IP Library › Granted Patent US 12,571,058
Granted Patent B2
US 12,571,058 · App. 18/406,057 · Granted Mar 10, 2026

Biosensors for detecting and/or neutralizing bioavailable uranium and related U-sensitive genetic molecular components, gene cassettes, vectors, genetic circuits, compositions, methods and systems

Inventor: Dan Mcfarland Park (Dublin, CA)
Assignee: LAWRENCE LIVERMORE NATIONAL SECURITY, LLC
C12Q1/6897C12N1/20C12N9/12C12N15/52C12N15/635C12N15/74C12Y207/13003G21F9/18G21F9/30C12N2830/002C12N2830/55C12N2840/002C12N2840/105G01N2520/00
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Quick Facts
Patent No.
US 12,571,058
App. No.
18/406,057
Granted
Mar 10, 2026
Kind
B2
Abstract

UO 2 F 2 biosensors, and related U-sensing and/or F-sensing genetic molecular components, genetic circuits, compositions, methods and systems are described, which in several embodiments can be used to detect and/or neutralize uranium and in particular bioavailable UO 2 F 2 .

Claims (95)

1. A UO 2 F 2 -biosensor comprising a genetically engineered bacterial cell capable of heterologously and/or natively expressing histidine kinase 1363, and U-sensitive transcriptional regulator 1362; the cell further capable of natively and/or heterologously expressing histidine kinase UzcS, and response regulator UzcS, wherein the bacterial cell is an engineered bacterial cell including a U-sensing genetic reportable molecular component and/or a U-sensing/U-neutralizing genetic molecular component, each comprising a U-sensitive promoter in a configuration wherein the U-sensitive promoter initiates expression of the U-sensing reportable molecular component and/or of the U-sensing U-neutralizing molecular component in presence of bioavailable U;

wherein the U-sensitive promoter of at least one U-sensing genetic reportable molecular component and/or a U-sensing/U-neutralizing genetic molecular component comprises

a U-sensitive transcriptional 1362 binding site having a DNA sequence

(SEQ ID NO: 1)

N 1 N 2 N 3 N 4 N 5 N 6 N 7 N 8 N 9 N 10 N 11 N 12 N 13 N 14 N 15 N 16 N 17 N 18 ,

in which

N 1 is C or T; N 2 is G or A; N 3 is T or C; N 4 is C; N 5 is A or G; N 6 is G or C; N 7 is C or G; N 8 is any nucleotide; N 9 is any nucleotide; N 10 is any nucleotide; N 11 is any nucleotide; N 12 is T or C; N 13 is G; N 14 is T or C; N 15 is C; N 16 is A or C; N 17 is G; and N 18 is C or G, and in which N 1 to N 17 selected independently

wherein the U-sensitive promoter of at least one U-sensing genetic reportable molecular component and/or a U-sensing/U-neutralizing genetic molecular component comprises

a U-sensitive transcriptional UzcR binding site having a DNA sequence:

(SEQ ID NO: 2)

CATTACN 7 N 8 N 9 N 10 N 11 N 12 TTAA

wherein N 7 -N 12 is independently any nucleotide,

the UzcR binding site inserted at a location downstream of a transcription start site of the U sensitive promoter

and wherein the genetically modified bacterial cell is an engineered bacterial cell further comprising an F-sensing riboswitch within at least one of

the U-sensing reportable genetic molecular component in a configuration wherein the U-sensing reportable genetic molecular component, is transcribed in presence of an effective amount of bioavailable fluoride,

an F-sensing reportable genetic molecular component in a configuration wherein the F-sensing reportable genetic molecular component is transcribed in presence of an effective amount of bioavailable fluoride, and

an F sensitive genetic circuit in which at least one molecular component is a reportable molecular component (and in particular one or more reportable genetic molecular component and/or one or more reportable cellular molecular component), the reportable molecular component expressed when the genetic circuit operates according to the circuit design in presence of bioavailable F.

2. The UO 2 F 2 biosensor of claim 1 , further comprising a UzcY gene and/or UzcZ gene inserted at a location downstream of a transcription start site of the UzcR U-sensitive promoter.

3. The UO 2 F 2 biosensor of claim 1 , wherein the bacteria are capable of natively expressing endogenous MarR family repressors and at least one gene of the endogenous MarR family, is knocked out.

4. The UO 2 F 2 biosensor of claim 1 , wherein the bacteria are capable of natively expressing endogenous urtAP genes and at least one gene of the endogenous urtAP is knocked out.

5. A UO 2 F 2 biosensor of claim 1 , wherein the F-sensing riboswitch comprises a crcB motif or an eric F motif.

6. A UO 2 F 2 biosensor of claim 1 , wherein the F-sensing riboswitch comprises a F-sensing riboswitch having sequence SEQ ID NO: 2509.

