WO2017040829A1 - Riboregulators regulated by external stimuli and methods of use thereof - Google Patents

Abstract

Provided herein are compositions and methods for inducing expression of a protein based on one or more external stimuli such as environmental stimuli.

Description

RIBOREGULATORS REGULATED BY EXTERNAL STIMULI

AND METHODS OF USE THEREOF

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Serial No.

62/213,217 filed on September 2, 2015 entitled "Riboregulators Regulated by External Stimuli and Methods of Use Thereof, the entire contents of which are incorporated by reference herein. BACKGROUND OF INVENTION

Riboregulators are sequences of RNA that effect changes in cells in response to a nucleic acid sequence. These RNA-based devices, which typically regulate protein translation or trigger mRNA degradation, have been used for a number of applications in synthetic biology, including sensitive control over gene expression, shunting of metabolic flux through different metabolic pathways, and synthetic control over cell death.

In riboregulators that control protein expression, repression of protein translation has relied on sequestration of the normally single-stranded ribosome binding site (RBS) within a duplex RNA region that is upstream of a gene of interest (GOI). An RNA in which the RBS is sequestered within a hairpin upstream of the GOI is thus a cis-repressed RNA (crRNA). A riboregulator based on an engineered crRNA can be constructed in which a trans-activating RNA (taRNA) binds to the crRNA and unwinds the repressing RNA duplex thereby exposing a now single- stranded RBS and activating translation of the downstream gene. In

riboregulators that decrease expression of the GOI, the RBS and initiation codon of the GOI are both exposed in the absence of the trigger RNA. However, a trans-repressing RNA (trRNA), which bears anti-sense sequence to the RBS and start codon or other sequence such as the trigger RNA itself, can bind to the riboregulator and strongly suppress translation of the downstream gene.

SUMMARY OF INVENTION

The invention provides toehold riboregulator systems that are induced by the presence of one or more external stimuli such as light, temperature, water or moisture, pH, and small molecules, among other things. These systems can be used to produce a particular protein of interest as a readout for the presence of the external stimulus (and thus act as a reporter

4950854_l.docx system of sorts), or they may take advantage of the external stimulus in order to induce the translation of the protein in instances in which it is known the external stimulus will exist or does exist and its presence is being exploited to introduce the protein into such an

environment.

Provided herein is a system comprising a host cell comprising an expression construct that encodes a switch RNA and one or more expression constructs that encode a trigger RNA, wherein a switch RNA comprises (i) a single-stranded toehold domain, (ii) a fully or partially double-stranded stem domain comprising an initiation codon, (iii) a loop domain comprising a ribosome binding site, and (iv) a coding domain, wherein the one or more trigger RNA(s) alone or combined hybridize to the single-stranded toehold domain of a switch RNA, and wherein expression of the switch RNA and the trigger RNA is induced in the presence of one or more external stimulus, or in the presence of one or more external stimuli and absence of one or more external stimuli, or in a combination of presence and absence of two or more external stimuli.

Also provided herein is a method of inducing translation of a protein by an external stimulus, comprising (1) providing a host cell comprising an expression construct that encodes a switch RNA and one or more expression constructs that encode a trigger RNA, wherein a switch RNA comprises (i) a single-stranded toehold domain, (ii) a fully or partially double-stranded stem domain comprising an initiation codon, (iii) a loop domain comprising a ribosome binding site, and (iv) a coding domain, wherein the one or more trigger RNA(s) alone or combined hybridize to the single-stranded toehold domain of a switch RNA, and wherein expression of the switch RNA and the trigger RNA is induced in the presence of one or more external stimulus, or in the presence of one or more external stimuli and absence of one or more external stimuli, or in a combination of presence and absence of two or more external stimuli, and (2) inducing translation of the coding domain by the presence of the external stimulus.

In certain embodiments, the coding domain encodes an enzyme or an enzyme cofactor. In certain embodiments, the coding domain encodes a reporter protein.

In certain embodiments, the external stimulus is light, heat, water or moisture, pH, or one or more small molecules.

In certain embodiments, the host cell is provided in a housing. In certain

embodiments, the host cell is provided in a housing and the coding domain encodes an enzyme that degrades the housing. In certain embodiments, the housing consists of paper, starch, cellulose and/or a hydrogel. In certain embodiments, the enzyme is a cellulose, an amylase, a proteases, a peptidase, a beta-glucosidase, an exoglucanase, or an endoglucanase.

In certain embodiments, the expression constructs comprise inducible promoters selected from the group consisting of cl-ts857, cspA, cadA, Lacl, TetR and AraC sensitive promoters, as well as any of the promoters provided in Table 1.

These and other aspects and embodiments of the invention will be described in greater detail herein.

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 depicts a schematic of the toehold switches for environmental sensors. Simple promoter swapping for switch and trigger RNAs allows for different environmental sensors. For example, cl-ts857 and cspA are temperature sensors, cadA is a pH sensor, and Lacl, TetR, and AraC are specific small molecule sensors.

FIG. 2 depicts simple heat sensors.

FIG. 3 depicts a heat sensor with toehold switches. In this example, there is a built-in redundant control using multiple AND gate triggers.

FIG. 4 shows a heat sensor with toehold switches with built-in redundant control using multiple AND gate triggers.

FIG. 5 depicts the reduction of false positives using toehold switches.

FIG. 6 shows complex evaluation of environmental inputs.

