WO2018140323A1

WO2018140323A1 - Novel biosensors and uses thereof

Info

Publication number: WO2018140323A1
Application number: PCT/US2018/014520
Authority: WO
Inventors: Cameron STRACHAN; Nina PATRICK; Will HORVAT
Original assignee: Prospect Bio, Inc.
Priority date: 2017-01-24
Filing date: 2018-01-19
Publication date: 2018-08-02

Abstract

In an aspect, the invention relates to the field of enzyme and metabolic pathway detection and discovery. In another aspect, the invention relates to nucleic acids encoding new biosensor polypeptides useful for detecting responses to stimuli, identifying new enzymes, and novel pathways in cells.

Description

NOVEL BIOSENSORS AND USES THEREOF

BACKGROUND OF THE INVENTION

[0001] Environmental microorganisms are an excellent source for making new enzymes, which can be used to engineer novel pathways into cells. However, environmental microorganisms can be difficult to culture in the laboratory let alone on an industrial scale. Accordingly, a number of metagenome screening methods have been developed to isolate useful genes from metagenomes, for example, metagenomic nucleotide sequencing methods (Okuta et al. Gene (1998) 212:221-228), and enzyme activity based screening (Henne et al. Appl. Environ. Microbiol. (1999) 65:3901-

3907). Specific enzyme activity based screening methods for metagenome screening, include Substrate-Induced Gene-Expression (SIGEX) screening (Uchiyama etal. Nature Biotechnology(2005) 23(l):88-93) and more recently Product-Induced Gene- Expression (PIGEX) screening (Uchiyama and Miyazaki Appl. Environ. Microbiol. (2010) 76(21):7029- 7035). Furthermore, several screening strategies have been developed to discover genetic elements that are activated in response to a metabolite, including intragenic genomic libraries and promoter traps (Uchiyama and Miyazaki PLOS ONE (2013) 8(9):e75795).

[0002] It is an object of the invention to provide a novel biosensor that can be used with methods of the invention to make and identify new and useful control regions and genes that respond to desired stimuli. It is also an object of the invention to make libraries of novel biosensors in metagenomic libraries that can be used to identify new and useful control regions and genes.

SUMMARY OF THE INVENTION

[0003] In an aspect, the present invention relates to novel biosensors for use in identifying targets for stimuli of interest, and methods for making the novel biosensors. The invention also relates to nucleic acids and polypeptides that encode the novel biosensors of the invention. In some embodiments, nucleic acids of the invention are comprised of a nucleic acid encoding a mobile genetic element, a nucleic acid encoding a ribosome binding site, a nucleic acid encoding a reporter polypeptide, and a nucleic acid encoding a selection marker. In some embodiments, the nucleic acid encoding the reporter and the nucleic acid encoding the selection marker are organized in an operon on the biosensor with each having a ribosome binding site. In some embodiments, the reporter is a moiety capable of being detected and includes, for example, a fluorescent reporter, a bioluminescent reporter, an X-ray reporter, a photoacoustic reporter, and/or an ultrasound reporter. In some embodiments, the selection marker encodes a polypeptide that confers a trait upon the host cell that is suitable for artificial selection. Examples of selection markers include nucleic acids encoding polypeptides that confer an antibiotic resistance to the host cell, nucleic acids encoding a polypeptide that complements an auxotrophic mutation in the host cell, or a polynucleotide that encodes a polypeptide that detoxifies a molecule. In some embodiments, the ribosome binding site is compatible with the host cell and initiates translation at a desired rate. In some embodiments, the mobile genetic element allows the nucleic acid encoding the biosensor polypeptides to randomly insert into nucleic acids (e.g., metagenomic inserts) in a host cell.

[0004] In some embodiments, host cells contain the nucleic acids and/or polypeptides of the invention. In some embodiments, the host cells are prokaryotic cells. In some embodiments, the prokaryotic cell is a species from Acidovorax, Agrobacterium, Alicyclobacillus, Anabaena, Anacystis, Arthrobacter, Azobacter, Bacillus, Brevibacterium, Chromatium, Clostridium, Corynebacterium, Enterobacter, Erwinia, Escherichia, Lactobacillus, Lactococcus, Mesorhizobium,

Methylobacterium, Microbacterium, Phormidium, Pseudomonas, Rhodobacter, Rhodopseudomonas, Rhodospirillum, Rhodococcus, Salmonella, Scenedesmun,

Serratia, Shigella, Staphlococcus, Strepromyces, Synnecoccus, and Zymomonas. In some embodiments, the host cell is E. coli. In some embodiments, the host cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is an algae specie and/or a photosynthetic microorganism from Agmenellum, Amphora, Anabaena,

Ankistrodesmus, Botryococcus, Boekelovia , Borodinella, Carteria, Chaetoceros, Chlamydomonas, Chlorella, Chlorococcum, Chlorogonium, Chrysosphaera,

Cricosphaera , Cryptomonas, Cyclotella , Dunaliella, Ellipsoidon, Eremosphaera, Euglena, Fragilaria, Gleocapsa, Gloeothamnion, Hymenomonas, Isochrysis,

Lepocinclis, Monoraphidium, Nannochloris, Nannochloropsis, Navicula,

Nephrochloris, Nitschia, Nitzschia, Ochromonas, Oocystis, Oscillatoria, Pascheria, Phagus, Phormidium, Platymonas, Pleurochrysis Prototheca, Pyrobotrys

Scenedesmus, Spirogyra, Tetraedron, Tetraselmis, or Volvox. In some embodiments, the host cell is Botryococcus braunii, Prototheca krugani, Prototheca moriformis, Prototheca portoricensis , Prototheca stagnora, Prototheca wickerhamii, or

Prototheca zopfli. In some embodiments, the eukaryotic cell is a fungi specie from Aspergillus, Candida, Chlamydomonas, Chrysosporium, Cryotococcus,

Debaromyces, Fusarium, Hansenula, Kluyveromyces, Neotyphodium, Neurospora, Penicillium, Pichia, Saccharomyces, Schizosaccharomyce, Trichoderma,

Xanthophyllomyces, Yarrowia, and Zygosaccharomyces. In some embodiments, the fungi is Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pompe, Kluyveromyces lactic, Hansenula polymorpha, or a filamentous fungi, e.g.

Trichoderma, Aspergillus sp., including Aspergillus niger, Aspergillus phoenicis, Aspergillus carbonarius.

[0005] In some embodiments, the host cells of the invention comprise nucleic acids from environmental sources cloned into metagenomic libraries. In some embodiments, the metagenomic library includes nucleic acids from a community of organisms found in hot springs, permafrost, soil, sea water, and/or hydrocarbon resource samples. In some embodiments, the metagenomic library includes nucleic acids from a community of organisms found in a particular environment. In some embodiments, the environment has a diverse community of microorganism such as those found in, for example, the human gut, the gut of other animals, the oral cavity of an animal, the rhizosphere, acid mine runoff, or geothermal hot springs. In some embodiments, the environment is an extreme environment. In some embodiments, the extreme environment is a hot spring, permafrost, hydrothermal vent, acid pool (or spring), alkaline hot spring, the Antarctic, etc. In some embodiments, the

environment is soil, sea water, hydrocarbon resource samples, or an aqueous body.

[0006] In some embodiments, metagenomic libraries are made from the organisms found in a particular environment. In some embodiments, the

metagenomic libraries are mixed with nucleic acids encoding the biosensors of the invention and the nucleic acids encoding the biosensors insert into the metagenomic library DNA. In some embodiments, the amplification of DNA from the

metagenomic library is tightly controlled by adjusting copy number in a host cell. In some embodiments, the nucleic acids encoding the biosensors randomly insert into the metagenomic library. In some embodiments, the metagenomic library with the randomly inserted biosensor nucleic acids is placed into appropriate host cells. In some embodiments, the host cells with the metagenomic library and biosensor nucleic acids are tested for expression of the biosensor nucleic acids. In some embodiments, metagenomic library clones with functionally expressed biosensors are identified. In some embodiments, the copy number of the metagenomic clone is changed from low copy to high copy (or vice versa) in the host cell. In some embodiments, functional biosensors in the metagenomic library are selected using the selectable marker so that only host cells with biosensors operably linked to an active control region are obtained. In some embodiments, expression from the control region is constitutive. In some embodiments, expression from the control region is inducible. In some embodiments, the basal (non-induced) level of transcription from an inducible control region is sufficient for selection using the selectable marker. In some embodiments, the induced level of transcription is sufficient for selection using the selectable marker. In some embodiments, the induced level of transcription results in sufficient levels of reporter for detection of the reporter.

[0007] In some embodiments, the host cells with metagenomic clones that functionally express the biosensors are tested with desired stimuli to find

biosensor/metagenomic clones that change expression levels of the reporter and/or selectable marker upon exposure to the stimuli. In some embodiments, metagenomic clones with biosensors that show changes in expression levels are isolated. In some embodiments, a biosensor identified as changing expression levels is isolated from the metagenomic library. In some embodiments, the stimulus is a terpene, a secondary metabolite, an aromatic compound, a fatty acid, or an alcohol.

[0008] In some embodiments, a library of metagenomic clones with functional biosensors is made. In some embodiments, compounds of known structure are tested against the library to determine which clones are activated by which structures. In some embodiments, the group of clones activated by a compound can be used to identify that compound and structurally similar compounds. In this embodiment, an unknown compound can be tested against the library and the clones activated are identified. The group of clones activated can be compared to a database for the library showing clones activated by known structures. This comparison of activated clones to a database for the library can provide structural information about the unknown molecule.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG. 1 is a diagram of a biosensor of the invention. [00010] FIG. 2 shows the range of response for selected biosensors to stimulus with a terpene.

[00011] FIG. 3 shows the DNA sequence for the biosensor. Underlined and italicized sequences are the mobile genetic element (ME), bold underlined sequences are the shine-dalgarno sequences, and underlined sequences are the open reading frames for the Gemini reporter and Spectinomycin resistance.

