Prokaryotic 2-component signaling pathways for use as logic gates in mammalian cells

Patent metadata

Field	Value
Publication number	US-10160978-B2
Application number	US-201515316169-A
Country	US
Kind code	B2
Filing date	Jun 4, 2015
Priority date	Jun 5, 2014
Publication date	Dec 25, 2018
Grant date	Dec 25, 2018

Abstract

The invention relates to mammalian cells comprising at least one prokaryotic two-component signaling (TCS) pathway comprised of an activator protein A, a response regulator (RR) protein B activated by said protein A, such activation leading to an activated RR protein B, and an output gene C operably linked to a promoter. Transcription from said promoter is activated by activated RR protein B, and the expression of output gene C defines at least a first state (0, no transcription) and a second state (1, detectable transcription). The invention further relates to logic gates designed from such cells, and methods for integrating a plurality of output signals based on the cells and logic gates of the invention.

First claim

The invention claimed is:

1. An isolated mammalian cell comprising at least one prokaryotic two-component signaling (TCS) pathway comprised of
i. an activator protein A,
ii. a response regulator (RR) protein B activated by said protein A, such activation leading to an activated RR protein B, and
iii. an output gene C operably linked to a promoter,

wherein transcription of the transcription apparatus in said isolated mammalian cell from said promoter is activated by activated RR protein B, and expression of output gene C defines at least a first state 0, which designates no transcription, and a second state 1, which designates detectable transcription, and
wherein said activator protein A is a sensor histidine kinase comprising a histidine residue, said RR protein B is a transcriptional regulator comprising an aspartic acid residue, and activation of said RR protein B by said activator protein A is effected by transfer of a phosphate from said histidine residue to said aspartic acid residue.

2. The isolated mammalian cell according to claim 1 , wherein pairs of the activator protein A and said RR protein B are selected from the group consisting of EnvZ-OmpR, NarX-NarL, DcuS-DcuR, PhoR-PhoB, SenX3-RegX3, PhoQ-PhoP, RstB-RstA, CpxA-CpxR, CssS-CssR, CreC-CreB, BaeS-BaeR, BasS-BasR,QseC-QseB, KdpD-KdpE, TorS-TorR, ArcB-ArcA, TctE-TctD, ResE-ResD, VicK-VicR, MprB-MprA, MtrB-MtrA, PrrB-PrrA, TrcS-TrcR, NrsS-NrsR, ManS-ManR, NblS-NblR, SasA-RpaAB, SaeS-SaeR, BceS-BceR, YxdK-YxdJ, NarQ-NarP, UhpB-UhpA, RcsC-RcsD-RcsB, BarA-UvrY, ComP-ComA, EvgS-EvgA, DegS-DegU, DesK-DesR, VraS-VraR, LiaS-LiaR, DevS-DevR, NreB-NreC, YdfH-YdfI, KinABCDE-Spo0FA, CitA-CitB, CitS-CitT, DctS-DctR, MalK-MalR, LytS-LytR, AlgZ-AlgR, NatK-NatR, AgrC-ArgA, ComD-ComE, GlnL-GlnG, NtrY-NtrX, HydH-HydG, AtoS-AtoC, PilS-PilR, GlrK-GlrR, PgtB-PgtA, DctB-DctD, KinB-AlgB, CheA-CheYBV, ChpA-ChB/PilGH, PixL-PixGH, WspE-WspRF, Cphl-Rcpl, PleC-PleD, CckA-CtrA/CpdR, LuxQN/CqsS-LuxU-LuxO, TtrS-TtrR, FlrB-FlrC, SLN1-YPD1-SSK1/SKN7, RpfC-RpfG, GlnK-GlnL, YesM-YesN, ChvG-ChvI, CiaH-CiaR, SalK-SalR, RegB-RegA and FixL-FixJ.

3. The isolated mammalian cell according to claim 1 , wherein said protein A and said RR protein B are codon-optimized for human cells.

4. The isolated mammalian cell according to claim 1 , wherein the output gene C encodes a fluorescent reporter, a protein or microRNA that affects the cell function or internal state.

5. The isolated mammalian cell according to claim 1 , wherein the output gene C is amcyan.

6. The isolated mammalian cell according to claim 1 , wherein the activator protein A can be constitutively active or activated by input signals.

7. The isolated mammalian cell according to claim 1 , wherein the input signals are selected from the group consisting of: quinones, nitrate, nitrite, citrate, isocitrate, fumarate, succinate, malate, indole, serine, aspartate, chemoattractants, oxygen, carbon monoxide, nitrous oxide, blue light, vancomycin, potassium, quorum sensing molecules, temperature change, sulfate ions, nicotinic acid, changes in osmolarity, toluene, O-xylene, glutamine, 2-ketoglutarate, and magnesium.

8. An engineered biological logic AND gate comprising the isolated mammalian cell according to claim 1 , wherein input 1 activates said activator protein A, input 2 activates said response regulator protein B and the output is the expression state of said output gene C.

9. The engineered biological logic AND gate according to claim 8 , wherein input 2 activates a second activator protein A′, which is able to activate a second RR protein B′ that enables transcription of the first response regulator protein B.

10. The engineered biological logic AND gate according to claim 8 , wherein input 1 and input 2 are stimuli selected from the group consisting of: quinones, nitrate, nitrite, citrate, isocitrate, fumarate, succinate, malate, indole, serine, aspartate, chemoattractants, oxygen, carbon monoxide, nitrous oxide, blue light, vancomycin, potassium, quorum sensing molecules, temperature change, sulfate ions, nicotinic acid, changes in osmolarity, toluene, O-xylene, glutamine, 2-ketoglutarate, magnesium and stimuli controlling expression of the components selected from transcription factors or microRNAs.

11. An engineered biological logic OR gate comprising the isolated mammalian cell according to claim 1 , wherein input 1 activates said activator protein A, input 2 activates a second activator protein A′ able to activate the same RR protein B as activator protein A and the output is the expression state of said output gene C.

12. An engineered biological logic NOR gate comprising the isolated mammalian cell according to claim 1 , wherein input 1 is an inhibitor A− of said activator protein A, input 2 is an inhibitor B− of said RR protein B and the output is the expression state of said output gene C.

13. An engineered biological logic NAND gate comprising the isolated mammalian cell according to claim 1 , wherein input 1 is an inhibitor A− of said activator protein A, input 2 is an inhibitor A′− of said activator protein A′ and the output is the expression state of said output gene C.

14. The isolated mammalian cell according to claim 1 , wherein pairs of the activator protein A and said RR protein B are selected from the group consisting of EnvZ-OmpR, NarX-NarL, and DcuS-DcuR.

Assignees

• Eth Zuerich

Inventors

• Benenson Yaakov
• Hansen Jonathan
• Kumar Swaminathan Krishna

Classifications

• C07K14/245

  Escherichia (G) · CPC title

• C12N9/12

  transferring phosphorus containing groups, e.g. kinases (2.7) · CPC title

• C12Y207/13003

  Histidine kinase (2.7.13.3) · CPC title

• C07K14/43595

  from coelenteratae, e.g. medusae · CPC title

• C12N15/635

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