Patent Application
20230204572
The content of the electronically submitted sequence listing in ASCII text file (Name 4831.014STR0_Seqlisting_ST26; Size: 103,411 bytes; and Date of Creation: Nov. 8, 2022) filed with the application is incorporated herein by reference in its entirety.
The long-term exposure of low levels of pollutants and chemicals in an environment can present a hazard to human and aquatic health. Early indications of exposure may help mitigate these long-term adverse effects (O'Malley, M., Lancet 1997, 349 (9059), 1161-1166; Zubiate, P. et al., Biosens. Bioelectron. X 2019, 2, 100026; Chiavaioli, F. et al., ACS Sensors 2018, 3 (5), 936-943). Chemical detection methods lack the sensitivity and specificity of mass spectrometry; however, mass spectrometry methods are high cost and lack the portability of chemical detection (Yang, L. et al., Anal. Sci. 2004, 20 (1), 199-203; Mishra, R. K. et al., ACS Sensors 2017, 2 (4), 553-561). Microbes have naturally evolved receptors to detect and respond to known human health hazards, like phenanthrene, lead, copper, and organophosphate pesticides (Wei H. et al., Int. J. Environ. Sci. Technol. 2014, 11 (3), 685-694; Peltola, P. et al., Sci. Total Environ. 2005, 350 (1-3), 194-203; Whangsuk, W. et al., Anal. Biochem. 2016, 493, 11-13; Khatun, M. A., et al., Anal. Chem. 2018, 90 (17), 10577-10584).
One environmental pollutant for which there is currently not a zero-power monitoring technology is the fungicide, 2-phenylphenol (2-PP). This molecule is used to preserve citrus fruits and vegetables and is used in the manufacturing of other fungicides, dyes, resins, and rubber chemicals (Yang, L., et al. Anal. Sci. 2004, 20 (1), 199-203; Wick, L. Y. et al. Environ. Sci. Technol. 1998, 32 (9), 1319-1328). According to the EPA, regulatory tolerance levels for 2-PP are as low as 10 ppm for certain fruits and the LC50 for some aquatic organisms can be as low as 0.32 mg/L (EPA. Reregistration Eligibility Decision for 2-Phenylphenol and Salts (Orthophenylphenol or OPP); 2006). While 2-PP has been found in urine, its toxicity to humans is unknown, but it is toxic to aquatic life (Ye, X. et al., Anal. Chem. 2005, 77 (16), 5407-5413). Current detection methods for 2-PP include high performance liquid chromatography with UV, electrochemical detection, and gas chromatography with mass spectrometric detection (Thompson, R. D. Determination of Phenolic Disinfectant Agents in Commercial Formulations by Liquid Chromatography; 2001; Vol. 84). These methods, which apply to detection of other pollutants or chemicals in an environment, require extractions, are labor intensive, require expensive equipment, or are low throughput and are not suitable for applications like personal protection and persistent environmental monitoring of large areas. Biosensors that are portable, deployable and require no power for detection of 2-PP, and other analytes such as pollutants or chemicals, in the environment are needed.
Provided is a polynucleotide comprising a Pseudomonas azelaica hbpR (hbpR) promoter operably linked to a polynucleotide encoding a HbpR protein and a heterologous promoter operably linked to a polynucleotide encoding a marker protein, wherein the marker protein creates an output signal. In some aspects, the output signal is in the visible spectrum.
In some aspects, the hbpR promoter and the heterologous promoter direct transcription in opposite directions.
In some aspects, the heterologous promoter is a Pseudomonas azelaica hbpC promoter.
In some aspects, the polynucleotide further comprises an amplification cassette comprising: a polynucleotide encoding a Pseudomonas syringae hrpR (HrpR) protein that is operably linked to the hbpC promoter.
In some aspects, the amplification cassette further comprises a polynucleotide encoding a Pseudomonas syringae hrpS (HrpS) protein downstream of the polynucleotide encoding the HrpR protein.
In some aspects, the polynucleotide further comprises a P. syringae hrpL (hrpL) promoter operably linked to a polynucleotide encoding a HrpR protein, a polynucleotide encoding a HrpS protein, and a polynucleotide encoding a marker protein.
In some aspects, the polynucleotide comprises a hrpL promoter operably linked to a polynucleotide encoding a HrpS protein, a polynucleotide encoding a HrpR protein, and a polynucleotide encoding a marker protein.
In some aspects, the HbpR protein encoded by the polynucleotide binds an analyte of interest.
In some aspects, the analyte of interest is a polyphenyl selected from the group consisting of 2-hydoxybiphenyl, 2,2′-dihydroxybiphenyl, 2-aminobiphenyl, and 2-hydroxybiphenylmethane.
In some aspects, the polynucleotide encoding the HbpR protein comprises at least one mutation compared to a polynucleotide encoding a wild-type HbpR protein.
In some aspects, the HbpR protein that comprises the at least one mutation binds a different analyte of interest than a wild-type HbpR protein. In some aspects, the different analyte of interest is a chlorinated polyphenyl. In some aspects, the analyte is a polychlorinated polyphenyl.
In some aspects, the marker protein is an amilCP protein.
Further provided is a polynucleotide comprising a P. syringae hrpL promoter operably linked to a polynucleotide encoding a HrpR protein and a polynucleotide encoding a marker. In some aspects, the polynucleotide comprises a hrpL promoter operably linked to a polynucleotide encoding a HrpS protein and a polynucleotide encoding a marker protein.
In some aspects, the polynucleotide comprises a P. syringae hrpL promoter operably linked to a polynucleotide encoding a HrpR protein, a polynucleotide encoding a HrpS protein, and a polynucleotide encoding a marker.
Also provide is a biocontainment material for containment and maintenance of a microbe comprising a polynucleotide as described herein.
In some aspects, the biocontainment material comprises a polymer-based hydrogel, a multi-layer hydrogel comprising a hydrogel and an elastomer outer layer, or a polymer-inorganic material hybrid hydrogel.
In some aspects, the polymer-based hydrogel comprises polyacrylamide alginate, chitosan, agarose, agar, gelatin, or pullulan.
In some aspects, the polyacrylamide alginate comprises alginate and polyacrylamide at a ratio of alginate to polyacrylamide of about 1:50.
In some aspects, the elastomer outer layer of the multi-layer hydrogel comprises a polyurethane skin.
In some aspects, the polymer-inorganic material hybrid hydrogel comprises alginate polyacrylamide and polycaprolactone.
Further provided is a method of preparing a biosensor system, wherein the method comprises (i) preparing a polymer solution; (ii) polymerizing the polymer solution; (iii) suspending a microbe comprising a polynucleotide as described herein in the polymer solution to prepare a polymer cell suspension; and (iv) cross-linking the polymer cell suspension to prepare a biosensor system.
In some aspects, the polymer used in the method is polyacrylamide alginate, chitosan, agarose, agar, gelatin, or pullulan.