DETAILED DESCRIPTION OF THE INVENTION

[00012] Before the various embodiments are described, it is to be understood that the teachings of this disclosure are not limited to the particular embodiments described, and as such can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present teachings will be limited only by the appended claims.

[00013] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present teachings, some exemplary methods and materials are now described.

[00014] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It is further noted that the claims can be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation. Numerical limitations given with respect to concentrations or levels of a substance are intended to be approximate, unless the context clearly dictates otherwise. Thus, where a concentration is indicated to be (for example) 10 μg, it is intended that the concentration be understood to be at least approximately or about 10 μg.

[00015] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which can be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present teachings. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

Definitions

[00016] In reference to the present disclosure, the technical and scientific terms used in the descriptions herein will have the meanings commonly understood by one of ordinary skill in the art, unless specifically defined otherwise. Accordingly, the following terms are intended to have the following meanings.

[00017] As used herein, "biomass" refers to material produced by growth and/or propagation of cells. Biomass may contain cells and/or intracellular contents as well as extracellular material.

[00018] As used herein, "codon optimized" refers to changes in the codons of the polynucleotide encoding a protein to those preferentially used in a particular organism such that the encoded protein is efficiently expressed in the organism of interest. Although the genetic code is degenerate in that most amino acids are represented by several codons, called "synonyms" or "synonymous" codons, it is well known that codon usage by particular organisms is nonrandom and biased towards particular codon triplets. This codon usage bias may be higher in reference to a given gene, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and the aggregate protein coding regions of an organism's genome.

[00019] As used herein, "consensus sequence" and "canonical sequence" refer to an archetypical amino acid sequence against which all variants of a particular protein or sequence of interest are compared. The terms also refer to a sequence that sets forth the nucleotides that are most often present in a DNA sequence of interest among members of related gene sequences. For each position of a gene, the consensus sequence gives the amino acid that is most abundant in that position in a multiple sequence alignment (MSA).

[00020] As used herein, "control sequence" refers to components, which are used for the expression of a polynucleotide and/or polypeptide of the present invention. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences may include, but are not limited to, some or all of the following: a promoter, an enhancer, an operator, an attenuator, a ribosome binding site (e.g., shine-dalgamo sequence), a leader, a polyadenylation sequence, a propeptide sequence, a signal peptide sequence, and a transcription terminator. At a minimum, the control sequences include a promoter and transcriptional signals, and where appropriate, translational start and stop signals.

[00021] As used herein, an "effective amount" refers to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological result.

[00022] As used herein, the terms "expression vector" or "expression construct" or "recombinant DNA construct" refer to a nucleic acid construct, that has been generated recombinantly or synthetically via human intervention, including by recombinant means or direct chemical synthesis, with a series of specified nucleic acid elements that permit transcription and/or translation of a particular nucleic acid in a host cell. The expression vector can be part of a plasmid, virus, or nucleic acid fragment. Typically, the expression vector includes a nucleic acid to be transcribed operably linked to a promoter. The expression vector can exist in a host cell as either an episomal or integrated vector/construct.

[00023] As used herein, "exogenous gene" refers to a nucleic acid that codes for the expression of an RNA and/or protein that has been introduced ("transformed") into a cell. A transformed cell may be referred to as a recombinant cell, into which additional exogenous gene(s) may be introduced. The exogenous gene may be from a different species (and so heterologous), or from the same species (and so

homologous), relative to the cell being transformed. Thus, an exogenous gene can include a homologous gene that occupies a different location in the genome of the cell or is under different control, relative to the endogenous copy of the gene. An exogenous gene may be present in more than one copy in the cell. An exogenous gene may be maintained in a cell as an insertion into the genome or as an episomal molecule.

[00024] As used herein, "heterologous" polynucleotide or polypeptide refers to any polynucleotide that is introduced into a host cell by laboratory techniques, or a polynucleotide that is foreign to a host cell. As such, the term includes

polynucleotides that are removed from a host cell, subjected to laboratory

manipulation, and then reintroduced into a host cell. In some embodiments, the introduced polynucleotide expresses a heterologous polypeptide. Heterologous polypeptides are those polypeptides that are foreign to the host cell being utilized. [00025] As used herein, "isolated polypeptide" refers to a polypeptide which is substantially separated from other components that naturally accompany it, e.g. , protein, lipids, and polynucleotides. The term embraces polypeptides which have been removed or purified from their naturally-occurring environment or expression system (e.g. , host cell or in vitro synthesis). The polypeptides of the invention may be present within a cell, present in the cellular medium, or prepared in various forms, such as lysates or isolated preparations.

[00026] As used herein "metagenomic" is meant to include any genetic material obtained from an environmental source, as opposed to a laboratory cultured source. In many cases the actual origin (i.e. species or strain) of organism from which the genetic material is obtained may not be known.

[00027] As used herein "metagenomic library" refers to a complex composition comprising a plurality of polynucleotides, of various origins and structure obtained from the whole genomes of a mixed population of microorganisms. Typically, the library comprises a plurality of unknown polynucleotides, i.e., of polynucleotides whose sequence and/or source and/or activity is not known or characterized. In addition to such unknown (or uncharacterized) polynucleotides, the library may further include known sequences or polynucleotides. In some embodiments, the polynucleotides are cloned into cloning constructs, allowing their maintenance and propagation in suitable host cells, (e.g., in E. coli). The polynucleotides in the library may be in the form of a mixture or separated from each other, in all or in part. It should be understood that one or more polynucleotides in the library may be present in various copy numbers.

[00028] As used herein "mobile genetic element" refers to any type of nucleic acid molecule that is capable of movement within a genome or from one genome to another. For example, mobile genetic elements include transposons or transposable elements (including retrotransposons, DNA transposons, and insertion sequences); and bacteriophage elements (including Mu). In some embodiments, a "mobile genetic element" refers to the sequences derived from transposons or viruses which facilitate the movement of other nucleic acids, e.g., the 5' and 3' LTRs of a nucleic acid that can be moved.

[00029] As used herein, "naturally-occurring" or "wild-type" refers to the form found in nature. For example, a naturally occurring or wild-type polypeptide or polynucleotide sequence is a sequence present in an organism that can be isolated from a source in nature and which has not been intentionally modified by human manipulation.

[00030] As used herein, "operably linked" and "operable linkage" refer to a configuration in which a control sequence or other nucleic acid is appropriately placed (i.e. , in a functional relationship) at a position relative to a polynucleotide of interest such that the control sequence or other nucleic acid can interact with the

polynucleotide of interest. In the case of a control sequence, operable linkage means the control sequence directs or regulates the expression of the polynucleotide and/or polypeptide of interest. In the case of polypeptides, operably linked refers to a configuration in which a polypeptide is appropriately placed at a position relative to a polypeptide of interest such that the polypeptide can interact as desired with the polypeptide of interest.

[00031] As used herein, "percentage of sequence identity" and "percentage homology" are used interchangeably herein to define to comparisons among polynucleotides or polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, where the portion of the

polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e. , gaps) as compared to the reference sequence for optimal alignment of the two sequences. The percentage may be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Alternatively, the percentage may be calculated by determining the number of positions at which either the identical nucleic acid base or amino acid residue occurs in both sequences or a nucleic acid base or amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Those of skill in the art appreciate that there are many established algorithms available to align two sequences. Optimal alignment of sequences for comparison can be conducted, e.g. , by the local homology algorithm of Smith and Waterman, Adv Appl Math. 2.482, 1981; by the homology alignment algorithm of Needleman and Wunsch, JMol Biol. 48:443, 1970; by the search for similarity method of Pearson and Lipman, Proc Natl Acad Sci. USA 85:2444, 1988; by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection (see generally, Current Protocols in Molecular Biology, F. M. Ausubel et al, eds., Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement). Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al, J. Mol. Biol. 215:403-410, 1990; and Altschul et al, Nucleic Acids Res. 25(17):3389-3402, 1977; respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. BLAST for nucleotide sequences can use the BLASTN program with default parameters, e.g. , a word length (W) of 11 , an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. BLAST for amino acid sequences can use the BLASTP program with default parameters, e.g. , a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc NatlAcad Sci. USA 89: 10915, 1989). Exemplary determination of sequence alignment and % sequence identity can also employ the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison WI), using default parameters provided.

[00032] As used herein, "recombinant" or "engineered" or "non-naturally occurring" refers to a cell, nucleic acid, protein or vector that has been modified due to the introduction of an exogenous nucleic acid or the alteration of a native nucleic acid. Thus, e.g., recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes differently than those genes are expressed by a non-recombinant cell. A "recombinant nucleic acid" is a nucleic acid made, in general, by the manipulation of nucleic acid, e.g., using polymerases and endonucleases, or otherwise into a form not normally found in nature. Recombinant nucleic acids may be produced, for example, to place two or more nucleic acids in operable linkage. Thus, an isolated nucleic acid or an expression vector formed in vitro by ligating DNA molecules that are not normally joined in nature, are both considered recombinant for the purposes of this invention. Once a recombinant nucleic acid is made and introduced into a host cell or organism, it may replicate using the in vivo cellular machinery of the host cell; however, such nucleic acids, once produced recombinantly, although subsequently replicated intracellularly, are still considered recombinant for purposes of this invention.

Similarly, a "recombinant protein" is a protein made using recombinant techniques, i.e., through the expression of a recombinant nucleic acid.

[00033] As used herein, "reference sequence" refers to a defined sequence used as a basis for a sequence comparison. A reference sequence may be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence. Generally, a reference sequence is at least 20 nucleotide or amino acid residues in length, at least 25 residues in length, at least 50 residues in length, or the full length of the nucleic acid or polypeptide. Since two polynucleotides or polypeptides may each (1) comprise a sequence (i.e. , a portion of the complete sequence) that is similar between the two sequences, and (2) may further comprise a sequence that is divergent between the two sequences, sequence comparisons between two (or more) polynucleotides or polypeptide are typically performed by comparing sequences of the two polynucleotides or polypeptides over a "comparison window" to identify and compare local regions of sequence similarity. In some embodiments, a "reference sequence" can be based on a primary amino acid sequence, where the reference sequence is a sequence that can have one or more changes to the primary sequence.

