US 12,331,298 B2
Phosphorylation-based miRNA sensor
Ron Weiss, Newton, MA (US); Ross D. Jones, Cambridge, MA (US); and Jin Huh, Watertown, MA (US)
Assigned to Massachusetts Institute of Technology, Cambridge, MA (US)
Filed by Massachusetts Institute of Technology, Cambridge, MA (US)
Filed on Jul. 11, 2022, as Appl. No. 17/811,593.
Application 17/811,593 is a continuation of application No. 16/528,772, filed on Aug. 1, 2019, abandoned.
Claims priority of provisional application 62/713,160, filed on Aug. 1, 2018.
Prior Publication US 2023/0235334 A1, Jul. 27, 2023
Int. Cl. C12N 15/52 (2006.01); C12Q 1/6876 (2018.01)
CPC C12N 15/52 (2013.01) [C12Q 1/6876 (2013.01); C12Q 2600/112 (2013.01); C12Q 2600/178 (2013.01)] 18 Claims
 
1. A cell state classifier, comprising:
(i) a first sensor circuit comprising a constitutive promoter operably linked to a nucleotide sequence encoding an activator, and a constitutive promoter operably linked to a nucleotide sequence encoding a kinase that phosphorylates the activator and produces a phosphorylated activator, and one or more target sites for a first microRNA;
(ii) a second sensor circuit comprising a constitutive promoter operably linked to a nucleotide sequence encoding a phosphatase that de-phosphorylates the phosphorylated activator, and one or more target sites for a second microRNA; and
(iii) a signal circuit comprising an activatable promoter that is activated by the phosphorylated activator, operably linked to a nucleotide sequence encoding an output molecule, and one or more target sites for the first microRNA,
optionally wherein the constitutive promoter of (i) and the constitutive promoter of (ii) are the same or wherein the constitutive promoter of (i) and the constitutive promoter of (ii) are different,
wherein the kinase, the phosphatase, and/or the activator are members of a bacterial two-component signaling system, wherein the bacterial two-component system comprises a histidine kinase comprising an amino acid sequence motif of HEXXN, HEXXT, or HDXXXP, wherein X is any amino acid, and a response regulator, and
wherein the phosphatase is a variant of the histidine kinase and comprises an amino acid substitution in the E or D of the HEXXN, HEXXT or HDXXXP motif.