Provided is a whole-cell biosensor system with robust biocontainment for field deployment and a strong visual reporter for readouts in the deployed environment. The engineered biosensors in an encapsulated and deployable system (eBEADS) demonstrate a portable, no power living sensor for detection of a phenylphenol compound, 2-PP, in the environment. In some aspects, a whole-cell living sensor to detect a phenylphenol compound is provided that is developed in Escherichia coli by utilizing the 2-PP degradation pathway with an amplification circuit to produce a visual colorimetric output. Such whole-cell biosensors can also be used to detect other analytes, including compounds that HbpR proteins bind to naturally or by engineering of the HbpR protein, e.g., by mutation of the protein. To enable field deployment, a physical biocontainment system is used. In some aspects, the biocontainment system comprises polyacrylamide alginate (PAA) beads to encapsulate the bacterial sensor strains, support long-term viability without supplemental nutrients, and allow permeability of the target analyte.
In some aspects, provided are polynucleotides, bacterial sensor cells, and biosensor systems engineered to sense analytes in an environment, e.g., an ecosystem such as in an urban, rural, or wild area. The biosensor system described herein comprises a bacterial cell that is engineered to express a marker protein upon contact with an analyte. In some aspects, the bacterial cell comprises a polynucleotide comprising a Pseudomonas azelaica HbpR protein expression cassette and a marker protein expression cassette under the control of a promoter that is inducible by a complex of HbpR protein and an analyte.
In some aspects, in the presence of the analyte, HbpR binds the analyte to form an HbpR/analyte complex, and the HbpR/analyte complex binds and activates the HbpR/analyte-inducible promoter leading to marker protein expression. In some aspects, the level of marker protein expressed correlates with the amount of HbpR/analyte complex bound to the inducible promoter, and, thus, the amount of analyte in contact with or that contacted the bacterial cell.
In some aspects, polynucleotides provided herein further comprise an amplification circuit that utilizes components of the Hrp Type III secretion system from Pseudomonas syringae. In some aspects, the amplification system comprises a polynucleotide encoding transcriptional activator proteins of P. syringae downstream of a HbpR/analyte complex inducible promoter. In some aspects, the HbpR/analyte complex inducible promoter is a hbpC promoter. In some aspects, the bacterial cell further comprises a polynucleotide encoding additional transcriptional activator proteins of P. syringae downstream of a hrpL promoter. Upon exposure of a bacterial cell to an analyte, the HbpR protein expressed by the HbpR expression cassette binds the analyte and the HbpR/analyte complex binds and activates the hbpC promoter to induce expression of the transcriptional activator proteins. The transcriptional activator protein in turn forms a hexameric complex and binds and activates the hrpL promoter that controls the expression of a marker protein and further transcriptional activator proteins, thereby amplifying the activation circuit and increasing marker protein expression. The amplification circuit leads to an amplified marker protein signal that can be detected with the naked eye.
Further provided is a biocontainment system that contains the engineered bacterial cells. The biocontainment system allows entry of water, nutrients and environmental analytes and prevents biosensor cells from leaking out of the biocontainment system.
The term “and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
It is understood that wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of” and/or “consisting essentially of” are also provided.
The term “approximately” or “about” as applied to one or more values of interest, refers to a value that is similar to a stated reference value and within a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). When the term “approximately” or “about” is applied herein to a particular value, the value without the term “approximately” or “about is also disclosed herein.
As described herein, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.
The terms “ug” and “uM” are used herein interchangeably with “μg” and “μM,” respectively.
Units, prefixes, and symbols are denoted in their Système International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. The headings provided herein are not limitations of the various aspects of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.
The terms “nucleic acids,” “nucleic acid molecules, “nucleotides,” “nucleotide(s) sequence,” and “polynucleotide” can be used interchangeably and refer to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNA molecules”, including mRNA) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; “DNA molecules”), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix. The term nucleic acid molecule, and in particular DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, supercoiled DNA and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences can be described herein according to the normal convention of giving only the sequence in the 5′ to 3′ direction along the non-transcribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA). A “nucleic acid composition” comprises one or more nucleic acids as described herein. RNA can be obtained by transcription of a DNA-sequence, e.g., inside a cell. In a prokaryotic cell, transcription of DNA usually results in premature RNA, which has to be processed into messenger RNA (mRNA). Processing of the premature RNA comprises a variety of different posttranscriptional-modifications such as splicing, 5′-capping, polyadenylation, export from the nucleus or the mitochondria and the like. The sum of these processes is also called maturation of RNA. The mature mRNA provides the nucleotide sequence that can be translated into an amino acid sequence of a particular peptide, or protein. Typically a mature mRNA comprises a 5′ cap, optionally a 5′ UTR, an open reading frame, optionally a 3′ UTR, and a poly(A) sequence.
The term “mRNA,” as used herein, refers to a single stranded RNA that encodes the amino acid sequence of one or more polypeptide chains.
As used herein, the terms “derived from” or “derivative” refer to a component that is isolated from or made using a specified molecule, or information (e.g., a nucleic acid sequence) from the specified molecule. For example, a polynucleotide sequence that is derived from another polynucleotide sequence can include a polynucleotide sequence that is identical or substantially similar to the polynucleotide sequence it derives from. In the case of polynucleotides, the derived species can be obtained by, for example, naturally occurring mutagenesis, artificial directed mutagenesis, or artificial random mutagenesis. The mutagenesis used to derive polynucleotides can be intentionally directed or intentionally random, or a mixture of both. The mutagenesis of a polynucleotide to create a different polynucleotide derived from the first polynucleotide can be a random event (e.g., caused by polymerase infidelity) and the identification of the derived polynucleotide can be made by appropriate screening methods known in the art. In some aspects, the screening methods comprise exposure to analytes and detection of growth of polynucleotide containing cells in the presence of the analytes. In some aspects, a polynucleotide sequence that is derived from a first polynucleotide sequence has a sequence identity of at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to the first polynucleotide sequence, respectively, wherein the derived polynucleotide sequence retains the biological activity of the original polynucleotide. The derived polynucleotide will not necessarily be derived physically from the nucleotide sequence of interest, but may be generated in any manner, including, but not limited to, chemical synthesis, replication, reverse transcription or transcription, which is based on the information provided by the sequence of bases in the region(s) from which the polynucleotide is derived. As such, it may represent either a sense or an antisense orientation of the original polynucleotide.
As used herein, the term “transfecting” or “transfection” refers to the transport of nucleic acids from the environment external to a cell to the internal cellular environment, with particular reference to the cytoplasm of a prokaryotic cell. Without being bound by any particular theory, it is to be understood that nucleic acids can be delivered to a cell either after being encapsulated within or adhering to one or more cationic polymer/nucleic acid complexes or being entrained therewith or by electroporation or calcium chloride. Nucleic acids include DNA and RNA as well as synthetic congeners thereof. Such nucleic acids include missense, antisense, nonsense, as well as protein producing nucleotides. In particular, but not limited to, they can be genomic DNA, cDNA, mRNA, tRNA, rRNA, hybrid sequences or synthetic or semi-synthetic sequences, and of natural or artificial origin. In addition, the nucleic acid can be variable in size, ranging from oligonucleotides to chromosomes. These nucleic acids can be of mammalian, bacterial, viral, or synthetic origin. They can be obtained by any technique known to a person skilled in the art.