[00034] As used herein, the term "reporter" or "reporter molecule" refers to a moiety capable of being detected indirectly or directly. Reporters include, without limitation, a chromophore, a fluorophore, a fluorescent protein, a receptor, a hapten, an enzyme, and a radioisotope.

[00035] As used herein, the term "reporter gene" refers to a polynucleotide that encodes a reporter molecule that can be detected, either directly or indirectly.

Exemplary reporter genes encode, among others, enzymes, fluorescent proteins, bioluminescent proteins, receptors, antigenic epitopes, and transporters.

[00036] As used herein, the term "reporter probe" refers to a molecule that contains a detectable label and is used to detect the presence (e.g., expression) of a reporter molecule. The detectable label on the reporter probe can be any detectable moiety, including, without limitation, an isotope (e.g., detectable by PET, SPECT, etc), chromophore, and fluorophore. The reporter probe can be any detectable molecule or composition that binds to or is acted upon by the reporter to permit detection of the reporter molecule.

[00037] As used herein, a "ribosome binding site" refers to a sequence of nucleotides upstream of the start codon of an mRNA transcript that is responsible for the recruitment of a ribosome during the initiation of protein translation.

[00038] As used herein, a "selection marker" refers to a gene introduced into a host cell that confers upon the host cell a trait suitable for artificial selection.

[00039] As used herein, "stringent hybridization conditions" refers to hybridizing in 50% formamide at 5XSSC at a temperature of 42 °C and washing the filters in 0.2XSSC at 60 °C. (1XSSC is 0.15M NaCl, 0.015M sodium citrate.) Stringent hybridization conditions also encompasses low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/O. 1% sodium dodecyl sulfate at 50 °C; hybridization with a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42 °C; or 50% formamide, 5XSSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5X Denhardt's solution, sonicated salmon sperm DNA (50 g ml), 0.1% SDS, and 10% dextran sulfate at 42 °C, with washes at 42 °C in 0.2XSSC (sodium chloride/sodium citrate) and 50% formamide at 55 °C, followed by a high-stringency wash consisting of 0.1XSSC containing EDTA at 55 °C.

[00040] As used herein, "substantial identity" refers to a polynucleotide or polypeptide sequence that has at least 80 percent sequence identity, at least 85 percent identity and 89 to 95 percent sequence identity. Substantial identity also encompasses at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 residue positions or a window of at least 30-50 residues, wherein the percentage of sequence identity is calculated by comparing the reference sequence to a sequence that includes deletions or additions or substitutions over the window of comparison. In specific embodiments applied to polypeptides, the term "substantial identity" means that two polypeptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using standard parameters, i.e., default parameters, share at least 80 percent sequence identity, preferably at least 89 percent sequence identity, at least 95 percent sequence identity or more (e.g. , 99 percent sequence identity).

[00041] As used herein, "substantially pure polypeptide" refers to a composition in which the polypeptide species is the predominant species present (i.e. , on a molar or weight basis it is more abundant than any other individual

macromolecular species in the composition), and is generally a substantially purified composition when the object species comprises at least about 50 percent of the macromolecular species present by mole or % weight. Generally, a substantially pure polypeptide composition will comprise about 60 % or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more of all macromolecular species by mole or % weight present in the composition. In some embodiments, the object species is purified to essential homogeneity (i.e. , contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species. Solvent species, small molecules (<500 Daltons), and elemental ion species are not considered macromolecular species.

Biosensors

[00042] In some embodiments, biosensors of the invention are encoded by nucleic acids comprised of polynucleotides encoding a mobile genetic element, a ribosome binding site, a reporter polypeptide, and a selection marker. In some embodiments, the reporter is a moiety capable of being detected which includes, for example, a fluorescent reporter, a bioluminescent reporter, an X-ray reporter, a photoacoustic reporter, and an ultrasound reporter. In some embodiments, the selection marker encodes a polypeptide that confers a trait upon the host cell that is suitable for artificial selection. Examples of selection markers include nucleic acids encoding polypeptides that confer an antibiotic resistance to the host cell, nucleic acids encoding a polypeptide that complements an auxotrophic mutation (e.g., auxotrophic mutations can inhibit the cell's ability to make an amino acid) in the host cell, or nucleic acids encoding a polypeptide that can detoxify a molecule. In some embodiments, the antibiotic resistance can be encoded by any of the resistance genes known in the art including, for example, those described in van Hoek et al, Front. Microbiol. 2: 1-27 (Sep. 2011). In some embodiments, the ribosome binding site is compatible with the host cell and has a high translation initiation rate in the host cell (e.g., a strong shine dalgarno sequence for ?, coli). In some embodiments, the ribosome binding site has a low translation initiation rate in the host cell. In some embodiments, the biosensor is made with a panel of ribosome binding sites of different strength to provide different windows of detection for the stimulus. In some embodiments, the mobile genetic element allows the nucleic acid encoding the biosensor polypeptides to randomly insert into nucleic acids in a host cell.

[00043] In some embodiments, the polynucleotide encoding the reporter and the polynucleotide encoding the selectable marker are organized in an operon so that both are expressed from the control region in the metagenomic nucleic acids. In some embodiments, the selection marker can be expressed at low levels from the control region to produce a detectable phenotype. In some embodiments, the selection marker gives the host cell resistance to an antibiotic. In some embodiments, low, constitutive expression from the control region in the metagenomic nucleic acids expresses enough antibiotic resistance polypeptide so that the host cell can withstand a low amount of antibiotic in the growth media. In some embodiments, the ribosome binding site for the selection marker is strong and provides a high level of translation initiation of the selection marker. In some embodiments, the polynucleotide encoding the reporter produces polypeptide that can be detected (directly or indirectly) when the control region in the metagenomic nucleic acid is transcribed at a higher rate than the low constitutive level. In some embodiments, the control region is induced to increase transcription and this higher level of transcription produces enough reporter polypeptide for detection.

[00044] In some embodiments, the biosensor as depicted in FIG. 1 has a mobile genetic element that is recognized by the Tn5 transposase. As depicted in FIG. 1, the mobile genetic element is made of two components, one is found at the 5' end of the biosensor construct and the other is found at the 3' end of the biosensor construct. In some embodiments, the mobile genetic elements of the biosensor have the sequences CTGTCTCTTATACACATCT (SEQ ID NO: 1) and AGATGTGTATAAGAGACAG (SEQ ID NO: 2). The ribosome binding site of FIG. 1 can be a ribosome binding site such as ACAGGAAAG (SEQ ID NO: 3), TAAGGAGGT (SEQ ID NO: 4), or many other ribosome binding sites that are well known in the art. The strength or amount of translation initiated from the ribosome binding site can be altered by changing the ribosome binding site in ways known in the art. The Gemini element of FIG. 1 is a reporter made of a fusion between a C-terminal portion of the alpha fragment of β- galactosidase and the N-terminus of full length green fluorescent protein, as described in Marin et al, PLoS ONE 4:e7569 (2009), which is incorporated by reference in its entirety for all purposes. The reporter element labeled as Gemini in FIG. 1 can be any other reporter gene described herein and/or known in the art. The Gemini reporter has a GFP portion that can be optically assayed to measure expression. The Gemini reporter also the alpha fragment of β-galactosidase that has enzymatic activity which can be complemented by the β-galactosidase omega fragment to produce enzymatic activity. The enzymatic activity of β-galactosidase is a more sensitive reporter than the fluorescence from GFP because the enzyme activity amplifies the expression signal.

[00045] The selection gene used in the construct of FIG. 1 is a Spectinomycin resistance gene as described in Clark et al, Antimicrob. Agents Chemotherap.

43: 157-160 (1999), which is incorporated by reference in its entirety for all purposes. The selection gene in FIG. 1 can also be any other selection gene described herein and/or known in the art. In the biosensor construct of FIG. 1, the nucleic acid encoding the Gemini reporter (reporter gene) and the nucleic acid encoding

Spectinomycin resistance (selection gene) are organized in an operon so that both nucleic acids are expressed when a functional control region is operably linked to the biosensor construct.

[00046] In an aspect of the invention, FIG. 3 shows a sequence for the biosensor of the invention (SEQ ID NO: 5) with the mobile genetic element being at sequences 1-19 and 3298-3316, the Gemini reporter is encoded at sequences 49-999, the ribosome binding site for the Gemini reporter is at sequences 34-42, the

Spectinomycin resistance is encoded at sequences 1085-1873, and the ribosome binding site for the Spectinomycin resistance is at sequences 1072-1077. These sequences are underlined (coding sequences), bold underlined (ribosome binding sites) and italicized underlined (mobile genetic element) in FIG. 3.

[00047] In some embodiments, the mobile genetic element can insert randomly or semi-randomly into the metagenomic nucleic acids. In some embodiments, the mobile genetic element is a transposon, a nucleic acid derived from a transposon, an LTR, a nucleic acid derived from an LTR, a Mu phage, a Mu phage vector, a nucleic acid derived from a Mu phage, or the like. In some embodiments, the transposon is a member of, for example, the DDE transposase family, the tyrosine (Y) transposase family, the serine (S) transposase family, the rolling circle (RC) transposase family, the Y2 transposase family, or the reverse transcriptase/endonuclease family.

[00048] In some embodiments, the mobile genetic element of the biosensor functions in an appropriate host cell. In some embodiments, the mobile genetic element of the biosensor functions in an appropriate in vitro environment. In some embodiments, the host cell is a bacterium, a fungal cell, a yeast cell, an insect cell, an algal cell, a plant cell, or a mammalian cell.

