Biological state machines
Provided herein are recombinase-based frameworks for building state machines in vitro and in vivo by using chemically controlled DNA excision and inversion operations to encode state in DNA sequence.
1. A system, comprising:
(a) n serine recombinases, wherein n is greater than 2; and
(b) an engineered nucleic acid comprising n(n−1) pairs of cognate recombination recognition sites (RRS),
wherein the n(n−1) pairs of RRSs of (b) are arranged in an overlapping configuration such that the two RRSs of each pair of the n(n−1) pairs of RRSs are separated from each other by at least one RRS of another pair of the n(n−1) pairs of RRSs, and wherein recombination between the two RRSs of each pair of the n(n−1) pairs of RRSs either inverts or excises at least one RRS of another pair of the n(n−1) pairs of RRSs.
2. The system of claim 1 , wherein n is greater than or equals 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.
3. The system of claim 1 , wherein the n serine recombinase is selected from Bxb1, Tp901, A118, PhlF and AraC.
4. The system of claim 1 , wherein the RRSs are selected from an attB site, an attP site, an attB site modified to include a CA dinucleotide, an attP site modified to include a CA dinucleotide, an attB site modified to include a GT dinucleotide, an attP site modified to include a GT dinucleotide, an attB site modified to include a AG dinucleotide, an attP site modified to include a AG dinucleotide, an attB site modified to include a TC dinucleotide, an attP site modified to include a TC dinucleotide, an attB site modified to include a AA dinucleotide, an attP site modified to include a AA dinucleotide, an attB site modified to include a GG dinucleotide, and an attP site modified to include a GG dinucleotide.
5. The system of claim 1 , wherein the system further comprises at least one engineered nucleic acid comprising at least one promoter operably linked to a nucleotide sequence encoding at least one of the n serine recombinases.
6. The system of claim 5 , wherein the at least one promoter is inducible.
7. The system of claim 6 , wherein the at least one promoter is selected from P PhlF , P BAD and P LtetO .
8. The system of claim 1 , wherein the engineered nucleic acid of (b) further comprises a nucleotide sequence encoding a detectable molecule.
9. The system of claim 8 , wherein the detectable molecule is a fluorescent molecule.
10. A system, comprising:
(a) three serine recombinases; and
(b) an engineered nucleic acid comprising six pairs of cognate recombinase recognition sites (RRSs),
wherein the six pairs of RRSs of (b) are arranged in an overlapping configuration such that the two RRSs of each pair of the six pairs of RRSs are separated from each other by at least one RRS of another pair of the six pairs of RRSs, and wherein recombination between the two RRSs of each pair of the six pairs of RRSs either inverts or excises at least one RRS of another pair of the six pairs of RRSs.
11. A system, comprising:
(a) four serine recombinases; and
(b) an engineered nucleic acid comprising twelve pairs of cognate recombinase recognition sites (RRSs),
wherein the twelve pairs of RRSs of (b) are arranged in an overlapping configuration such that the two RRSs of each pair of the twelve pairs of RRSs are separated from each other by at least one RRS of another pair of the twelve pairs of RRSs, and wherein recombination between the two RRSs of each pair of the twelve pairs of RRSs either inverts or excises at least one RRS of another pair of the twelve pairs of RRSs.
12. An isolated cell comprising the system of claim 1 .
13. The isolated cell of claim 12 , wherein the isolated cell is a bacterial cell or a mammalian cell.
14. The isolated cell of claim 12 , wherein the isolated cell is a stem cell.