“Percent (%) sequence identity” or “percent identity” with respect to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to the nucleic acids or amino acids in the reference polynucleotide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid or amino acid sequence identity can be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, percent sequence identity values can be generated using the sequence comparison computer program BLAST.
By “level” is meant a level or activity of a protein, or mRNA encoding the protein, optionally as compared to a reference. The reference can be any useful reference, as defined herein. By a “decreased level” or an “increased level” of a protein is meant a decrease or increase in protein level, as compared to a reference. A level of a protein can be expressed in mass/vol (e.g., g/dL, mg/mL, g/mL, ng/mL) or percentage relative to total protein or mRNA in a sample.
By a “reference” is meant any useful reference used to compare protein or mRNA levels or activity. The reference can be any sample, standard, standard curve, or level that is used for comparison purposes. The reference can be a normal reference sample or a reference standard or level.
The term “recombinant” as used herein to describe a nucleic acid molecule means a polynucleotide of genomic, cDNA, viral, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation, is not associated with all or a portion of the polynucleotide with which it is associated in nature. The term “recombinant” as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide. In general, the gene of interest is cloned and then expressed in transformed cells. The cell expresses the foreign gene to produce the protein under expression conditions.
The terms “recombinant host cells”, “host cells,” “cells”, “cell lines,” “cell cultures”, and other such terms denoting microorganisms cultured as unicellular entities refer to cells which can be, or have been, used as recipients for recombinant nuclei acid, and include the original progeny of the original cell which has been transfected.
“Operably linked” refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, a given promoter operably linked to a coding sequence is capable of effecting the expression of the coding sequence when the proper enzymes are present. Expression is meant to include the transcription of mRNA from a DNA or RNA template and can further include translation of a protein from an mRNA template. The promoter need not be contiguous with the coding sequence, so long as it functions to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked” to the coding sequence.
As used herein, the term “promoter” refers to DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In some aspects, a coding sequence is located 3′ to a promoter sequence. Promoters can be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters can direct the expression of a gene in different cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters.” Promoters that cause a gene to be expressed in a specific cell type are commonly referred to as “cell-specific promoters.”
Promoters that are induced and cause a gene to be expressed following exposure or treatment of the cell with an agent, biological molecule, chemical, ligand, light, or the like that induces the promoter are commonly referred to as “inducible promoters” or “regulatable promoters.” Inducible promoter include any promoter whose activity is affected by a cis or trans-acting factor. In some aspects, an inducible promoter includes a promoter that is induced by binding, e.g., of a transcription factor/analyte complex. In some aspects, the inducible promoter is one derived from an inducible promoter present in nature. In some aspects, an inducible promoter is one generated by DNA synthesis or cloning techniques. In some aspects, an inducible promoter can combine promoter elements originating from several different naturally occurring promoters.
The term “hbpR promoter,” as used herein refers to a region upstream of a HbpR protein encoding region of Pseudomonas azelaica HBP1, a soil bacterium that is able to grow on the fungicide 2-hydroxybiphenyl as sole source of carbon and energy. The hbpR promoter drives expression of a 63 kDa Hrp regulatory protein. In some aspects, the hbpR promoter comprises the nucleic acid sequence of SEQ ID NO: 1.
The term “hbpC promoter,” as used herein refers to a region upstream of a hbpC gene which is the first gene of the 2-hydroxybiphenyl degradation pathway in Pseudomonas azelaica HBP1. In some aspects, the hbpC promoter comprises the nucleic acid sequence of SEQ ID NO: 2.
The term “hrpL promoter,” as used herein refers to a hrpL promoter of Pseudomonas syringae. In some aspects, the hrpL promoter comprises the nucleic acid sequence of SEQ ID NO: 3.
It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths can have identical promoter activity.
The terms “transcriptional regulatory protein,” “transcriptional regulatory factor,” and “transcription factor” are used interchangeably herein, and refer to a protein that binds a DNA response element and thereby transcriptionally regulates the expression of an associated gene or genes. Transcriptional regulatory proteins generally bind directly to a DNA response element, however in some cases binding to DNA can be indirect by way of binding to another protein or small molecule and the protein/small molecule complex in turn binds to, or is bound to a DNA response element. In some cases, transcriptional regulatory proteins bind to a DNA response element only when in a complex with an activating moiety. In some aspects, the activating moiety can be an analyte.
The terms “coding sequence” or a sequence “encoding” a particular molecule (e.g., a selectable marker protein) as used herein refer to a nucleic acid that is transcribed (in the case of DNA) or translated (in the case of mRNA) into polypeptide, in vitro or in vivo, when operably linked to an appropriate regulatory sequence, such as a promoter. The boundaries of the coding sequence are determined by a start codon at the 5′ (amino) terminus and a translation stop codon at the 3′ (carboxy) terminus. A coding sequence can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and synthetic DNA sequences. A transcription termination sequence will usually be located 3′ to the coding sequence.
The term “termination signal sequence” as used herein refers to any genetic element that causes RNA polymerase to terminate transcription, such as for example a polyadenylation signal sequence. A polyadenylation signal sequence is a recognition region necessary for endonuclease cleavage of an RNA transcript that is followed by the polyadenylation consensus sequence AATAAA. A polyadenylation signal sequence provides a “polyA site,” i.e., a site on a RNA transcript to which adenine residues will be added by post-transcriptional polyadenylation.
The term “mutation” as used herein refers to any changing of the structure of a gene, resulting in a variant (also called “mutant”) form that can be transmitted to subsequent generations. Mutations in a gene can be caused by the alternation of single base in DNA, or the deletion, insertion, or rearrangement of larger sections of genes or chromosomes.
The term “modified” as used herein refers to a changed state or structure of a molecule of the disclosure. Molecules can be modified in many ways including chemically, structurally, and functionally. In some aspects, the modification is relative to a reference wild-type molecule.
The term “synthetic” as used herein refers to produced, prepared, and/or manufactured by the hand of man. Synthesis of polynucleotides or polypeptides or other molecules of the present disclosure can be chemical or enzymatic.
The term “polypeptide” as used herein is intended to encompass a singular “polypeptide” as well as plural “polypeptides,” and comprises any chain or chains of two or more amino acids. Thus, as used herein, a “peptide,” a “peptide subunit,” a “protein,” an “amino acid chain,” an “amino acid sequence,” or any other term used to refer to a chain or chains of two or more amino acids, are included in the definition of a “polypeptide,” even though each of these terms can have a more specific meaning. The term “polypeptide” can be used instead of, or interchangeably with any of these terms.
The term “hydrogel” as used herein refers to a cross-linked hydrophilic polymer that is water soluble before cross-linking and includes, but is not limited to, a polyacrylamide hydrogel; polyacrylic acid hydrogel, polymethacrylic acid hydrogel, polyvinylalcohol hydrogel, polyethyleneglycol hydrogel, polylactic acid hydrogel; polyglycolic acid hydrogel, poly(lactic-co-glycolic acid) hydrogel, alginate polyacrylamide hydrogel; alginate polyacrylic acid hydrogel, alginate polymethacrylic acid hydrogel, alginate polyvinylalcohol hydrogel, alginate polyethyleneglycol hydrogel, alginate polylactic acid hydrogel; alginate polyglycolic acid hydrogel, alginate poly(lactic-co-glycolic acid) hydrogel; carboxymethyl cellulose hydrogel, hydroxyethyl cellulose hydrogel, and a starch-acrylic acid copolymer hydrogel. After cross-linking the hydrogel may degrade in water over time where the degradation rate depends on the composition of the hydrogel.