Reporters

[00049] In some embodiments, the reporter is selected from, for example, fluorescent reporters, bioluminescent reporters, and enzyme reporters. Other reporters are well known in the art and can be used as the reporter in the biosensors of the invention. For example, the biosensor could encode a polypeptide that activates the expression of another gene which provides a detectable reporter signal. In some embodiments, the reporter is selected because it will produce a detectable signal when expressed at the desired level from a candidate control region. In some embodiments, the reporter is selected to provide a signal that distinguishes high

transcription/expression rates from the candidate control regions from medium and low expression rates for the candidate control regions. In some embodiments, the reporter is selected to provide a signal that distinguishes medium

transcription/expression rates from low expression rates for the candidate control regions. In some embodiments, the reporter is selected to provide a signal that distinguishes between low level rates of expression for the candidate control regions.

[00050] In some embodiments, the fluorescent reporter includes, for example, green fluorescent protein from Aequorea victoria or Renilla reniformis, and active variants thereof (e.g., blue fluorescent protein, yellow fluorescent protein, cyan fluorescent protein, etc.); fluorescent proteins from Hydroid j ellyfishes, Copepod, Ctenophora, Anthrozoas, and Entacmaea quadricolor, and active variants thereof; and phycobiliproteins and active variants thereof.

[00051] In some embodiments herein where the reporter is a bioluminescent reporter, any number of bioluminescent proteins can be used as the reporter. These include, for example, aequorin (and other Ca⁺² regulated photoproteins), luciferase based on luciferin substrate, luciferase based on Coelenterazine substrate (e.g., Renilla, Gaussia, and Metridina), and luciferase from Cypridina, and active variants thereof.

[00052] In some embodiments, the reporter is an enzyme including for example, acetylcholinesterase, alkaline phosphatase, chloramphenicol

acetyltransferase, peroxidase, or β-lactamase. Many other enzymes are well-known in the art and can be used as reporters for the biosensors of the invention.

[00053] In some embodiments, reactants paired with aceytlcholinesterase include, for example, acetylthiocholine, or ThioStar® (which is commercially available from Arbor Assays or Kamiya Biomedical Co.) In some embodiments, reactants paired with alkaline phosphatase include, for example, / aminophenyl phosphate (commercially available from Sigma Aldrich), PNPP '-Nitrophen l Phosphate, Disodium Salt), CSPD® chemiluminescent reactant, 1 ,2-dioxetane chemiluminescent reactant, DynaLight™ Substrate with RapidGlow™ Enhancer, which are all commercially available from ThermoFisher Scientific. In some embodiments, reactants paired with chloramphenicol acetyltransferase include, for example, FAST CAT® Green (deoxy), which is commercially available from

ThermoFisher Scientific. In some embodiments, redox reactants paired with peroxidase, include, for example, hydroquinone, hydroxymethyl ferrocene, osmium complex, p-aminophenol, m-aminophenol, and o-aminophenol (o-AP). In some embodiments, other reactants for peroxidase include, for example, ABTS (2,2'- Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt), OPD (o- phenylenediamine dihydrochloride), TMB (3,3',5,5'-tetramethylbenzidine),

SuperSignal ELISA Pico Chemiluminescent Substrate, QuantaBlu NS/K Fluorogenic Substrate, QuantaRed Enhanced Chemifluorescent HRP Substrate (ADHP), Amplex Red reagent, all of which are commercially available from ThermoFisher Scientific. In some embodiments, reactants paired with β-lactamase, include, for example, C3' thiolate-substituted cephalosporins. In some embodiments, other reactants for β- lactamase include, for example, CCF2-FA, CCF2-AM, CCF4-AM, Fluorocillin™ Green reagent, LyticBLAzer™_h-BODIPY® FL Substrate, which is commercially available from ThermoFisher Scientific.

[00054] In some embodiments, the reporter is detectable by multiple imaging modalities, for example tyrosinase which has been shown to yield photoacoustic imaging (PAI), MRI and PET (with a suitable radiotracer) signals (see, e.g., Qin, C. et al, "Tyrosinase as a multifunctional reporter gene for photoacoustic/MRI/PET triple modality molecular imaging," Scientific Rep. 3 : 1490 (2013), incorporated herein by reference in its entirety for all purposes). Alternatively, the reporter gene can be a fusion protein comprising two or more reporters linked together (e.g., a luciferase- GFP-thymidine kinase triple fusion reporter). (Ray P. et al, "Imaging tri-fusion multimodality reported gene expression in living subjects," Cancer Res. 64: 1323- 1330 (2004), incorporated herein by reference in its entirety for all purposes).

Mobile Genetic Elements

[00055] In some embodiments, mobile genetic elements are capable of moving, with or without duplication, from one site in a genome to another, or from one cell to another. In some embodiments, mobile genetic elements include viruses, transposable genetic elements, short interspersed elements (SINES), long interspersed elements (LINES), LTRs, and the like. In some embodiments, the mobile genetic element is characterized by inverted terminal repeats located 5 ' and 3' of the nucleic acid that is moved. In some embodiments, an enzyme, transposase catalyzes the transposition or movement of the mobile genetic element. In some embodiments, the transposase is encoded by the mobile genetic element. In some embodiments, the transposase is encoded in nucleic acids outside of the mobile genetic element. In some

embodiments, the mobile genetic element is just the sequences that facilitate the movement of other nucleic acids (e.g., the LTRs).

[00056] In some embodiments, mobile genetic elements of the invention have the following properties (1) small size (length in base pairs), (2) easy to manipulate by recombinant methods, (3) a simple transposition mechanism, (4) the transposase can catalyze all of the steps of transposition, and (5) the transposition activity is highly specific and does not stimulate resistance mechanisms in the host.

[00057] In some embodiments, the mobile genetic element is a transposable element. In some embodiments, the transposable element is characterized by inverted terminal repeats located at distal 5' and 3 ' positions. In some embodiments, the mobile genetic element is just the inverted terminal repeats. Transposable elements have been found in both prokaryotes and eukaryotes. In some embodiments, transposable elements are grouped into two classes: class I retrotransposons and class II DNA transposons. [00058] In some embodiments, the class I Retrotransposons, are grouped in two subclasses, the long terminal repeat (LTR) and the non-LTR retrotransposons. LTR retrotransposons have direct LTRs that range from -100 bp to over 5 kb in size. LTR retrotransposons are further sub-classified into the Tyl-copia-like (Pseudoviridae), Ty3-gypsy-like (Metaviridae), and BEL-Pao-like groups based on both their degree of sequence similarity and the order of encoded gene products. Tyl-copia and Ty3- gypsy groups of retrotransposons are commonly found in high copy number (up to a few million copies per haploid nucleus) in animals, fungi, protista, and plants genomes. BEL-Pao like elements have so far only been found in animals.

[00059] In some embodiments, retroviruses are classified separately from LTR transposons, however, retroviruses share many features with LTR retrotransposons. In some embodiments, a major difference between retrotransposons and retroviruses is that retroviruses have an Envelope protein (ENV). In some embodiments, a retrovirus is transformed into an LTR retrotransposon through inactivation or deletion of the domains that enable extracellular mobility. If such a retrovirus infects and subsequently inserts itself in the genome in germ line cells, it may become transmitted vertically and become an Endogenous Retrovirus (ERV).

[00060] In some embodiments, eukaryotic class II elements include five families, namely P, PiggyBac, hAT, helitron, and Tcl-mariner. In some

embodiments, prokaryotic mobile genetic elements include transposons (e.g., Tn 3, Tn 5, and Tn 10) and insertion sequences (e.g., IS1, IS2, and IS10). In some embodiments, the mobile genetic elements are Tn 5 transposase recognition sequences. In some embodiments, the Tn 5 transposase recognition sequences are SEQ ID NOs: 1 and 2.

[00061] In some embodiments, IS 1 is 768 bp long, and is present in 4 to 19 copies in the E. coli chromosome, IS2 is present in 0 to 12 copies on the E. coli chromosome and in one copy on the F plasmid, and IS 10 is found in a class of plasmids called R plasmid. In some embodiments, IS sequences of the invention end in inverted terminal repeats of 9-41 base pairs. In some embodiments, the inverted terminal sequences of a nucleic acid to be moved are perfect inverted repeats.

[00062] In some embodiments, Long Interspersed Nuclear Elements (LINE) are a group of genetic elements that are found in large numbers in eukaryotic genomes, comprising 17% of the human genome. In some embodiments, the LINE are grouped into several subgroups, such as LI, L2 and L3. In some embodiments, a LINE begins with an untranslated region (UTR) that includes an RNA polymerase II promoter, two non-overlapping open reading frames (ORF1 and ORF2), and ends with another UTR. In some embodiments, ORFl encodes an RNA binding protein and ORF2 encodes a protein having an endonuclease (e.g. RNase H) as well as a reverse transcriptase. In some embodiments, the reverse transcriptase has a higher specificity for the LINE RNA than other RNA, and makes a DNA copy of the RNA that can be integrated into the genome at a new site.

[00063] In some embodiments, Short Interspersed Nuclear Elements (SINES) are short DNA sequences (e.g., less than 500 bases) that represent reverse-transcribed RNA molecules originally transcribed by RNA polymerase III into transfer RNA, 5S ribosomal RNA, and other small nuclear RNAs. In some embodiments, SINEs do not encode a functional reverse transcriptase protein and rely on other mobile elements for transposition. In some embodiments, SINES have their own endonuclease that will allow them to cleave their way into the genome. In some embodiments, SINEs integrate at chromosomal breaks using random DNA breaks to prime reverse transcriptase.

Selectable Markers

[00064] Selectable markers are well-known in the art for prokaryotic and eukaryotic cells, including host cells of the invention. Generally, the selection gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the construct containing the selection gene will not survive in the culture medium. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, or (b) complement auxotrophic deficiencies.

[00065] Selectable markers may confer resistance (or ability to grow) to a number of different antibiotics or toxins including, for example, ampicillin, erythromycin, chloramphenicol, kanamycin, methotrexate, neomycin, spectinomycin, or tetracycline. Other selectable markers suitable for use in the invention may be found at the Antibiotic resistance genes database at ardb.cbcb.umd.edu, which is incorporated by reference in its entirety for all purposes.

