Modular nucleic acid-based circuits for counters, binary operations, memory, and logic

Patent metadata

Field	Value
Publication number	US-9624554-B2
Application number	US-201314135009-A
Country	US
Kind code	B2
Filing date	Dec 19, 2013
Priority date	Dec 22, 2008
Publication date	Apr 18, 2017
Grant date	Apr 18, 2017

Abstract

We have created novel engineered genetic counter designs and methods of use thereof that utilize DNA recombinases to provide modular systems, termed single invertase memory modules (SIMMs), for encoding memory in cells and cellular systems. Our designs are easily extended to compute to high numbers, by utilizing the >100 known recombinases to create subsequent modules. Flexibility in our engineered genetic counter designs is provided by daisy-chaining individual modular components, i.e., SIMMs together. These modular components of the engineered genetic counters can be combined in other network topologies to create circuits that perform, amongst other things, logic and memory. Our novel engineered genetic counter designs allow for the maintenance of memory and provide the ability to count between discrete states by expressing the recombinases between their cognate recognition sites.

First claim

We claim:

1. A single invertase memory module (SIMM) engineered nucleic acid molecule comprising a nucleic acid sequence encoding: a forward recombinase recognition site (RRS for ), an inverted promoter sequence (iP inv ), a recombinase sequence (RC), a reverse recombinase recognition site (RRS rev ), [RRS for -iP inv -RC-RRS rev ],a ribosome binding site (RBS), a protein degradation tag sequence (D), and a transcriptional terminator sequence (T), where the recombinase encoded by the recombinase sequence is specific for the forward and reverse recombination recognition sites.

2. An inducer engineered genetic counter nucleic acid molecule comprising a nucleic acid sequence comprising an inducible promoter sequence (iP A ), at least one single invertase memory module (SIMM), and an output nucleic acid sequence encoding an output product (OP), where the SIMM comprises a forward recombinase recognition sequence (RRS for ), an inverted inducible promoter sequence (iP 1,inv ), a ribosome binding site (RBS), a recombinase gene sequence (RC), a degradation tag sequence (D), a transcriptional terminator sequence (T), and a reverse recombinase recognition sequence (RRS rev ), such that the inducer engineered genetic counter nucleic acid molecule comprises a nucleic acid molecule encoding the following components:
iP A -[RRS 1,for -iP 1,inv -RBS-RC 1 -D-T-RRS 1,rev ] n -OP,
wherein iP A and the iP 1 of each SIMM are responsive to the same inducer, wherein the recombinase encoded by each at least one SIMM is specific for the forward and reverse recombinase recognition site of that SIMM, and wherein n is an integer value≧1 and wherein the engineered genetic counter nucleic acid molecule is introduced into a cellular or non-cellular system.

3. The engineered genetic counter of claim 2 , wherein the recombinase sequence and the forward and reverse recombinase recognition sites of at least one SIMM comprise a Cre recombinase sequence of SEQ ID NO: 1002, and LoxP recombinase recognition sites comprising the sequence of SEQ ID NO:1 and SEQ ID NO:2.

4. The engineered genetic counter of claim 2 , wherein the recombinase sequence and the forward and reverse recombinase recognition sites of one SIMM comprise a Flp recombinase sequence of SEQ ID NO:3 or SEQ ID NO: 1002 and FRT recombinase recognition sites comprising the sequence of SEQ ID NO: 4.

5. The engineered genetic counter of claim 2 , wherein the recombinase sequence and the forward and reverse recombinase recognition sites of one SIMM comprise a Flp recombinase sequence of SEQ ID NO:3 or SEQ ID NO: 1002 and FRT recombinase recognition sites comprising the sequence of SEQ ID NO: 5.

6. The engineered genetic counter of claim 2 , wherein the recombinase sequence and the forward and reverse recombinase recognition sites of one SIMM comprise a FimB recombinase of SEQ ID NO: 9 and recombinase recognition sites comprising the sequence of SEQ ID NO: 7 and SEQ ID NO: 8.

7. The engineered genetic counter of claim 2 , wherein the recombinase sequence and the forward and reverse recombinase recognition sites of one SIMM comprise a FimE recombinase of SEQ ID NO: 10 and recombinase recognition sites comprising the sequence of SEQ ID NO: 7 and SEQ ID NO: 8 respectively.

8. The engineered genetic counter of claim 2 , wherein the recombinase sequence of one SIMM comprises a Cre recombinase of SEQ ID NO: 1002, and the LoxP recombinase recognition sites of that SIMM comprise a sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17.