The term “hybrid” material, as used herein, refers to a material that contains at least two different components including, but not limited to, two materials with different tensile strengths and/or transparency.
The term “elastomer skinned polymer-based hydrogel,” as used herein, refers to a hydrogel that comprises a polymer-based component and an elastomer component that surrounds the polymer-based component in a skin-like manner.
The terms “analyte” or “analyte of interest,” as used herein, refers to a compound or substance that is capable of detection using the whole-cell biosensor systems described herein, where an HbpR protein expressed by the bacterial sensors is capable of binding the analyte and is capable of activating the transcriptional activator protein HbpR. Example analytes described herein include, but are not limited to, phenylphenol compounds, such as 2-PP, chlorinated polyphenyl compounds, and chlorinated biphenyl compounds.
The term “phenylphenol” refers to a diphenyl-based compound that may contain a substitution in one of the phenyl rings and is capable of interacting with a HpR protein as disclosed herein. An example phenylphenol described herein includes “hydroxyl-phenylphenol,” “OH-phenylphenol,” “substituted diphenyl,” “2-phenylphenol,” “2-PP,” “2-HBP,” “2-OH-PP,” or “2-OH-BP,” which are used interchangeably.
The term “whole cell biosensor,” as used herein, refers to an analyte detection system that uses whole cells, e.g., whole bacterial cells for the expression of an analyte detection system that enables detection of an analyte through, e.g., a color change of the whole cell biosensor.
Provided are polynucleotides comprising an HbpR/analyte complex-inducible marker protein expression system for biosensing analytes in an environment.
The polynucleotides comprise components of the 2-phenylphenol and 2,2′-biphenol degradation pathway of Pseudomonas azelaica HBP1. This pathway is regulated by the transcriptional activator protein HbpR.
In some aspects, a polynucleotide comprises a Pseudomonas azelaica HbpR protein expression cassette comprising a P. azelaica hbpR promoter operably linked to a polynucleotide encoding a P. azelaica HbpR protein. In some aspects, the hbpR promoter comprises SEQ ID NO: 1. In some aspects, the polynucleotide further comprises a promoter that is inducible by a HbpR/analyte complex operably linked to a polynucleotide encoding a marker protein.
In some aspects, the polynucleotide further comprises a polynucleotide encoding a P. syringae HrpR transcriptional activator protein. In some aspects, the polynucleotide further comprises a polynucleotide encoding a P. syringae HrpS transcriptional activator protein. In some aspects, the polynucleotide encoding the P. syringae HrpR transcriptional activator protein and the polynucleotide encoding a P. syringae HrpS transcriptional activator protein are under the control of a hbpC promoter. In some aspects, the hbpC promoter comprises SEQ ID NO: 2. In some aspects, the polynucleotide encoding the P. syringae HrpR transcriptional activator protein is operably linked to the hbpC promoter and the polynucleotide encoding the P. syringae HrpS transcriptional activator protein is downstream of the polynucleotide encoding the P. syringae HrpR transcriptional activator. In some aspects, the polynucleotide encoding the P. syringae HrpS transcriptional activator protein is operably linked to the hbpC promoter and the polynucleotide encoding the P. syringae HrpR transcriptional activator protein is downstream of the polynucleotide encoding the P. syringae HrpS transcriptional activator protein.
In some aspects, the polynucleotide encoding a P. syringae HrpR transcriptional activator protein and the polynucleotide encoding a P. syringae HrpS transcriptional activator protein are linked through an IRES.
In some aspects, the polynucleotide encoding a P. syringae HrpR transcriptional activator protein and the polynucleotide encoding a P. syringae HrpS transcriptional activator protein are linked through a self-cleaving peptide.
In some aspects, provided is a polynucleotide comprising a P. syringae HrpL promoter operably linked to a marker protein, a P. syringae HrpR protein, and a P. syringae HrpS protein. In some aspects, the hrpL promoter comprises SEQ ID NO: 3. When co-expressed, HrpR and HrpS proteins form a hetero-hexameric complex that binds to and activates the P. syringae HrpL promoter, thereby inducing enhanced expression of the complex components, HrpR and HrpS protein, and of the marker protein. Therefore, the polynucleotide provides an amplification of the marker protein signal.
In some aspects, the P. azelaica HbpR expression cassette and the HbpR/analyte complex-inducible promoter-HrpR-HrpS-marker protein expression cassette are present in the same polynucleotide. In some aspects, the P. azelaica HbpR expression cassette promoter and the HbpR/analyte complex-inducible-HrpR-HrpS-marker protein expression cassette are transcribed in opposite directions.
In some aspects, the P. azelaica HbpR expression cassette and the HbpR/analyte complex-inducible-HrpR-HrpS-marker protein expression cassette are present in different polynucleotides.
In some aspects, the hrpL promoter-HrpR-HrpS-marker protein expression cassette, the P. azelaica HbpR expression cassette, and the HbpR/analyte complex-inducible promoter-HrpR-HrpS-marker protein expression cassette are present in one polynucleotide.
In some aspects, the hrpL promoter-HrpR-HrpS-marker protein expression cassette and the HbpR/analyte complex-inducible-HrpR-HrpS-marker protein expression cassette are present in one polynucleotide and the P. azelaica HbpR expression cassette.
In some aspects, the hrpL promoter-HrpR-HrpS-marker protein expression cassette and the P. azelaica HbpR expression cassette are present in one polynucleotide and the HbpR/analyte complex-inducible-HrpR-HrpS-marker protein expression cassette is present in a separate polynucleotide.
In some aspects, the marker protein is a LacZ gene, a green fluorescent protein (GFP) gene, a red fluorescent protein (RFP) gene, a yellow fluorescent protein (YFP) gene, or an amilCP protein. In some aspects, the marker protein is amilCP and the purple-blue color produced by the expressed amilCP causes a whole cell biosensor containing the amilCP-expressing bacteria to turn purple blue, wherein the purple blue color of the biosensor can be detected with the naked eye.
In some aspects, the P. azelaica HbpR expression cassette encodes a wild-type HbpR. In some aspects, the wild-type HbpR binds a hydroxylated biphenyl. In some aspects, the wild-type HbpR binds a 2-hydroxy-biphenyl, 1,2-diphenyl hydrazine, 1-bromo-4-phenoxy benzene, 1-chloro-4-phenoxy benzene, 2-chloronaphtalene, benzidine, or 1-fluoro-4-phenoxy-benzene.
In some aspects, the HbpR encoding polynucleotide comprises at least one mutation compared to a wild-type HbpR encoding polynucleotide, wherein the at least one mutation changes the analyte affinity and/or specificity of the HbpR protein.