[00066] Selectable markers may also complement auxotrophic deficiencies including, for example, amino acid auxotrophies caused by the loss of an enzyme activity needed to make the amino acid (such auxotrophs can be complemented by a nucleic acid encoding an enzyme with an activity that can replace the lost activity), carbon utilization auxotrophs, vitamin or cofactor auxotrophs, etc.

Nucleic Acids

[00067] In some embodiments, the present invention relates to the nucleic acids that encode, at least in part, the metagenomic libraries with the biosensors of the present invention. In some embodiments, the nucleic acids encode an individual biosensor operably linked to a control region of the metagenomic library. In some embodiments, the nucleic acids of the invention include the gene from which the control region is obtained. In some embodiments, the nucleic acids may be natural, synthetic or a combination thereof. In some embodiments, the nucleic acids of the invention may be RNA, mRNA, DNA or cDNA.

[00068] In some embodiments, the nucleic acids of the invention include expression constructs, such as plasmids, or viral vectors, or linear vectors, or vectors that integrate into chromosomal DNA. Expression constructs can contain a nucleic acid sequence that enables the construct to replicate in one or more selected host cells (e.g., an origin of replication). Such sequences are well known for a variety of cells. E.g., the origin of replication from the plasmid pBR322 is suitable for most Gram- negative bacteria. In eukaryotic host cells, e.g., mammalian cells, the expression construct can be integrated into the host cell chromosome and then the construct replicates with the host chromosome. Similarly, constructs can be integrated into the chromosome of prokaryotic cells.

[00069] In general, expression constructs containing replication and control sequences that are derived from species compatible with the host cell are used in connection with a suitable host cell. The expression construct ordinarily carries a replication site, as well as marking sequences that are capable of providing phenotypic selection of the construct in transformed cells. For example, E. coli is typically transformed using pBR322, a plasmid derived from an E. coli species (see, e.g., Bolivar et al., (1977) Gene, 2: 95). pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells.

[00070] In some embodiments, the constructs used can be stimulated to increase (or decrease) copy number in a suitable host cell. This copy control can be used to change the window of detection/selection for the biosensors that are cloned in the constructs, e.g., fosmid clones. For example, the CopyControl Cloning System vectors which are sold by Epicentre can be used in the invention to make fosmid clones whose copy number can be inducibly changed (using arabinose). These copy number controllable constructs may be used in conjunction with the EPI300 E. coli strain which is also sold by Epicentre. In some embodiments, the CopyControl Cloning System is used to induce a high copy number for fosmid clones in the Metagenomic library.

[00071] Expression constructs also generally contain a selection gene, also termed a selectable marker. Selectable markers are well-known in the art for prokaryotic and eukaryotic cells, including host cells of the invention. Generally, the selection gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the construct containing the selection gene will not survive in the culture medium.

Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g. , ampicillin, neomycin, methotrexate, spectinomycin,

chloramphenicol, kanamycin, or tetracycline, (b) complement auxotrophic deficiencies, e.g. , the gene encoding D-alanine racemase for Bacilli unable to make D-alanine because of a mutant D-alanine racemase. In some embodiments, an exemplary selection scheme utilizes a drug to arrest growth of a host cell. Those cells that are successfully transformed with a heterologous gene produce a protein conferring drug resistance and thus survive the selection regimen. Other selectable markers for use in bacterial or eukaryotic (including mammalian) systems are well- known in the art.

[00072] The expression construct for producing the polypeptides of the invention contain a suitable control region that is recognized by the host organism and is operably linked to the nucleic acid encoding the polypeptide of interest. Promoters used in the constructs of the invention include cis-acting transcriptional control elements and regulatory sequences that are involved in regulating or modulating the timing and/or rate of transcription of a gene. For example, a promoter can be a cis- acting transcriptional control element, including an enhancer, a promoter, a transcription terminator, an origin of replication, a chromosomal integration sequence, 5' and 3' untranslated regions, or an intronic sequence, which are involved in transcriptional regulation. These cis-acting sequences can interact with proteins or other biomolecules to carry out (turn on/off, regulate, modulate, etc.) transcription. "Constitutive" promoters are those that drive expression continuously under most environmental conditions and states of development or cell differentiation.

"Inducible" or "regulatable" promoters direct expression of the nucleic acid of the invention under the influence of environmental conditions or developmental conditions. Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions, elevated temperature, drought, or the presence of light.

[00073] Promoters suitable for use with prokaryotic hosts include the beta- lactamase and lactose promoter systems (Chang et al, (1978) Nature, 275: 615;

Goeddel et al, (1979) Nature, 281 : 544), the arabinose promoter system (Guzman et al, (1992) J. Bacteriol., 174: 7716-7728), alkaline phosphatase, a tryptophan (trp) promoter system (Goeddel, (1980) Nucleic Acids Res., 8: 4057 and EP 36,776) and hybrid promoters such as the tac promoter (deBoer et al, (1983) Proc. Natl. Acad. Sci. USA, 80: 21-25). Other exemplary bacterial promoters include lacl, lacZ, T3, T7, gpt, lambda PR, and PL. Other bacterial promoters suitable for expression vectors are also well known in the art. Exemplary eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein I. The nucleotide sequences of these and many other promoters have been published, thereby enabling a skilled worker to operably ligate them to DNA encoding the polypeptide of interest (Siebenlist et al, (1980) Cell, 20: 269) using linkers or adaptors to supply any required restriction sites. See also, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); and Current Protocols in Molecular Biology, Ausubel et al, eds, Green Publishers Inc. and Wiley and Sons, N.Y (1994), both of which are incorporated by reference in their entirety for all purposes.

[00074] Control regions for use in bacterial systems also generally contain a

Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding the polypeptide of interest. The Shine-Dalgarno sequence and the initiating ATG codon are used in the initiation of translation by the ribosome in bacterial systems.

[00075] Expression constructs of the invention typically have promoter elements, e.g., enhancers, to regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 base pairs upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 base pairs apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription.

[00076] The present invention also provides nucleic acids that encode polypeptides. The nucleic acid encoding a polypeptide can be easily prepared from an amino acid sequence of the polypeptide of interest using the genetic code. The nucleic acid encoding a polypeptide can be prepared using a standard molecular biological and/or chemical procedure. For example, based on the base sequence, a nucleic acid can be synthesized, and the nucleic acid of the present invention can be prepared by combining DNA fragments which are obtained from a cell or other nucleic acid using a polymerase chain reaction (PCR).

[00077] The nucleic acid of the present invention can be linked to another nucleic acid so as to be expressed under control of a suitable promoter. The nucleic acid of the present invention can be also linked to, in order to attain efficient transcription of the nucleic acid, other regulatory elements that cooperate with a promoter or a transcription initiation site, for example, a nucleic acid comprising an enhancer sequence, or a terminator sequence. In addition to the nucleic acid of the present invention, a gene that can be a marker for confirming expression of the nucleic acid (e.g. a drug resistance gene, a gene encoding a reporter enzyme, or a gene encoding a fluorescent protein) may be incorporated.

[00078] When the nucleic acid of the present invention is introduced into a host cell, the nucleic acid of the present invention may be combined with a substance that promotes transference of a nucleic acid into a cell, for example, a reagent for introducing a nucleic acid such as a liposome or a cationic lipid, in addition to the aforementioned excipients. Alternatively, a construct carrying the nucleic acid of the present invention is also useful.

Environmental Sources and Metagenomic Libraries [00079] In some embodiments, metagenomic libraries are made by extracting DNA from environmental samples, cloning that DNA in bulk into suitable constructs, and placing the constructs into an appropriate host cell. In some embodiments, metagenomic libraries are used for identification of microorganisms in the environment, detection and cloning of genes of interest, and discovery of biosynthetic pathways that can be used to make new metabolites and/or engineer new synthetic (or degradative) capabilities into other organisms. In some embodiments, the metagenomic library is an expression library allowing expression of the metagenomic DNA in an appropriate host organism. In some embodiments, the metagenomic library is combined with biosensors of the invention to make metagenomic biosensor libraries capable to responding to a variety of desired stimuli.

[00080] In some embodiments, the Meta-G-Nome™ DNA Isolation Kit commercially sold by Epicenter is used to extract nucleic acids from environmental samples. (Epicentre Catalog No. MGN0910). The Meta-G-Nome™ DNA Isolation Kit isolates randomly-sheared, high-molecular- weight (HMW) metagenomic DNA, free of humic and fulvic acid, directly from unculturable or diffi cult-to-culture microbial species present in environmental samples. The DNA isolated using this kit is approximately 40 kb in size and is ready for immediate use in end-repair reactions and subsequent cloning into fosmid vectors. The Meta-G-Nome™ DNA Isolation Kit instructions are incorporated by reference in their entirety for all purposes.

[00081] In some embodiments, the CopyControl™ Fosmid Library Production Kit with pCClFOS™ Vector and/or the CopyControl™ Fosmid Library Production Kit with pCC2FOS™ Vector are used to make fosmid clones of the metagenomic DNA. In some embodiments the DNA obtained from the Meta-G-Nome™ DNA Isolation Kit is used as inserts for the CopyControl™ vectors. The CopyControl

Cloning System combines the clone stability afforded by single-copy cloning with the advantages of high yields of DNA obtained by "on-demand" induction of the clones to high-copy number. For example, CopyControl BAC (Bacterial Artificial

Chromosome) clones can be induced to 10-20 copies per cell and CopyControl Fosmid and PCR clones can be induced from single-copy to 10-200 copies per cell. The CopyControl™ Fosmid Library Production Kit instructions are incorporated by reference in their entirety for all purposes. [00082] In some embodiments, the methods found in W099/45154,

W096/34112, U.S. Patent No. 7,910,522, Thomas et al, Microb. Informal Exper. 2:3- 15 (2012), Daniel Directed Molecular Evolution of Proteins (Ed. S. Brakmann, K. Johnsson) Wiley-VCH Verlag GmbH & Co., KGA, ISBNs: 3-527-30423-1 (2002), Mewis et al., J. Visualized Exper. 48, doi: 10.3791/2461 (2011), Strachan et al, Proc. Natl Acad. Sci. 111 : 10143-48 (2014), USSN 15/115,644 filed July 29, 2016, and USSN 15/191,482 filed June 23, 2016, which are all incorporated by reference in their entirety for all purposes, are used to make metagenomic libraries, including expression libraries using a pool of expression constructs where each expression construct contains DNA which is operably associated with one or more regulatory regions that drive expression of genes in an appropriate host. Many other methodologies for making metagenomic libraries are well known to those of ordinary skill in the art.

