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

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;

wherein the bacterial cell is an engineered bacterial cell including a 1362 U-sensing genetic reportable molecular component and/or a 1362 U-sensing/U-neutralizing genetic molecular component, each comprising a U-sensitive promoter in a configuration wherein the U-sensitive promoter directly 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 is

a 1362 U-sensitive prompter 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 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 are selected independently

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 , wherein the U-sensitive promoter further comprises a 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.

3. The UO 2 F 2 biosensor of claim 2 , 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.

4. The UO 2 F 2 biosensor of claim 2 , wherein the bacteria are capable of natively expressing endogenous MarR family repressors such as MarR1 and/or MarR2 genes and at least one gene of the endogenous MarR family, preferably all genes of the endogenous MarR family, is knocked out.

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

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

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: 2509.

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: 2510 or SEQ ID NO: 2512.

9. 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.

10. 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.

11. 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.

12. 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.

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

N 1 is C; and/or

N 2 is G; and/or

N 3 is T; and/or

N 5 is A; and/or

N 6 is G; and/or

N 14 is T; and/or

N 16 is A.

14. 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.

15. The UO 2 F 2 biosensor of claim 1 , wherein the U-sensitive promoter 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).

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

17. The UO 2 F 2 biosensor of claim 1 , wherein the U-sensitive promoter is P1361 or Pphyt.

18. The UO 2 F 2 biosensor of claim 1 , wherein biosensor comprises, the F-sensing reportable genetic molecular component and the 1362 F-sensing genetic reportable molecular component and/or a 1362 U-sensing/U-neutralizing genetic molecular component, and the F-sensing reportable genetic molecular component are operatively connected to a same or a different reportable molecular component in U-sensing genetic circuit.

19. The UO 2 F 2 biosensor of claim 15 , wherein

the cell is further natively and/or heterologously expressing histidine kinase UzcS, and response regulator UzcS, and

the U-sensing genetic circuit further comprises a UzcR U-sensing genetic reportable molecular component and/or a UzcR U-sensing/U-neutralizing genetic molecular component, each comprising a UzcR U-sensitive promoter in a configuration wherein the U-sensitive promoter directly initiates expression of the U-sensing reportable molecular component and/or of the U-sensing U-neutralizing molecular component in presence of bioavailable U,

the UzcR U sensitive promoter comprising a UzcR binding site having 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, and in some embodiments any one of N 7 -N 11 can independently be A.

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

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

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

23. The UO 2 F 2 biosensor of claim 20 , wherein two or more in series AND gates and/or in parallel AND gates are connected by activating, inhibiting, binding, or converting reactions.

24. The UO 2 F 2 biosensor of claim 20 , 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.

25. 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.

26. 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.

27. 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 signal.

28. 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.

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

30. The UO 2 F 2 biosensor of claim 29 , wherein the proteobacterial cell is an alphaproteobacteria, a betaproteobacteria, or a gam maproteobacteria.

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

32. The UO 2 F 2 biosensor of claim 29 , wherein the proteobacterial cell is a Caulobacter crescentus cell.

33. The UO 2 F 2 biosensor of claim 32 , wherein the Caulobacter crescentus cell is a member of a strain selected from the group consisting of NA1000, CB15, and OR37.

34. 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.

35. A method of detecting and reporting and/or neutralizing bioavailable U, the method 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.

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

one or more U sensitive promoters comprising a U-sensitive 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 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 are selected independently

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.

37. UO 2 F 2 -sensing genetic reportable component of claim 36 , wherein the one or more U sensitive promoters comprise

a U sensitive 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.

38. A UO 2 F 2 -sensing genetic reportable component of claim 36 , 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 and wherein the UO 2 F 2 -sensing gene cassette is an expression cassette.

39. A UO 2 F 2 -sensing genetic reportable component of claim 38 , wherein the UO 2 F 2 -sensing gene cassette is comprised within a vector.

40. The UO 2 F 2 -sensing genetic reportable component of claim 36 , wherein

N 1 is C; and/or

N 2 is G; and/or

N 3 is T; and/or

N 5 is A; and/or

N 6 is G; and/or

N 14 is T; and/or

N 16 is A.