In some aspects, the mutant HbpR binds a chlorinated biphenyl. In some aspects, the mutant HbpR binds a polychlorinated biphenyl. In some aspects, the polychlorinated biphenyl is polychlorinated biphenyl-1 (PCB-1). In some aspects, the polychlorinated biphenyl is polychlorinated biphenyl-3 (PCB-3). In some aspects, the mutant HbpR does not bind 2-phenylphenol and does bind PCB-1. In some aspects, the mutant HbpR does not bind 2-phenylphenol and does bind PCB-3. In some aspects, the mutant HbpR binds 2-phenylphenol with low affinity and PCB-1 with high affinity. In some aspects, the mutant HbpR binds 2-phenylphenol with low affinity and PCB-3 with high affinity.
In some aspects, the mutant HbpR protein is encoded by a polynucleotide comprising SEQ ID NO: 5. In some aspects, the mutant HbpR protein is encoded by a polynucleotide comprising SEQ ID NO: 6. In some aspects, the mutant HbpR protein is encoded by a polynucleotide comprising SEQ ID NO: 7.
In some aspects, the polynucleotides described herein are present in a bacterial cell. In some aspects, the bacteria are transformed to contain at least one polynucleotide as described herein. In some aspects, the bacteria are transformed to contain at least two polynucleotides as described herein.
In some aspects, the bacterial cell is a soil bacterium. In some aspects, the soil bacterium includes, but is not limited to, the genera Rhizobium, Bacillus, Mycobacterium, Streptomyces, Xanthomonas, Arthrobacter, Micrococcus, Pseudomonas, Corynebacterium, Agrobacterium, Flavobacterium, Alcaligenes, Clostridium, or Azospirillum.
In some aspects, the polynucleotides used with the soil bacteria comprise modifications. In some aspects, the modifications comprise changes to promoters, ribosomal binding sites, or other genetic regulatory elements that can be used in the respective soil bacterium, and combinations thereof.
In some aspects, the bacterial cell is Escherichia co/i.
In some aspects, the bacterial cells are whole cell biosensors that express a marker protein upon being contacted with an analyte. In some aspects, the expression levels of the marker protein in the bacterial cell are directly correlated to the amount of analyte the bacterial cell was contacted with.
In some aspects, the bacterial whole cell biosensor is contained within a biocontainment element such that expression of the marker protein by the bacterial cell induces a color change of the biocontainment element.
Provided are materials and method of using the same to contain a bacterial whole cell biosensor such that entry of the bacterial cell into the environment is prevented. In some aspects, the integration of biocontainment materials and sensing bacterial strains provides a deployable end-to-end living whole cell biosensor system. In some aspects, the biosensor system comprises a β-galactosidase reporter. In some aspects, the biosensor system comprises an amplification circuit. In some aspects, the biosensor system enables an up to 66-fold increase in β-galactosidase reporter output with the addition of the amplification circuit. In some aspects, the whole cell biosensor responds to as little as about 1 μM 2-PP when unencapsulated. In some aspects, the whole cell biosensor system when contained in a biocontainment element responds to about 10 μM 2-PP.
In some aspects, the biocontainment material comprises pores that are large enough to allow entry of water, nutrients and analytes into the interior of the biocontainment element and small enough to prevent whole cell biosensor bacteria to leave the biocontainment element. In some aspects, the biocontainment materials comprise ion crosslinked hydrogels. In some aspects, the biocontainment materials comprise several components that are selected based on their capability of forming pores of desired diameters upon crosslinking. In some aspects, the biocontainment elements are customized according to the size of whole cell biosensor bacteria they are to contain and according to the environment in which the biocontained whole cell sensors are to be deployed. In some aspects, when the biocontained whole cell sensors are to be deployed in a dry environment, the biocontainment materials are selected to enable sufficient hydration and may include a hydration retention additive such as, e.g., glycerol.
In some aspects, the biocontainment material comprises a polymer-based hydrogel, a polymer-based multilayer hydrogel, an elastomer skinned polymer-based hydrogel, or a polymer-inorganic material hybrid hydrogel.
In some aspects, the polymer is a polyacrylamide-alginate, alginate, chitosan, agarose, agar, gelatin, or pullulan.
In some aspects, the polymer-based hydrogel is a polyacrylamide-alginate hydrogel and comprises alginate, polyacrylamide and N,N′-Methylenebisacrylamide (MBAA).
In some aspects, the alginate polyacrylamide hydrogel comprises alginate and polyacrylamide in a ratio of alginate to polyacrylamide of about 1:40, about 1:42, about 1:44, about 1:46, about 1:48, about 1:50, about 1:52, about 1:54, about 1:56, about 1:58 or about 1:60. In some aspects, the ratio of alginate to polyacrylamide is from about 1:40 to about 1:50; about 1:40 to about 1:60; or about 1:42 to about 1:60; about 1:44 to about 1:58; about 1:46 to about 1:56; about 1:48 to about 1:54; or about 1:50 to about 1:52.
In some aspects, the ratio of alginate to polyacrylamide is from about 1:40 to about 1:60. In some aspects, the ratio of alginate to polyacrylamide is from about 1:45 to about 1:55. In some aspects, the ratio of alginate to polyacrylamide is about 1:45. In some aspects, the ratio of alginate to polyacrylamide is about 1:55.
In some aspects, the ratio of alginate to polyacrylamide is about 1:50.
In some aspects, the alginate polyacrylamide hydrogel comprises about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0% of MBAA. In some aspects, the alginate polyacrylamide hydrogel comprises about 1.0 to about 1.4; about 1.5 to about 1.7; or about 1.8 to about 2.0% MBAA. In some aspects, the alginate polyacrylamide hydrogel comprises about 1.0 to about 2.0; about 1.1 to about 1.9; about 1.2 to about 1.8, about 1.3 to about 1.7, or about 1.4 to about 1.6% MBAA.
In some aspects, the alginate polyacrylamide hydrogel comprises about 1.65, about 1.66, about 1.67, about 1.68, about 1.69, about 1.75, about 1.85, or about 1.95% of MBAA. In some aspects, the alginate polyacrylamide hydrogel comprises about 1.65 to about 1.67, about 1.68 to about 1.75, or about 1.76 to about 1.95% MBAA. In some aspects, the alginate polyacrylamide hydrogel comprises about 1.65 to about 1.95, about 1.66 to about 1.9, about 1.67 to about 1.85, about 1.68 to about 1.8, or about 1.69 to about 1.75% of MBAA.
In some aspects, the polymer-based hydrogel comprises an elastomer skin. In some aspects, the elastomer skinned polymer-based hydrogel comprises a polyurethane skin.
In some aspects, the polymer-inorganic material hybrid hydrogel comprises an inorganic nanomaterial reinforced hydrogel. In some aspects, the inorganic nanomaterial reinforced hydrogel comprises alginate polyacrylamide and polycaprolactone.
In some aspects, the inorganic nanomaterial reinforced hydrogel comprises between about 0.1 mg/ml and about 10 mg/ml of polycaprolactone. In some aspects, the inorganic nanomaterial reinforced hydrogel comprises about 0.1 mg/ml to about 1 mg/ml; about 1.1 mg/ml to about 2 mg/ml; about 2.1 mg/ml to about 3 mg/ml; about 3.1 mg/ml to about 4 mg/ml; about 4.1 mg/ml to about 5 mg/ml; about 5.1 mg/ml to about 6 mg/ml; about 6.1 mg/ml to about 7 mg/ml; about 7.1 mg/ml to about 8 mg/ml; about 8.1 mg/ml to about 9 mg/ml; about 9.1 mg/ml to about 10 mg/ml of polycaprolactone.
