US 11,959,127 B2
Thermal bioswitches and related genetic circuits, vectors, cells, compositions, methods and systems
Mikhail G. Shapiro, Los Angeles, CA (US); Dan Piraner, Pasadena, CA (US); Mohamad H. Abedi, Azusa, CA (US); Brittany Moser, Irvine, CA (US); and Audrey Lee-Gosselin, Pasadena, CA (US)
Assigned to California Institute of Technology, Pasadena, CA (US)
Filed by CALIFORNIA INSTITUTE OF TECHNOLOGY, Pasadena, CA (US)
Filed on Mar. 17, 2021, as Appl. No. 17/204,782.
Application 17/204,782 is a continuation of application No. 15/384,254, filed on Dec. 19, 2016, granted, now 10,975,420.
Claims priority of provisional application 62/269,715, filed on Dec. 18, 2015.
Prior Publication US 2021/0277453 A1, Sep. 9, 2021
Int. Cl. C12Q 1/68 (2018.01); B01L 7/00 (2006.01); C12N 15/10 (2006.01); C12N 15/63 (2006.01); C12N 15/82 (2006.01); C12Q 1/6825 (2018.01); G01N 33/53 (2006.01); C12N 15/00 (2006.01)
CPC C12Q 1/6825 (2013.01) [B01L 7/52 (2013.01); C12N 15/1055 (2013.01); C12N 15/63 (2013.01); C12N 15/8217 (2013.01); G01N 33/53 (2013.01); C12N 15/00 (2013.01); C12Q 2525/30 (2013.01)] 22 Claims
 
1. 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.