[00083] In some embodiments, the metagenomic library includes nucleic acids from communities of organisms found in hot springs, permafrost, soil, sea water, and hydrocarbon resource samples. In some embodiments, the environmental source of microorganisms for the metagenomic library are a soil sample, the rhizosphere, acid mine runoff, geothermal hot springs, hydrothermal vents, acid pools (or springs), alkaline hot springs, permafrost, the Antarctic, seawater, hydrocarbon resource samples (in the soil), the human gut, the gut of other animals, or the oral cavity of an animal. In some embodiments, the environment is an aqueous body. Many other environmental sources are well-known to the person of ordinary skill in the art and are contemplated as within the scope of the invention.

Host Cells

[00084] In the present invention, various host cells can be used with the polynucleotides and polypeptides of the invention. The host cell may be any of the host cells familiar to those skilled in the art, including prokaryotic cells and eukaryotic cells, such as bacterial cells, fungal cells, yeast cells, mammalian cells, insect cells, or plant cells. Suitable prokaryotic host cells for expression of the biosensors and/or metagenomic libraries of the invention are well known in the art. Suitable prokaryote host cells include bacteria, e.g., eubacteria, such as Gram- negative or Gram-positive organisms, for example, any species of Acidovorax, Agrobacterium, Alicyclobacillus, Anabaena, Anacystis, Arthrobacter, Azobacter, Bacillus, Brevibacterium, Chromatium, Clostridium, Corynebacterium, Enterobacter, Erwinia, Escherichia, Lactobacillus, Lactococcus, Mesorhizobium,

Methylobacterium, Microbacterium, Phormidium, Pseudomonas, Rhodobacter, Rhodopseudomonas, Rhodospirillum, Rhodococcus, Salmonella, Scenedesmun, Serratia, Shigella, Staphlococcus, Strepromyces, Synnecoccus, and Zymomonas, including, e.g., E. coli, B. subtilis, P. aeruginosa, Salmonella typhimurium, Bacillus cereus, Pseudomonas fluorescens, Serratia marcescens, Clostridium acetobutylicum, Clostridium Beijerinckii, Clostridium saccharoperbutylacetonicum, Clostridium saccharobutylicum, Clostridium aurantibutyricum, or Clostridium tetanomorphum.

[00085] One example of an E. coli host is E. coli 294 (ATCC 31,446). Other strains such as E. coli B, E. coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are also suitable. These examples are illustrative rather than limiting. Strain W3110 is a typical host because it is a common host strain for recombinant DNA product fermentations. In one aspect of the invention, the host cell should secrete minimal amounts of proteolytic enzymes. For example, strain W3110 may be modified to effect a genetic mutation in the genes encoding proteins, with examples of such hosts including E. coli W3110 strains 1 A2, 27 A7, 27B4, and 27C7 described in U.S. Pat. No. 5,410,026 issued Apr. 25, 1995, which is incorporated by reference in its entirety for all purposes.

[00086] In some embodiments the host cells are plant cells. In some embodiments the plant cells are cells of monocotyledonous or dicotyledonous plants, including, but not limited to, alfalfa, almonds, asparagus, avocado, banana, barley, bean, blackberry, brassicas, broccoli, cabbage, canola, carrot, cauliflower, celery, cherry, chicory, citrus, coffee, cotton, cucumber, eucalyptus, hemp, lettuce, lentil, maize, mango, melon, oat, papaya, pea, peanut, pineapple, plum, potato (including sweet potatoes), pumpkin, radish, rapeseed, raspberry, rice, rye, sorghum, soybean, spinach, strawberry, sugar beet, sugarcane, sunflower, tobacco, tomato, turnip, wheat, zucchini, and other fruiting vegetables (e.g. tomatoes, pepper, chili, eggplant, cucumber, squash etc.), other bulb vegetables (e.g., garlic, onion, leek etc.), other pome fruit (e.g. apples, pears etc.), other stone fruit (e.g., peach, nectarine, apricot, pears, plums etc.), Arabidopsis, woody plants such as coniferous and deciduous trees, an ornamental plant, a perennial grass, a forage crop, flowers, other vegetables, other fruits, other agricultural crops, herbs, grass, or perennial plant parts (e.g., bulbs; tubers; roots; crowns; stems; stolons; tillers; shoots; cuttings, including un-rooted cuttings, rooted cuttings, and callus cuttings or callus -generated plantlets; apical meristems etc.). The term "plants" refers to all physical parts of a plant, including seeds, seedlings, saplings, roots, tubers, stems, stalks, foliage and fruits.

[00087] In other embodiments, the host cells are algal and/or photosynthetic, including but not limited to algae or photosynthetic cells of the genera Agmenellum, Amphora, Anabaena, Ankistrodesmus, Botryococcus, Boekelovia , Borodinella, Botryococcus, Carteria, Chaetoceros, Chlamydomonas, Chlorella, Chlorococcum, Chlorogonium, Chrysosphaera, Cricosphaera , Cryptomonas, Cyclotella , Dunaliella, Ellipsoidon, Eremosphaera, Euglena, Fragilaria, Gleocapsa, Gloeothamnion, Hymenomonas, Isochrysis, Lepocinclis, Monoraphidium, Nannochloris,

Nannochloropsis, Navicula, Nephrochloris, Nitschia, Nitzschia, Ochromonas, Oocystis, Oscillatoria, Nitzschia, Pascheria, Phagus, Phormidium, Platymonas, Pleurochrysis Prototheca, Pyrobotrys Scenedesmus, Spirogyra, Tetraedron,

Tetraselmis, or Volvox. In some embodiments, the host cell is Botryococcus braunii, Prototheca krugani, Prototheca moriformis, Prototheca portoricensis, Prototheca stagnora, Prototheca wickerhamii, or Prototheca zopfli.

[00088] In some embodiments, the eukaryotic cells are fungi cells, including, but not limited to, fungi of the genera Aspergillus, Candida, Chlamydomonas, Chrysosporium, Cryotococcus, Debaromyces, Fusarium, Hansenula, Kluyveromyces, Neotyphodium, Neurospora, Penicillium, Pichia, Saccharomyces,

Schizosaccharomyce, Trichoderma, Xanthophyllomyces, Yarrowia, and

Zygosaccharomyces. Exemplary fungi cells include Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Saccharomyces lactis, Schizosaccharomyces pompe, Kluyveromyces lactis, Pichia pastoris, Hansenula polymorpha, or filamentous fungi, e.g. Trichoderma, Aspergillus sp., including Aspergillus niger, Aspergillus phoenicis, Aspergillus carbonarius.

[00089] Exemplary insect cells include any species of Spodoptera or

Drosophila, including Drosophila S2 and Spodoptera Sf9. Exemplary animal cells include CHO, COS or Bowes melanoma or any appropriate mouse or human cell line known to person of skill in the art.

Introduction of Polynucleotides to Host Cells [00090] In some embodiments, the nucleic acids encoding the biosensors of the present invention is/are inserted into a construct(s) encoding a metagenomic library, and the metagenomic library with the biosensor is introduced into a plurality of cells. In some embodiments, the nucleic acids of the invention encode a biosensor operably linked to a control region from the metagenomic library. In some embodiments, the nucleic acids of the invention encode the gene from which the control region is derived. In some embodiments, the nucleic acid(s) of the invention is/are introduced to the eukaryotic cell by transfection (e.g., Gorman, et al. Proc. Natl. Acad. Sci. 79.22 (1982): 6777-6781, which is incorporated by reference in its entirety for all purposes), transduction (e.g., Cepko and Pear (2001) Current Protocols in Molecular Biology unit 9.9; DOI: 10.1002/0471142727.mb0909s36, which is incorporated by reference in its entirety for all purposes), calcium phosphate transformation (e.g., Kingston, Chen and Okayama (2001) Current Protocols in Molecular Biology Appendix 1C; DOI: 10.1002/0471142301.nsa01cs01, which is incorporated by reference in its entiret ' for all purposes), cell-penetrating peptides (e.g., Copolovici, Langel, Eriste, and Langel (2014) ACS Nano 2014 8 (3), 1972-1994; DOI: 10.1021/nn4057269, which is incorporated by reference in its entirety for all purposes), electroporation (e.g Potter (2001) Current Protocols in Molecular Biology unit 10.15; DOI:

10.1002/0471142735.iml015s03 and Kim et al (2014) Genome 1012-19.

doi: 10.1101/gr.171322.113, Kim et al. 2014 describe the Amaza Nucleofector, an optimized electroporation system, both of these references are incorporated by reference in their entirety for all purposes), microinjection^. g., McNeil (2001) Current Protocols in Cell Biology unit 20.1 ; DOI: 10.1002/0471143030.cb2001sl 8, which is incorporated by reference in its entirety for all purposes), liposome or cell fusion (e.g.,Hawley-Nelson and Ciccarone (2001) Current Protocols in Neuroscience Appendix IF; DOI: 10.1002/04711423 Ol .nsaOlfs 10, which is incorporated by reference in its entirety for all purposes), mechanical manipulation (e.g. Sharon et al. (2013) PNAS 2013 110(6); DOI: 10.1073/pnas. l218705110_r which is incorporated by reference in its entirety for all purposes) or other well-known techniques for delivery of nucleic acids to host cells. Once introduced, the nucleic acids of the invention can be expressed episomally, or can be integrated into the genome of the host cell using well known techniques such as recombination (e.g., Lisby and Rothstein (2015) Cold Spring Harb Perspect Biol. Mar 2;7(3). pii: a016535. doi: 10.1101/cshperspect.a016535, which is incorporated by reference in its entirety for all purposes), non-homologous integration (e.g., Deyle and Russell (2009) Curr Opin Mol Ther. 2009 Aug; 11 (4): 442-7, which is incorporated by reference in its entirety for all purposes) or transposition (as described above for mobile genetic elements). The efficiency of homologous and non-homologous recombination can be facilitated by genome editing technologies that introduce targeted double-stranded breaks (DSB). Examples of DSB-generating technologies are CRISPR/Cas9, TALEN, Zinc- Finger Nuclease, or equivalent systems (e.g., Cong et al . Science 339.6121 (2013): 819-823, Li et al. Nud. Acids Res (2011): gkrl 88, Gajet al. Trends in Biotechnology 31.7 (2013): 397-405, all of which are incorporated by reference in their entirety for all purposes), transposons such as Sleeping Beauty (e.g., Singh et al (2014) Immunol Rev. 2014 Jan;257(l): 181-90. doi: 10.1111/imr. l2137, which is incorporated by- reference in its entirety for all purposes), targeted recombination using, for example, FLP recombinase (e.g., O'Gorman, Fox and Wahl Science (1991)

15:251(4999): 1351-1355, which is incorporated by reference in its entirety for all purposes), CRE-LOX (e.g., Sauer and Henderson PNAS (1988): 85; 5166-5170), or equivalent systems, or other techniques known in the art for integrating the nucleic acids of the invention into the eukaryotic cell genome.