In some aspects, the inorganic nanomaterial reinforced hydrogel comprises about 1 mg/ml of polycaprolactone.
In some aspects, the polymer-inorganic material hybrid hydrogel comprises a polycrystalline polymer. In some aspects, the polymer-inorganic material hybrid hydrogel comprises a polycrystalline polymer that is shear thinning. In some aspects, the shear thinning polycrystalline polymer hybrid hydrogel can be used for 3D extrusion printing.
In some aspects, the polymer-inorganic material hybrid hydrogel comprises clay. In some aspects, the polymer-inorganic material hybrid hydrogel comprises laponite. In some aspects, the polymer-inorganic material hybrid hydrogel comprises silica.
Provided is a biosensor system comprising a bacterial cell described herein and a biocontainment material described herein encapsulating the bacterial cell.
In some aspects, the biosensor system further comprising a hydration retention additive. In some aspects, the hydration retention additive is glycerol.
In some aspects, the biosensor system is designed such that the biocontainment material contains the bacterial cells within the biosensor system and allows nutrients, water and analytes to penetrate the biocontainment material. In some aspects, the bacterial cell does not leak out of the engineered biocontainment material for at least about nine days. In some aspects, the bacterial cell does not leak out of the engineered biocontainment material for about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, or 56 days. In some aspects, the bacterial cell does not leak out of the engineered biocontainment material for about 10 to about 15, about 16 to about 20, about 21 to about 25, about 26 to about 30, about 31 to about 35, about 36 to about 40, about 41 to about 45, about 46 to about 50, about 51 to about 56 days; or about 10 to about 56, about 12 to about 54, about 14 to about 52, about 16 to about 50, about 18 to about 48, about 20 to about 46, about 22 to about 42, about 24 to about 40, about 26 to about 38, about 28 to about 36, or about 30 to about 34 days.
In some aspects, the bacterial cell remains viable in the biosensor system for about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, or 56 days. In some aspects, the bacterial cell remains viable in the biosensor system for about 10 to about 15, about 16 to about 20, about 21 to about 25, about 26 to about 30, about 31 to about 35, about 36 to about 40, about 41 to about 45, about 46 to about 50, about 51 to about 56 days; or about 10 to about 56, about 12 to about 54, about 14 to about 52, about 16 to about 50, about 18 to about 48, about 20 to about 46, about 22 to about 42, about 24 to about 40, about 26 to about 38, about 28 to about 36, or about 30 to about 34 days.
In some aspects, the bacterial cell of the biosensor system remains viable for between about 9 days and about 56 days.
In some aspects, the biosensor system comprises a bacterial cell that expresses an amilCP marker protein from a polynucleotide as described herein and the biosensor system adopts a purple-blue color when contacted with an analyte.
In some aspects, the biosensor system adopts a purple-blue color between about 6 hours and 24 hours after being contacted with an analyte. In some aspects, the biosensor system adopts a purple-blue color between about 2 hours and 6 hours after being contacted with an analyte.
In some aspects, the biosensor system is contained in a particle. In some aspects, the biosensor system is contained in a microparticle. In some aspects, the microparticle comprising the biosensor system comprising bacterial sensor cells adopts a blue-purple color when contacted with an analyte. In some aspects, the microparticle comprises more than one biosensor system.
In some aspects, the microparticle comprises a polymer.
In some aspects, the microparticle comprises a polymer-based hydrogel. In some aspects the microparticle hydrogel comprises polyacrylamide alginate, chitosan, agarose, agar, gelatin, or pullulan.
In some aspects, the microparticle comprises biosensor systems with bacterial sensor cells comprising polynucleotides encoding different analyte-binding transcriptional activator proteins for detection of different analytes. In some aspects, the marker proteins contained within the different biosensor systems of a microparticle generate a same color and the intensity of the color change of the microparticle is a measure for the presence of different analytes.
In some aspects, the marker proteins contained within the different biosensor systems are different and the colors generated by each biosensor system are a measure for the analyte detected by the respective biosensor system. In some aspects, the different colors generated by the different marker proteins create a distinct combination color indication. In some aspects, one marker protein contained in the biosensor system generates a red fluorescent protein upon being contacted with an analyte and a second marker protein contained in the biosensor system generates a green fluorescent protein upon being contacted with an analyte and, when exposed to light of a wavelength that excites the red and green marker proteins, the biosensor system generates a yellow color if both marker proteins are generated at about equal amounts. In some aspects, the biosensor system generates a green-yellow color when more of the second marker protein is generated. In some aspects, the biosensor system generates an orange color when more of the first marker protein is generated.
In some aspects, a marker protein contained in the biosensor system is amilGFP and generates a yellow color upon being contacted with an analyte. In some aspects, a marker protein contained in the biosensor system is fwYellow and generates a yellow color upon being contacted with an analyte.
In some aspects, a marker protein contained in the biosensor system is meffRFP and generates a red color upon being contacted with an analyte. In some aspects, a marker protein contained in the biosensor system is eforCP and generates a red color upon being contacted with an analyte.
In some aspects, a first marker protein contained in the biosensor system is selected from the group consisting of amilGFP and fwYellow and generates a yellow color upon being contacted with an analyte. In some aspects, a second marker protein contained in the biosensor system is selected from the group consisting of meffRFP and eforCPfwYellow and generates a red color upon being contacted with an analyte.
In some aspects, the marker proteins are generated when contacted with the same analyte. In some aspects, the marker proteins are generated when contacted with different analytes. In some aspects, the biosensor system generates a yellow color when contacted with a first analyte and a red color when contacted with a second analyte. In some aspects, when contacted with both analytes the biosensor generates an orange color. In some aspects, when contacted with more of the first analyte than the second analyte, the biosensor generates a yellow-orange color. In some aspects, when contacted with more of the second analyte than the first analyte, the biosensor generates a red-orange color.
In some aspects, the biosensor system comprises an agent that is cytotoxic to the bacterial sensor cells. In some aspects, the biosensor system further comprises a trigger release system. In some aspects, the release of the cytotoxic agent is triggered by an environmental condition. In some aspects, the release is caused by UV light, magnetic field, pH or temperature. In some aspects, upon triggering the release of the cytotoxic agent within the biosensor system, the bacterial cells of the biosensor system are neutralized.
Provided is a method of preparing a biosensor system as described herein.
In some aspects, the method comprises: (i) preparing a polymer solution; (ii) polymerizing the polymer solution; and (iii) suspending a bacterial cell comprising a polynucleotide as described herein to prepare a polymer cell suspension.
In some aspects, the method further comprises mixing a polymer cell suspension with a mechanically hard polymer. In some aspects, the mechanically hard polymer is polycaprolactone.
In some aspects, the ratio of cross-linked polymer cell suspension and mechanically hard polymer is from about 50:1 to about 1:50, or any ratio in between.
