Thermal bioswitches and related genetic circuits, vectors, cells, compositions, methods and systems

A method to engineer a TlpA dimer, the method comprising:

• providing a TlpA dimer having a melting temperature Tm0 and a bioswitch temperature Tbs0 with Tbs0=Tm0=0° C. to 5° C., the TlpA dimer comprising two TlpA monomers, each having SEQ ID NO:
• 461, and comprising
• a TlpA DNA binding domain of SEQ ID NO:
• 472 and
• a TlpA coiled coil temperature sensing domain having a temperature sensing amino acid sequence comprising alpha-helical heptad repeats having SEQ ID NOs:
• 473 to 484 and SEQ ID NO:
• 486, each alpha-helical heptad repeat having at least one amino acid residue at each register position a, b, c, d, e, f, and g,
• replacing in the TlpA coiled coil temperature sensing domain of at least one TlpA monomer, at least one of
• a polar amino acid in at least one of position b, position e, and position g of at least one alpha-helical heptad repeat, with a hydrophobic amino acid;
• an amino acid in at least one of position a and position d of at least one alpha-helical heptad repeat, with an amino acid configured to increase or decrease hydrophobic packing between amino acid residues in positions a and/or d of corresponding alpha-helical heptad repeats in the TlpA dimer, and
• an amino acid in at least one of position e and g of at least one alpha-helical heptad repeat with an amino acid configured to increase or decrease coulombic repulsion between corresponding amino acid residues in positions a, d, e and/or g of corresponding heptad repeats in the TlpA dimer;
• wherein the replacing obtains an engineered TlpA dimer variant having a melting temperature Tmm lower or higher than Tm0 and a bioswitch temperature Tbsm lower or higher than Tbs0.