[00091] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes,

nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle). Other methods of state-of-the-art targeted delivery of nucleic acids are available, such as delivery of polynucleotides with targeted nanoparticles or other suitable sub-micron sized delivery system.

[00092] Techniques for transforming a wide variety of higher plant species are well known and described in the technical and scientific literature. See, e.g., Weising (1988) Ann. Rev. Genet. 22:421-477; U.S. Pat. No. 5,750,870, which are both incorporated by reference in their entirety for all purposes.

Methods for Making Metagenomic Clones with Biosensors

[00093] In some embodiments, the biosensors of the invention are comprised of a nucleic acid encoding a mobile element, a nucleic acid(s) encoding a ribosome binding site(s), a nucleic acid encoding a reporter polypeptide, and a nucleic acid encoding a selection marker. In some embodiments, the nucleic acid encoding the reporter polypeptide and the nucleic acid encoding the selection marker are in an operon arrangement so that when a control region is operably linked to the biosensor both reporter and selection marker are expressed from the control region. In some embodiments, the nucleic acid encoding the mobile genetic element represent the inverted terminal repeat sequences that allow the nucleic acids in between the ITRs (the reporter and selection marker) to be moved within the host cell. In some embodiments, the mobile genetic element is other sequences that allow nucleic acids (the reporter and selection marker) to be moved within the host cell. In some embodiments, the nucleic acids encoding the mobile genetic element also include nucleic acids encoding a transposase. In some embodiments, the transposase is encoded by nucleic acids that are not part of the biosensor construct.

[00094] In some embodiments, a nucleic acid encoding the mobile element, ribosome binding site(s), reporter polypeptide, and selection marker is located on a vector or a construct. In some embodiments, this vector or construct is transformed into a plurality of host cells that contain a metagenomic library. In this embodiment, a transposase is supplied either by the construct or vector with the biosensor construct or by the host cell. In this embodiment, the biosensor construct is transposed from the construct or vector into the nucleic acids of the host cell, including the metagenomic library. In some embodiments, metagenomic clones (e.g., in fosmids) are mixed in vitro with biosensor constructs and an appropriate transposase (or other recombination enzyme) and biosensor constructs are inserted in the metagenomic clones in vitro. In some embodiments, the ratio of biosensor constructs and metagenomic clones is such that one biosensor integration will be obtained per metagenomic construct (e.g., per fosmid). In some embodiments, the ratio of biosensor constructs and metagenomic clones is such that 2, 3, 4, 5, 6, 7, 8, 9, 10 or more biosensor integrations will be obtained per metagenomic construct (e.g., per fosmid).

[00095] In some embodiments, functional biosensors are identified by expression of the selection marker. In some embodiments, the selection marker is an antibiotic resistance and functional biosensors are found by selecting for host cells that are resistant to the antibiotic. In some embodiments, functional biosensors are found by screening for the selection marker and/or the reporter. In some embodiments, the selected or screened clones have one functional biosensor integration per metagenomic construct (or per host cell). In some embodiments, the selected or screened clones have 2, 3, 4, 5, 6, 7, 8, 9, 10 or more functional biosensor integrations per metagenomic construct. In some embodiments, a metagenomic library is obtained by selection or screening for functional biosensors in each metagenomic construct.

[00096] In some embodiments, functional biosensors are identified by constitutive expression from a control region. In some embodiments, this constitutive expression provides a low level of the selection marker in the host cell. In some embodiments, this low level of the selection marker can be used to select for host cells that grow in the presence of a low concentration of a selection agent (e.g., an antibiotic).

Methods for Using Biosensors

[00097] In some embodiments, the biosensors of the library are used to identify clones that express the biosensor in response to a desired stimulus (small molecule or physical factor of interest). These clones can be used to identify genes that can be used in biosynthetic pathways, catabolic pathways, energy metabolism, regulatory functions, or the like in a host cell. These clones can also be used to find control regions and associated transcription factors that respond directly or indirectly to the stimulus. In some embodiments the biosensor clone itself is used to as sensor for the stimulus. In some embodiments, the biosensors of the library are used to identify genes whose expression is increased in the presence of certain molecules or stimuli. In some embodiments, the selection marker is used to identify constitutive (or basal) expression from the control region, and the reporter is used to identify control regions that increase expression in response to a desired stimulus. In some embodiments, the stimulus is a small molecule, a substrate, a metabolic intermediate, a macromolecule, a polypeptide, a carbohydrate, a lipid, a nucleic acid, and the like. In some embodiments, the stimulus is a physical parameter such as, for example, pH, temperature, light, pressure, and the like.

[00098] In some embodiments, functional biosensors are selected by placing the biosensor constructs into an appropriate host cell and plating the cells on a low concentration of selective agent (e.g., 20 μg/ml spectinomycin for Spectinomycin resistance). [00099] In some embodiments, the stimulus is the small molecule including for example, a terpene, an amino acid derivative, a hydrocarbon (e.g., alkanes, cycloalkanes, alkenes, cycloalkenes, alkynes, aromatic hydrocarbons), alcohols, ethers, aldehydes, ketones, carboxylic acids, esters, chlorides, amides, nitriles, lipids, carbohydrates, proteins, nucleic acids, secondary metabolites, alkaloids, non-protein amino acids, amines, cyanogenic glycosides, alkamides, lectins, peptides, steroids, saponins, flavonoids, tannins, phenylpropanoids, lignins, coumarics, polyacetylenes, fatty acids, waxes, organic acids, metals, heteroatoms, pyridines and pyrimindines. In some embodiments, the stimulus is a terpene, a secondary metabolite, an aromatic compound, a fatty acid, or an alcohol. In some embodiments, a metagenomic library with functional biosensors is screened for reporter activity upon exposure of clones of the library to a stimulus. In some embodiments, the clones of the library represented by individual clones in individual wells or unique locations on a substrate. The clones can be assayed in any way known in the art, including, for example, a multiple well plate format (e.g., 96 well plates, 384 well plates, 1536 well plates, etc.), fluorescent activated cell sorting (FACS) format, a microfluidics format, or a

droplet/microfluidics format (e.g., Mazutis et al, Nat. Protoc. 8: 870-891 (2013), which is incorporated by reference in its entirety for all purposes).

[000100] In some embodiments, the copy number of the biosensor is changed (e.g., increased) to change the response curve of the biosensor to the stimulus. For example, increasing the copy number of the biosensor increases the number of biosensors in the cell. In some embodiments, the cells with the biosensor are grown with high amounts of spectinomycin (e.g., 200-500 μg/ml) with the stimulus. The higher spectinomycin reduces the background level of reporter expression in the control samples and allows detection of reporter activity at lower amounts of stimulus. The higher spectinomycin and copy number can be used individually or together to change the response of the biosensor to a stimulus. In some embodiments, the one or both are used to increase the sensitivity of detection with low amounts of a stimulus. In some embodiments, one or both are used to increase the dynamic range of stimulus detection by the biosensor. In some embodiments, the some clones that show changed sensitivity in response to a copy number change and/or growth in the presence of high spectinomycin are isolated and referred to as selection modulated clones. Selection modulated biosensors are biosensors that modulate their reporter response curves in response to selective pressure (e.g., Spectinomycin) and/or copy number of the biosensor.

[000101] In some embodiments, the clones with functional biosensors are screened for reporter activity in cell free protein synthesis format. Such cell free systems include, for example, those described in Chong, Curr. Protoc. Mol. Biol.

108: 16.30.1 - 16.30.11 (2015); Swartz, Nat. Biotechnol. 27:731-732 (2009); Zemella et al, ChemBioChem 16:2420-2431 (2015), all of which are incorporated by reference in their entirety for all purposes. Such cell free systems can be used when the stimulus screened against the library does not effectively get into the host cells harboring the functional biosensors. For example, the stimulus could be a small molecule that does not cross the cell membrane and for which there is no transporter. In some embodiments, the host cells with the biosensors may be modified by addition of a recombinant transporter capable of transporting a desired small molecule stimulus into the host cell. The recombinant transporter can be a broad specificity transporter capable of transporting a number of different ligands. Transporters that could be used in the invention include, for example, AcrB, AcrAB, TolC, AcrAB- TolC, MexB, MexAB-OprM, MexXY, MexXY-OprM, OpcPl/OpcP2, AmrAB- OprA, BpeAB-OprB, BpeEF-OprC, OmpF/OmpC, OmpA-AB, AdeABC, AdelJK, MtrCDE, SmeZ, SmeJK, OqxAB, norA, or mepA.