7. A UO 2 F 2 biosensor of claim 1 , wherein the F-sensing riboswitch comprises a F-sensing riboswitch having sequence SEQ ID NO: 2510 or SEQ ID NO: 2512.

8. A UO 2 F 2 biosensor of claim 1 , wherein the F-sensing riboswitch comprises a F-sensing riboswitch having sequence SEQ ID NO: 2511 or SEQ ID NO: 2513.

9. A UO 2 F 2 biosensor of claim 1 , wherein the F-sensing riboswitch comprises a F-sensing riboswitch having any one of SEQ ID NO: 205 to SEQ ID NO: 1988 and from SEQ ID NO: 1999 to SEQ ID NO: 2231.

10. The UO 2 F 2 biosensor of claim 1 , wherein the F-sensing riboswitch comprises a F-sensing riboswitch Sphingomonas sp. MM-1, having sequence SEQ ID NO: 2514 or an F-sensing riboswitch from Sphingomonas sp, 67-36 having sequence SEQ ID NO: 2525 or the F-sensing riboswitch is the F-sensing riboswitch from Pseudomonas Syringae having sequence SEQ ID NO: 2526.

11. A UO 2 F 2 biosensor of claim 1 , wherein the bacterial cell comprises an endogenous F-sensing riboswitch and the endogenous F-sensing riboswitch is knocked out.

12. The UO 2 F 2 biosensor of claim 1 , wherein in the sequence SEQ ID NO:1:

N 1 is C; N 2 is G; N 3 is T; N 5 is A; N 6 is G; N 14 is T; and/or N 16 is A.

13. The UO 2 F 2 biosensor of claim 1 , wherein the U-sensitive transcriptional 1362 binding site has a sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27.

14. The UO 2 F 2 biosensor of claim 1 , wherein the U-sensitive promoter comprising the U-sensitive transcriptional 1362 binding site further comprises nucleotides N 19 N 20 N 21 , downstream of SEQ ID NO: 1 wherein N 19 is any nucleotide; N 20 is any nucleotide; and N 21 is G (SEQ ID NO: 83).

15. The UO 2 F 2 biosensor of claim 14 , wherein N 18 of the regulator direct repeat is located about −17 to about −40 upstream of a transcription start site.

16. The UO 2 F 2 biosensor of claim 1 , wherein the U-sensitive promoter is P 1361 or P phyt .

17. The UO 2 F 2 biosensor of claim 1 , wherein the F-sensing reportable genetic molecular component the U-sensing genetic reportable molecular component and/or the U-sensing/U-neutralizing genetic molecular component, are operatively connected to a same or a different reportable molecular component in U-sensing genetic circuit.

18. The UO 2 F 2 biosensor of claim 17 , wherein the U-sensitive genetic circuit comprises one or more AND gates.

19. The UO 2 F 2 biosensor of claim 18 , wherein at least one of the AND gates is an in-series AND gate.

20. The UO 2 F 2 biosensor of claim 18 , wherein at least one of the AND gates is an in-parallel AND gate.

21. The UO 2 F 2 biosensor of claim 18 , wherein two or more in series AND gates and/or in parallel AND gates are connected by activating, inhibiting, binding, or converting reactions.

22. The UO 2 F 2 biosensor of claim 18 , wherein at least one of the AND gates is selected from the group consisting of an HRP AND gate, a bacterial two-hybrid AND gate, a tripartite GFP AND gate, and a FRET sensor AND gate.

23. The UO 2 F 2 biosensor of claim 1 , wherein the UO 2 F 2 biosensor is configured to detect and/or neutralize bioavailable U present in a target environment at a concentration of 100 nM or greater, between 100 nM and 1 μM, or greater than 1 μM.

24. The method of claim 1 , wherein the UO 2 F 2 biosensor is configured to detect bioavailable F present in a target environment at a concentration 10 μM, or greater, between 50 μM and 60 μM, greater than 1 nanomolar and between 100 nanomolar and 1 mM.

25. The UO 2 F 2 biosensor of claim 1 , wherein the reportable molecular component is capable of being detected using fluorescence, luminescence, chemiluminescence, colorimetric analysis, radioactivity, or electrical.

26. The UO 2 F 2 biosensor of claim 1 , wherein the U-neutralizing molecular component is configured to decrease or eliminate toxicity of U by bioreduction, biomineralization, bioaccumulation, and/or biosorption.

27. The UO 2 F 2 biosensor of claim 1 , wherein the bacterial cell is a proteobacterial cell.

28. The UO 2 F 2 biosensor of claim 27 , wherein the proteobacterial cell is an alphaproteobacteria, a betaproteobacteria, or a gammaproteobacteria.

29. The UO 2 F 2 biosensor of claim 27 , wherein the proteobacterial cell is a Caulobacteridae cell.

30. The UO 2 F 2 biosensor of claim 27 , wherein the proteobacterial cell is a Caulobacter crescentus cell.

31. The UO 2 F 2 biosensor of claim 30 wherein the Caulobacter crescentus cell is a member of a strain selected from the group consisting of NA1000, CB15, and OR37.

