of interest when one of the following occurs:
• a) a decrease in FRET signal is detected when the biosensor is an OFF biosensor;
• b) an increase in FRET signal is detected when the biosensor is an ON biosensor.