In some aspects, the method further comprises coating a cross-linked polymer cell suspension with an elastomer skin. In some aspects, the elastomer skin is a polyurethane skin.
In some aspects, the method further comprises co-printing a cross-linked cell polymer suspension using a 3D printer. In some aspects, the 3D printed cross-linked polymer cell suspension is in the form of a film. In some aspects, the 3D printed cross-linked polymer cell suspension is deposited on a particle. In some aspects, the 3D printed cross-linked polymer cell suspension film is deposited on an adhesive structure. In some aspects, the 3D printed cross-linked polymer cell suspension film is deposited on a device deployable into an environment. In some aspects, the 3D printed cross-linked polymer cell suspension film is deposited on a bead. In some aspects, the 3D printed cross-linked polymer cell film is deposited on a microbead.
The engineered biosensors described herein are encapsulated and deployable systems (eBEADS) comprising an amplification circuit that enhances sensor output and, by expressing an amilCP marker protein when exposed to micromolar quantities of, e.g., 2-PP, generate a purple-blue color that is visible to the human eye.
Provided are methods for detecting the presence of an analyte in an environment, the method comprising: (i) deploying a biosensor system described herein or a microparticle described herein in an environment; and (ii) detecting an output signal of the biosensor system or microparticle.
In some aspects, provided is a method for measuring a quantity of an analyte in an environment, the method comprising: (i) deploying a biosensor system described herein or a microparticle described herein in an environment; (ii) detecting an output signal of the biosensor system or microparticle; (iii) quantifying the output signal of the biosensor system or microparticle using an output signal scale that indicates a quantity of an analyte for a specified output signal intensity. In some aspects, the output signal scale provides output signal intensities for specified or known quantities of analyte such that an output signal intensity detected with an eBEAD can be quantified.
In some aspects, the quantification of the output signal is after about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24 hours or after about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days of deployment.
All chemicals including 2-phenylphenol (2-PP), ethanol, isopropyl β-D-1-thiogalactopyranoside (IPTG), acrylamide, N,N′ methylenebisacrylamide (MBAA), alginic acid sodium salt from brown algae (alginate, medium viscosity), and calcium chloride were obtained from MilliporeSigma (St. Louis, Mo.).
Unless otherwise noted, bacteria were grown at 30° C. in Lysogeny Broth (LB) media (10 g NaCl, 10 g tryptone, and 5 g yeast extract per liter) with carbenicillin (100 μg/mL) and/or kanamycin (50 μg/mL) for selection of plasmids. Plasmids were directly transformed into chemically competent Escherichia coli (E. coli) NEB5α cells (New England Biolabs, NEB; Ipswich, Mass.). Strains containing plasmid for plasmid preparation were incubated at 37° C.
Cultures were grown overnight to saturation and then diluted to an OD600 of 0.2. Cultures were incubated at 30° C. with shaking until an OD600 of 0.7-1 before inducing cultures with 2-phenylphenol dissolved in 100% ethanol. Cultures were incubate for 3 hours or 1 mL samples were taken every hour. For experiments where 2-PP was removed after incubating for 3 hours, cultures were divided in two tubes and centrifuged. One half of the cultures were washed with 1×PBS to remove 2-phenylphenol and resuspended in fresh medium with antibiotics and without 2-phenylphenol. The other half was resuspended in their original medium without washing. Aliquots were taken every hour. Cells were harvested, washed with 1×PBS, and resuspended in one volume of 1×PBS. One hundred μl were used for analysis using the Beta-Glo Assay System (Promega; Madison, Wis.) according to the manufacturer's instructions. Statistical significance of results was determined using a Student's two-tailed t-test. The limit of detection (LOD) was calculated using GraphPad Prism software and is defined as the blank plus three times the standard deviation of the blank.
Visual Reporter Demonstration with Amil CP on Plates
Strains containing AmilCP reporters were incubated overnight to saturation at 30° C. with shaking. Three μl of culture were pipetted onto 6 cm LB-agar plates containing 0.2 mM IPTG, 100 μg/mL carbenicillin, 50 μg/mL kanamycin, and 0 μM, 1 μM, or 10 μM of 2-phenylphenol. Images of the plates were taken at time points 0, 6, 12, 24, and 48 hours with a Nikon SMZ25 stereoscope.
Using the same encapsulation protocol as the sensing strain beads, positive control strain cultures containing plasmids pBL_IMP_25 and pBL_IMP_26 that constitutively express the reporter were used to fabricate triplicate samples. Negative control beads lacking cells were also fabricated. The sensing strain microbeads were cultured at 30° C., 200 RPM in LB medium with no salt. OD600 measurements of the LB medium surrounding the beads were taken with a NanoDrop One MicroVolume UV-Vis Spectrometer (Thermo Scientific; Waltham, Mass.) every 24 hours for a nine-day period.
The encapsulation matrix's ability to support cell viability and long-term shelf life was assessed using the Live/Dead BacLight Bacterial Viability assay (ThermoFisher). Briefly, cells constitutively expressing AmilCP were encapsulated in hydrogel formulations as described above and stored at 4° C. or 25° C. without supplemental nutrients or liquid in a closed 50 mL conical tube in biological triplicate “batches”. Cell viability was evaluated for each storage condition immediately after encapsulation, 24 hours, and weekly for one month. At each time point and storage condition, a single bead from each replicate “batch” was stained with 1 ml of 3 l/ml equal parts SYTO9 (live stain) and propidium iodide (dead stain) for 30 minutes, washed with 1×PBS, and imaged as an end point measurement on Zeiss Confocal Microscope (LSM 900 Airy scan 2). Images were processed using ImageJ.
The encapsulated sensing strain's ability to detect low concentrations of 2-PP and produce a visible output signal were evaluated by time series of microscope images. Briefly, the positive control strain and 2-PP with amplification circuit strain were encapsulated in the PAA formulation as previously described above. The microbeads were placed on LB agar plates containing 0.2 mM IPTG, 100 μg/mL carbenicillin, and 50 μg/mL kanamycin supplemented with 0, 1 or 10 μM 2-phenylphenol and incubated at 30° C. Images of the sensing strain microbeads were taken at time points 0, 6, 12, 24, and 48 hours with a Nikon SMZ25 stereoscope.
The plasmids constructed are listed in Table 1. HbpR, its 654 bp upstream region containing the hbpC promoter (Accession U73900), and lacZα were cloned into pAKgfp132 to make pBL_IMP_11. LacZα was replaced with the amilCP reporting gene to make pBL_IMP_31. For the amplification plasmids, hrpR and hrpS were inserted downstream of the hbpC promoter and upstream of either the lacZα or amilCP reporter to make pBL_IMP_32 and pBL_IMP_28, respectively. A ribosome binding site (BBa_B0034, Registry of Standard Biological Parts) was inserted upstream of each hrpR, hrpS, and the reporter gene. For the second amplification plasmid, the entire hrpR, hrpS, and either lacZα or amilCP were cloned downstream of the hrpL promoter into the pVLT3333 plasmid backbone to make pBL_IMP_33 and pBL_IMP_25, respectively. For the positive control plasmids that constitutively express the reporter, the hbpC promoter in pBL_IMP_28 was replaced with the tac promoter to produce pBL_IMP_26. All genetic components were amplified using Q5 polymerase and assembled using NEBluilder® HiFi DNA Assembly Master Mix (NEB). All PCR primers and gBlocks were purchased from Integrated DNA Technologies (IDT; Coralville, Iowa).
