Anti-sigmas for programmable transcriptional regulation
The invention relates to anti-sigma factors (“anti-sigmas”) that bind to sigma factors and block activation of transcription. Anti-sigmas and their cognate sigma factors provide a highly effective mechanism for regulating gene expression in genetic circuits.
1. A recombinant synthetic genetic circuit comprising:
an extracytoplasmic function (ECF) sigma factor;
an anti-sigma factor that is the cognate of the ECF sigma factor; and
a promoter that is recognized by the ECF sigma factor,
wherein the ECF sigma factor and anti-sigma factor are orthogonal, and
wherein the ECF sigma factor and/or the anti-sigma factor are genetically engineered.
2. The recombinant synthetic genetic circuit of claim 1 , wherein the ECF sigma factor and/or the promoter are chimeric, and/or wherein the recombinant genetic circuit is expressed within a host cell and wherein the host cell is a prokaryotic cell or a eukaryotic cell, and/or wherein the ECF sigma factor and/or anti-sigma factor are codon-optimized for expression in the host cell, and/or wherein the ECF sigma factor is selected from the group consisting of ECF01-ECF43, and/or wherein the anti-sigma factor is selected from the group of anti-sigma factors contained within Table 1.
3. The recombinant synthetic genetic circuit of claim 1 , wherein the recombinant synthetic genetic circuit comprises a combination of logic gates.
4. The recombinant synthetic genetic circuit of claim 3 , wherein the logic gates are selected from the group consisting of AND, NAND, NOR, OR, NOT, XOR, EQUALS, AND, IMPLIES, and ANDN gates.
5. The recombinant synthetic genetic circuit of claim 4 , wherein the AND gates comprises an ECF sigma factor and a promoter that is recognized by the ECF sigma factor.
6. The recombinant synthetic genetic circuit of claim 1 , wherein the recombinant synthetic genetic circuit is a component of a synthetic genetic switch.
7. The recombinant synthetic genetic circuit of claim 6 , wherein the synthetic genetic switch is bistable.
8. The recombinant synthetic genetic circuit of claim 1 , wherein the recombinant synthetic genetic circuit is a component of a pulse generator.
9. A system comprising a plurality of recombinant synthetic genetic circuits of claim 1 .
10. A host cell comprising a heterologous genetic circuit comprising
an extracytoplasmic function (ECF) sigma factor;
an anti-sigma factor that is the cognate of the ECF sigma factor; and
a promoter that is recognized by the ECF sigma factor,
wherein the ECF sigma factor and anti-sigma factor are orthogonal.
11. The host cell of claim 10 , wherein the ECF sigma factor, the anti-sigma factor and/or the promoter are genetically engineered, and/or wherein the ECF sigma factor and/or the promoter are chimeric, and/or wherein the host cell is a prokaryotic host cell, and/or wherein the genetic circuit comprises one or more logic gates selected from the group consisting of AND, NAND, NOR, OR, NOT, XOR, EQUALS, AND, IMPLIES, and ANDN gates.
12. The host cell of claim 11 , wherein the AND gates comprises a sigma factor and a sigma factor target DNA sequence, and/or wherein two or more logic gates are combined by having the output promoter of an upstream gate serve as the input promoter of a downstream gate.
13. A library comprising two or more anti-sigma factors, wherein each anti-sigma factor selectively binds to one or more cognate ECF sigma factors, wherein the anti-sigma factor and the one or more cognate ECF sigma factors are orthogonal.
14. The library of claim 13 , further comprising one or more cognate ECF sigma factors.
15. The library of claim 14 , further comprising one or more promoters that are recognized by the one or more cognate ECF sigma factors.
16. The library of claim 15 , wherein the anti-sigma factors, cognate ECF sigma factor and/or promoter are genetically engineered, and/or wherein the cognate ECF sigma factor and/or promoter is chimeric, and/or wherein the cognate ECF sigma factors and/or anti-sigma factors are codon-optimized for expression in a host cell, and/or wherein the cognate ECF sigma factors are under the control of an inducible promoter, and/or wherein the cognate ECF sigma factor is selected from the group consisting of ECF01-ECF43, and/or wherein the anti-sigma factor is selected from the anti-sigma factors within Table 1.