9. The engineered genetic counter of claim 2 , wherein at least one inducible promoter comprises a sequence selected from the group consisting of a PLtet0-1 promoter of SEQ ID NO: 33, a PBAD promoter of SEQ ID NO: 34, a PTrc-2 promoter of SEQ ID NO: 35, a PLiaco-i promoter of SEQ ID NO: 36, a PAiLaco-i promoter of SEQ ID NO: 37, a Piac/ara-i promoter of SEQ ID NO: 38, and a PLSICOΠ promoter of SEQ ID NO: 39.

10. The engineered genetic counter of claim 2 , wherein at least one inducible promoter comprises a sequence selected from the group consisting of SEQ ID NO: 320-SEQ ID NO: 842.

11. The engineered genetic counter of claim 2 , wherein the RBS sequence of at least one SIMM comprises a sequence that is selected from the group consisting of SEQ ID NO: 843-SEQ ID NO: 850.

12. The engineered genetic counter of claim 2 , wherein the RBS sequence of at least one SIMM comprises a sequence that is selected from SEQ ID NO: 851-SEQ ID NO: 994.

13. The engineered genetic counter of claim 2 , wherein the protein degradation tag sequence of at least one SIMM comprises a sequence that is selected from the group consisting of sequences that encode for the peptides of SEQ ID NO: 995-SEQ ID NO: 1001.

14. A single-inducer engineered genetic counter nucleic acid molecule comprising a nucleic acid sequence encoding: an inducible promoter sequence (iP A ), one single invertase memory module (SIMM), and an output nucleic acid sequence encoding an output product (OP), where the SIMM comprises a forward recombinase recognition sequence (RRS for ), an inverted inducible promoter sequence (iP 1,inv ), a ribosome binding site (RBS), a recombinase gene sequence (RC), a degradation tag sequence (D), a transcriptional terminator sequence (T), and a reverse recombinase recognition sequence (RRS rev ), such that the single-inducer engineered genetic counter nucleic acid molecule comprises a nucleic acid sequence encoding the following components:
iP A -[RRS 1,for -RBS-RC 1 -D-T-RRS 1,rev ]-OP,
wherein iP A and iP 1 are responsive to the same inducer, and wherein the recombinase encoded by the SIMM is specific for the forward and reverse recombinase recognition site of the SIMM, and
wherein the engineering genetic counter nucleic acid molecule is introduced into a cellular or non-cellular system.

15. The single-inducer engineered genetic counter of claim 14 , wherein the inducible promoter sequences iPA and iPi are responsive to arabinose.

16. The single-inducer engineered genetic counter of claim 14 , wherein the inducible promoter sequences iPA and iPi are PBAD promoter sequences.

17. The single-inducer engineered genetic counter of claim 14 , wherein the recombinase gene sequence (RCi) encodes an FIp recombinase and the forward (RRSfor) and reverse (RRSrev) recombinase recognition sites are FRTF and FRTR sites.

18. The single-inducer engineered genetic counter of claim 14 , wherein the output nucleic acid sequence encodes green fluorescent protein.

19. A single-inducer engineered genetic counter nucleic acid molecule comprising a nucleic acid sequence encoding: an inducible promoter sequence (iP A ), two single invertase memory modules (SIMMs), and an output nucleic acid sequence encoding an output product (OP), where each SIMM comprises a forward recombinase recognition sequence (RRS for ), an inverted inducible promoter sequence (iP inv ), a ribosome binding site (RBS), a recombinase gene sequence (RC), a degradation tag sequence (D), a transcriptional terminator sequence (T), and a reverse recombinase recognition sequence (RRS rev ), such that the single-inducer engineered genetic counter nucleic acid molecule comprising a nucleic acid sequence encoding the following components:
iP A -[RRS 1,for -iP 1,inv -RBS-RC 1 -D-T-RRS 1,rev ]-[RRS 2,for -iP 1,inv -RBS-RC 2 -D-T-RRS 2,rev ]-OP,
wherein iP A and iP 1 are responsive to the same inducer, and wherein the recombinase encoded by each SIMM is specific for the forward and reverse recombinase recognition site o

Assignees

• Collins James J
• Lu Timothy Kuan-Ta
• Univ Boston
• Massachusetts Inst Technology

Inventors

• Collins James J
• Lu Timothy Kuan-Ta

Classifications

• C12Q1/6897 Primary

  involving reporter genes operably linked to promoters · CPC title

• C12N15/635

  Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline · CPC title

• C12N15/63

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title

• G06N3/123

  DNA computing · CPC title

• C12Q2521/507

  Recombinase · CPC title