32. A UO 2 F 2 -sensing system comprising:

one or more of the UO 2 F 2 — biosensors of claim 1 operatively connected to an electronic signal transducer adapted to convert a UO 2 F 2 — biosensor reportable molecular component output into an electronic output.

33. A method of detecting and reporting and/or neutralizing bioavailable U comprising:

contacting one or more of the UO 2 F 2 biosensors of claim 1 , with a target environment comprising one or more target ranges of U concentration, the contacting performed for a time and under conditions to detect and report and/or neutralize bioavailable U in the target environment.

34. A UO 2 F 2 -sensing genetic reportable component comprising:

one or more U sensitive promoters comprising

a U-sensitive transcriptional 1362 binding site having a DNA sequence

(SEQ ID NO: 1)

N 1 N 2 N 3 N 4 N 5 N 6 N 7 N 8 N 9 N 10 N 11 N 12 N 13 N 14 N 15 N 16 N 17 N 18 ,

in which

N 1 is C or T;

N 2 is G or A;

N 3 is T or C;

N 4 is C;

N 5 is A or G;

N 6 is G or C;

N 7 is C or G;

N 8 is any nucleotide;

N 9 is any nucleotide;

N 10 is any nucleotide;

N 11 is any nucleotide;

N 12 is T or C;

N 13 is G;

N 14 is T or C;

N 15 is C;

N 16 is A or C;

N 17 is G; and

N 13 is C or G,

and in which N 1 to N 17 selected independently

the one or more U sensitive promoters further comprising

a U sensitive transcriptional UzcR binding site, having a DNA sequence:

(SEQ ID NO: 2)

CATTACN 7 N 8 N 9 N 10 N 11 N 12 TTAA

wherein N 7 -N 12 is independently any nucleotide

together with

a U-sensing reportable molecular component,

wherein at least one of the one or more U-sensitive promoters and the U-sensing reportable molecular component, comprises an F-sensing riboswitch in a single output configuration wherein the U-sensing reportable genetic molecular component, is transcribed in presence of an effective amount of bioavailable fluoride,

and wherein the one or more U sensitive promoters and the U-sensing reportable molecular component are in a configuration wherein the one or more U sensitive promoters directly initiate expression of the U-sensing reportable molecular component in presence of bioavailable U and bioavailable Fluoride.

35. A UO 2 F 2 -sensing genetic reportable component of claim 34 wherein the one or more U-sensitive promoters and the U-sensing reportable molecular component are configured to provide a UO 2 F 2 -sensing gene cassette, a UO 2 F 2 -sensing gene cassette being an expression cassette.

36. A UO 2 F 2 -sensing genetic reportable component of claim 35 , wherein the UO 2 F 2 -sensing gene cassette is comprised within a vector.

37. The UO 2 F 2 biosensor of claim 1 , wherein the U-sensitive 1362 binding site and the UzcR binding site, are on a same U-sensing genetic reportable molecular component and/or U-sensing/U-neutralizing genetic molecular component.

38. The UO 2 F 2 biosensor of claim 3 , wherein all genes of the endogenous MarR family, are knocked out.

39. The UO 2 F 2 biosensor of claim 4 , wherein all genes of the endogenous urtAP are knocked out.

40. The UO 2 F 2 biosensor of claim 1 , wherein the bacterial cell natively expresses a fluoride efflux pump and the gene encoding the native efflux pump is deleted.

41. The UO 2 F 2 biosensor of claim 40 , wherein the fluoride efflux pump is crcB.

42. The UO 2 F 2 biosensor of claim 41 , wherein the fluoride efflux pump is ericF.

43. The UO 2 F 2 biosensor of claim 27 , wherein the bacterial cell natively expresses a fluoride efflux pump and the gene encoding the native efflux pump is deleted.

Assignments (3)
CONFIRMATORY LICENSE Recorded Mar 6, 2024
From: LAWRENCE LIVERMORE NATIONAL SECURITY, LLC
To: US DEPARTMENT OF ENERGY
Reel/Frame 066664/0223 →
CONFIRMATORY LICENSE Recorded Mar 6, 2024
From: LAWRENCE LIVERMORE NATIONAL SECURITY, LLC
To: US DEPARTMENT OF ENERGY
Reel/Frame 066664/0267 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Feb 21, 2024
From: PARK, DAN MCFARLAND
To: LAWRENCE LIVERMORE NATIONAL SECURITY, LLC
Reel/Frame 066520/0437 →
Continuity (3)
Continuation 16781950 · Feb 4, 2020
Provisional Application 62801077 · Feb 4, 2019
Related Publication 20240376556A1 · Nov 14, 2024
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