[000102] In some embodiments, the clones of the library are multiplexed and each well or location on a substrate contains multiple clones from the metagenomic library with functional biosensors at different locations in the metagenomic constructs. In some embodiments the multiplexed library as 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 1,000 or more clones per multiplexed well or location on a substrate. In the multiplexed embodiment, groups of clones that have one or more clones that increase expression in response to a stimulus are selected for deconvolution to identify the clone which responds to the stimulus. In some embodiments, the pool of clones is grown and then separated into individual clones to identify the clone of interest. In some embodiments, groups of clones are plated onto media with the selection agent and the stimulating agent such that growth of host cells requires elevated expression levels of the selection marker by the biosensor. Clones from this selection are grown individually and tested for reporter expression in response to the stimulus to confirm that the clone increases expression in response to the stimulus. In some embodiments, clones in a well or location have different reporters so that expression of a particular reporter identifies the clone, narrows the identification of the clone to a subset of the clones in the well or location, or allows for selection of the clone.

[000103] The inventions disclosed herein will be better understood from the experimental details which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the inventions as described more fully in the claims which follow thereafter. Unless otherwise indicated, the disclosure is not limited to specific procedures, materials, or the like, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

EXAMPLES

Example 1. Functional Biosensors

[000104] A metagenomic library made from environmental samples obtained from hots springs, permafrost, sea water, soil, and hydrocarbon resource samples (from soil) was made using the Meta-G-Nome™ DNA Isolation Kit and

CopyControl™ Fosmid Library Production Kit obtained from Epicentre. 300-400 fosmid clones from this library were obtained and mixed with a Tn5 transposase and sufficient biosensor construct (FIG. 3) was added to give about 10 biosensor inserts per fosmid. The transposase randomly integrates the biosensor construct into the fosmid vector, and the modified fosmid library with biosensor constructs was transformed into E. coli strain EPI300™ (commercially sold by Epicentre). The E. coli EPI300™ transformed with the modified fosmid library was plated on LB media with 20 μg/ml spectinomycin and 0.01% arabinose. This selects for fosmid clones that have a basal or constitutive level of biosensor transcription sufficient to express enough Spec^r so the host cells could grow on media with 20 μg/ml spectinomycin.

[000105] About 20,000 fosmid clones with biosensors were found to have sufficient basal transcription in E. coli EPI300™ to provide spectinomycin resistance. The individual clones were picked and placed into 96 well plates with 20 μg/ml spectinomycin. Some of these clones were checked for basal expression of the Gemini reporter and no detectable fluorescence was found for the tested clones. [000106] The functional biosensors were interrogated with a terpene and clones that produced reporter fluorescence above control were selected. The results from this test are shown in FIG. 2 that depicts the mean difference between a clone without terpene and the same clone with terpene. FIG. 2 shows that about 500 of the tested clones showed increased fluorescence in response to terpene, indicating that these clones have biosensors integrated at sites showing upregulation in response to this stimulus.

[000107] In an alternative embodiment, the functional biosensors were interrogated with a low concentration of terpene (e.g., 50 μΜ to 400 μΜ) and grown in media with a high concentration of Spectinomycin (e.g., 200 - 500 μg/ml) to screen for biosensor clones that can respond to the lower terpene concertation. In a further alternative embodiment, IPTG, high Spectinomycin, and low terpene (e.g., 50 μΜ to 400 μΜ terpene) was added to the media to express the β-galactosidase omega fragment from the host cell and the Gemini reporter (with the β-galactosidase alpha fragment) from induced biosensors. The omega fragment complements the alpha fragment of the Gemini reporter to produce β-galactosidase activity as a measure of induction of a biosensor from the terpene. The β-galactosidase activity is measured as described in Martin et al, PLoS ONE 4;e7569 (2009), which is incorporated by reference in its entirety for all purposes. In another alternative embodiment, IPTG along with terpene (e.g., 500 μΜ to 1.5 mM terpene) was added to the media to express the β-galactosidase omega fragment from the host cell and the Gemini reporter (with the β-galactosidase alpha fragment) from induced biosensors.

[000108] In some embodiments, the control region and/or coding region are sequenced for the Spec^r clones to identify the gene which responds to a terpene. The sequences obtained are analyzed using sequence comparison tools to identify potential functions of the identified genes. Novel genes identified can be used to investigate novel cellular signaling pathways, and to investigate novel biochemical pathways. Example 2. Identification of Structure with a Library of Biosensors

[000109] The biosensor library made in Example 1 is used in both multiplex or single clone approaches. Clones from the biosensor library are placed into multiple 96 well plates and grown on media with 20 μg/ml of spectinomycin to select for constitutive expression of the biosensor. [000110] The panel of clones or multiplexed clones is interrogated with a panel of compounds and/or conditions. Each compound or condition will produce a pattem of reporter positive clones that serve as a functional ID for each compound or condition. Compounds or conditions that stimulate expression from the same clones (have the same or similar pattems of reporter expression) can be functionally grouped together.

[000111] An unknown compound or compounds can be interrogated against the biosensor library and the pattern of reporter expression for the unknown compared to the pattems obtained from known compounds. This comparison can identify the structure of the unknown or share structural features that the unknown has with a panel of known compounds. This novel method of detecting a chemical of interest can allow for detection of chemicals are similar to known chemical compounds.

[000112] Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Numeric ranges are inclusive of the numbers defining the range. The word "comprising" is used herein as an open-ended term, substantially equivalent to the phrase "including, but not limited to", and the word "comprises" has a corresponding meaning. Citation of references herein is not an admission that such references are prior art to an embodiment of the present invention. Any priority document(s) and all publications, including but not limited to patents and patent applications, cited in this specification are incorporated herein by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein and as though fully set forth herein. The invention includes all embodiments and variations substantially as hereinbefore described and with reference to the examples and drawings.

Claims

CLAIMS We claim:

1. A polynucleotide comprising a nucleic acid encoding a reporter, a nucleic acid encoding a selection marker, a nucleic acid encoding a first ribosome binding site, a nucleic acid encoding a second ribosome binding site, and a mobile genetic element, wherein the nucleic acid encoding the first ribosome binding site is operably linked to the nucleic acid encoding the reporter, wherein the nucleic acid encoding second ribosome binding site is operably linked to the nucleic acid encoding the selection marker, and wherein the nucleic acid encoding the reporter and the nucleic acid encoding the selection marker can be expressed from the same promoter whereby both the selection marker and the reporter are expressed.

2. The polynucleotide of claim 1, wherein the selection marker is selected from the group consisting of an antibiotic resistance gene, a polypeptide that complements an auxotrophic deficiency, and a polypeptide that makes a critical nutrient.

3. The polynucleotide of claim 2, wherein the antibiotic resistance is a spectinomycin resistance.

4. The polynucleotide of claim 1 , wherein the reporter is selected from the group consisting of an optical reporter, a fluorescent reporter, a bioluminescent reporter, a fusion protein reporter, an enzyme, and combinations of the foregoing.

5. The polynucleotide of claim 4, wherein the reporter is a Gemini reporter.

6. The polynucleotide of claim 1 , wherein the mobile genetic element is derived from a mobile genetic element selected from the group consisting of a virus, a transposon, an insertion sequence, a short interspersed element, a long interspersed element, a long terminal repeat from a transposon, and an inverted terminal repeat.

7. The polynucleotide of claim 6, wherein the mobile genetic element is SEQ ID NOs: 1 and 2.

8. The polynucleotide of claim 1 , wherein the nucleic acids encoding the first and second ribosome binding sites are selected from the group consisting of a SEQ ID NO: 3, a SEQ ID NO: 4, and combination of the SEQ ID NO: 3and the SEQ ID NO:4.

9. A polynucleotide construct comprising an origin of replication, a nucleic acid encoding a selectable marker, a nucleic acid obtained from a metagenomic source, and a nucleic acid encoding a biosensor, wherein the nucleic acid encoding the biosensor is operably linked to a control region in the nucleic acid obtained from the metagenomic source, and wherein the nucleic acid encoding the biosensor comprises a nucleic acid encoding a reporter and a nucleic acid encoding a selection marker, and a first and a second ribosome binding site, and a mobile genetic element, wherein the first ribosome binding site is operably linked to the nucleic acid encoding the reporter, and the second ribosome binding site is operably linked to the nucleic acid encoding the selection marker, and wherein the nucleic acid encoding the reporter and the nucleic acid encoding the selection marker are organized in an operon whereby both the selection marker and the reporter are expressed from the control region.

10. The polynucleotide of claim 9, wherein the antibiotic resistance is a spectinomycin resistance.

11. The polynucleotide of claim 10, wherein the reporter is a Gemini reporter.

12. A library of polynucleotides comprising, a plurality of constructs of claim 9.

13. The polynucleotides of claim 12, wherein the antibiotic resistance is a spectinomycin resistance.

14. The polynucleotides of claim 12, wherein the reporter is a Gemini reporter.

15. A host cell comprising the polynucleotide of claim 9.

16. The host cell of claim 15, wherein the host cell is an Escherichia coli.

17. A plurality of host cells comprising the plurality of polynucleotides of claim 12, wherein each member of the library is contained in a different host cell.

18. The plurality of host cells of claim 17, wherein the bacteria are a plurality of

Escherichia coli.

19. A method for finding a control region responsive to a stimulus, comprising the steps of: obtaining a plurality of host cells of claim 17, interrogating the host cells with a stimulus, identifying host cells that increase expression of the reporter or the selection marker or both in response to the stimulus.

20. The method of claim 19, further comprising the step of isolating the host cells that increase expression of the reporter in response to a stimulus.

21. The method of claim 19, wherein the host cell comprises an inducible polynucleotide encoding a polypeptide that can change the copy number of the polynucleotide of claim 17, and further comprising the step of changing the copy number of the polynucleotide of claim 21 in the host cell.

22. The method of claim 20, further comprising the step of sequencing the control region.

23. The method of claim 20, wherein the stimulus is a small molecule.

24. The method of claim 23, wherein the small molecule is selected from the group consisting of a terpene, a secondary metabolite, an aromatic compound, a fatty acid, and an alcohol.