Bacterial sensor cells were prepared by electroporation of the plasmids described above into the bacteria and bacteria were grown overnight as described above.
The bacterial sensing cells were entrapped in a semi-interpenetrating network of alginate and polyacrylamide hydrogel microbeads. To this end, alginate, acrylamide, and MBAA were dissolved in deionized water. The total weight percent of alginate and acrylamide was 33.3% (w/v) at a ratio of 1:50 (w/w) alginate to acrylamide. MBAA was added to a final concentration of 1.67% (w/v). The polymer solution was polymerized in a Branson 1800 sonicator bath containing water at 60° C. for 3 hours and allowed to cool overnight. Overnight cultures of bacteria containing the plasmids (grown as described above) were pelleted and resuspended into the polymer solution at an OD600 of 3 per ml. The polymer cell suspension was then extruded through a sterile 18G needle into 1 M calcium chloride bath and allowed to cross-link for 30 minutes in the bath. The sensing strain microbeads were then filtered and rinsed twice with sterile deionized water.
In the bacterial sensor cells when exposed to 2-PP, the HbpR promoter is activated by 2-PP and HbpR protein is expressed from the transfected plasmids. HbpR protein binds to 2-PP to form a HbpR/2-PP complex that binds to and activates the hbpC promoter also present in the transfected plasmids. Activation of the hbpC promoter results in expression of a lacZα reporter gene (
To enhance reporter production, an amplification circuit was added to the bacterial sensor cells. HrpR and HrpS are transcriptional activators that form a hetero-hexameric complex to activate the hrpL promoter. An amplification circuit was generated using two plasmids. The first plasmid was derived from pBL_IMP_11 and contained the hrpR and hrpS genes downstream of the hbpC promoter, before the reporter gene (
Inclusion of the amplification circuit resulted in an increase in reporter activity for all concentrations of 2-PP greater than 1 μM 2-PP. Increases in reporter gene expression ranged from 17 to 66-fold after three hours of incubation (
In order to test the response of the reporter over time, the sensor strains were treated with various amounts of 2-PP and reporter activity was measured every hour for five hours. After two hours, there was an increase in reporter activity for the 5 μM, 10 μM, and 50 μM 2-PP treatments for strains that lacked an amplification circuit when compared to the 0 μM 2-PP control (
To demonstrate that this sensor strain could be used for visual detection without the use of any additional equipment, the lacZα gene in all plasmids was replaced with amilCP, a purple-blue chromoprotein from Acropora millepora (Table 1).
After 12-24 hours on agar plates containing 10 μM 2-phenylphenol, the strains with amplification circuit produced enough AmilCP purple reporter to become just barely visible. After 48 hours on agar plates containing 1 μM and 10 μM 2-phenylphenol, the AmilCP purple color became faintly visible in the strain without amplification circuit while strains with the amplification circuit produced a vibrant purple color (
Addition of the amplification circuit allowed for earlier detection of 2-PP by eye and produced a more vibrant color than strains without the amplification circuit as seen on agar plates as well as in liquid culture (
An important consideration when designing a living sensor is the persistence of the reporter output. In some use cases, it is desirable to have a living sensor that does not need to be exposed to the analyte of interest for an extended period for production of a detectable signal output. To determine the continuous production of the reporter with the addition of the amplification circuit, the sensor strain was exposed to the analyte for a limited period of time and then removed as described in the methods. For the strain without amplification circuit, (3-galactosidase reporter activity continued to increase for the samples that still contained 2-PP, whereas the strains where 2-PP was removed, the signal immediately began to decrease (
These results are consistent with what was expected based on the reporter design. For the non-amplification strain, once the analyte is removed, there is nothing to drive expression from the hbpC promoter, so the signal will no longer increase and will eventually decrease depending on the stability of the reporter protein. For the amplification strain, after removing the analyte, the HrpR and HrpS proteins are still present to induce expression from the hrpL promoter and continue to produce more HrpR and HrpS proteins. This cycle should continue to produce reporter protein as long as the degradation of HrpR and HrpS is not faster than the production of the proteins in the amplification circuit. Addition of the amplification circuit to this living sensor adds stability and does not require constant analyte input to sustain activation of the amplification circuit.
With a robust, visual reporting sensing strain developed, a containment system for environmental delivery was necessary. A polyacrylamide-alginate (PAA) hydrogel was selected for cell compatibility. To validate that entrapment of the sensing strain cells in the semi-interpreting hydrogel network, microbeads containing sensing strain cells were incubated in salt free, Lysogeny Broth media at 30° C. The OD600 of the surrounding media was observed every 24 hours for a nine day incubation period. The OD600 remained zero for the duration of nine days indicating no cell leakage (
Long term viability of cells encapsulated in the PAA beads was evaluated in a series of live/dead assays on beads stored without additional nutrients at 25° C. and 4° C. over the course of one month. Confocal imaging of the beads showed the majority of cells are alive (green) after one month of storage at both temperatures (
To generate biosensor systems that detect additional analytes, HbpR mutants were generated using a HbpR codon optimized library sequence (SEQ ID NO: 4) and a plasmid containing the same (SEQ ID NO: 8) and selection against different analytes. Mutant HbpR proteins that selectively bind analytes other than 2-PP were obtained through this process. For example, mutant HbpR proteins P6-A-8 (SEQ ID NO: 5), P6-F-8 (SEQ ID NO: 6), and P6-B-9 (SEQ ID NO: 7) showed selective lacZ reporter expression in the presence of polychlorinated biphenyl-1 (PCB-1) (
To demonstrate eBEADS can detect 2-phenylphenol at low concentrations, the sensor strain with amplification circuit was encapsulated and exposed to 0 to 10 μM 2-PP for 48 hours at 30° C. The encapsulated strain showed a purple color response within 24 hours of exposure to 10 μM 2-PP (
To enhance mechanical stability of the biosensor systems described herein, polymer inorganic material hybrid nanoparticles were generated.
To this end, hydrogels were loaded with an inorganic reinforcement material. Electron microscopy images of polymeric, lipid, and mesoporous silica nanoparticles are shown in
eBEADS is an extensible system for deploying whole-cell living sensors with no need for equipment. The engineered living sensor utilizes the HbpR transcriptional activator and an amplification circuit to detect as little as 1 μM 2-PP. This whole-cell living sensor produces a long-lasting amplified signal up to 66 times with minimal background. The signal amplification allows a timely and clear visual response, which is vital for in field readouts. Physical biocontainment and long-term viability of whole-cell living sensors in PAA microbeads was demonstrated. eBEADS is an end-to-end living sensor for the zero-power detection of 2-PP in the environment. This system serves as a tailorable platform to detect additional environmental pollutants and enables new sensor form factors to support applications like remote deployment or wearables.
This application claims the benefit of U.S. Provisional Application No. 63/277,264, filed Nov. 9, 2021, the contents of which are incorporated by reference herein in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63277264 | Nov 2021 | US |