FIG. 7A and 7B provide flow cytometry data obtained using a temperature-controlled toehold switch system. FIG. 7A provides histograms of GFP fluorescence for a temperature- controlled toehold switch/trigger system at different temperatures compared to negative and positive controls. Negative controls are constructs lacking GFP and positive controls are constructs that constitutively express GFP. The temperature-controlled toehold switches and triggers were both controlled by the thermo-labile lambda repressor, and these show temperature responsiveness as the temperature is increased from 30 °C to 37°C. In FIG. 7A, the plots correspond (from top to bottom) to negative control 30°C, negative control 33°C, negative control 37°C, positive control 30°C, positive control 33°C, positive control 37°C, temperature-controlled toehold switch and trigger 30°C, temperature-controlled toehold switch and trigger 33°C, and temperature-controlled toehold switch and trigger 37°C. FIG. 7B provides a plot of GFP mode fluorescence levels from the same data set as FIG. 7A. The data evidence that a temperature-controlled toehold switch and trigger system can be used to temperature-control the production of a protein of interest, such as for example a reporter protein as in this example.

DETAILED DESCRIPTION OF INVENTION

The invention provides compositions controlled by environmental stimuli, and methods of using such compositions to detect or exploit such environmental stimuli. The compositions are referred to collectively as riboregulators. They are nucleic acid based genetic systems that comprise a two or more RNA molecules, at least one of which encodes a protein (and is referred to herein as a "switch") and at least one of which is a trigger that hybridizes to the switch thereby activating it and causing translation of the encoded protein.

The switches comprise a regulatory region and a coding region. The regulatory region typically comprises a hairpin structure that prevents translation of the coding sequence. The hairpin structure comprising a stem and loop structure, and also optionally a single-stranded "bubble" within the stem structure. A ribosome binding sequence (RBS) is typically located in the loop structure, and a start codon AUG is typically located in the bubble structure, as illustrated in FIG. 1. The coding sequence begins at the AUG although typically the majority of this sequence is downstream of the stem structure. The "coding region", as used herein, refers to the coding sequence downstream of the stem structure.

The switch typically also comprises a single stranded region referred to as a toehold domain or sequence (referred to herein as a "toehold"). The toehold may be present at the single-stranded loop or the single- stranded bubble, or at another location. In the exemplary switch illustrated in FIG. l, the toehold is located upstream of the stem structure and denoted "a". The toehold, as its name implies, is the entry point for system triggers to bind to the switch and cause the unwinding of the hairpin, thereby allowing binding of the ribosome to the RBS and subsequent translation of the coding sequence. The toehold sequence is therefore complementary to a sequence in the trigger (or a combination or complex of triggers, as discussed below). The trigger (or combination or complex of triggers) typically comprises additional sequence. At least part of this additional sequence may be

complementary to sequence in the switch, such as sequence that is adjacent to the toehold sequence. In the switch illustrated in FIG. 1, this additional sequence is labeled as "b". The trigger is labeled as "trigger RNA" and comprises sequences complementary to "a" and "b", such complementary sequences denoted "a*" and "b*". Switches comprising such toehold sequences are referred to herein as "toehold switches".

A toehold domain of at least 5 or 6 nts in length is preferable for initial binding of the trigger. The toehold domain can therefore be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleotides in length. Moreover, it was also found that the trigger need only unwind two-thirds of the stem in order to allow translation of the downstream protein. Based on these findings, the stem domain may be as small as 12 bps for adequate repression in the switch. The stem domain may however be longer than 12 bps, including 13, 14, 15, 16, 17, 18, 19, 20, or more base pairs in length. Furthermore, expanding the loop length to 12-nts and replacement of the RBS with a slightly stronger version with the canonical Shine- Dalgarno sequence does not decrease the degree of repression by the switch. Accordingly, the length of the loop domain may be 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides.

The switches may have additional features. In some instances, the top three bases of the hairpin stem may be A-U base pairs. In some instances, the bottom three base pairs of the stem may comprise two strong G-C base pairs and one A-U base pair. In some instances, the length of the toehold may range from about 12- to about 15-nts. This latter feature may in some instances strengthen the initial binding between a trigger RNA and its switch RNA. In some instances, the size of the hairpin loop may range from about 11- to about 15-nts to enhance translation of the output protein upon switch activation. In some instances, the loop size is 15-nts. In yet other instances, a cognate trigger may be used that unwinds the first 15 of the 18 bases in the switch stem. In some instances, one or more, including all, of these features may be used simultaneously.

Opening of the switch in order to activate translation of the coding sequence may require the presence of a single trigger, or if may require the presence of two or more triggers. In some instances, the switch is opened in the presence of any one of a number of triggers. In that case, the system may be referred to as having an "OR gate" intending that the opening (de-repression) of the switch requires the presence of any one of a number of triggers (e.g., trigger A or trigger B). In some instances, the switch is opened in the presence of two or more triggers. In that case, the system may be referred to as having an "AND gate" intending that the opening of the switch requires the presence of two triggers (e.g., trigger A and trigger B). The AND gate is illustrated in FIG. 4. In an AND gate, the two or more triggers combine with each other such as for example via hybridization to form a composite sequence that is able to open the switch. As shown in FIG. 4, trigger 1 and trigger 2 complex with each other to generate a structure having single stranded region "c*" adjacent to single stranded region "b*", and this "composite" sequence is complementary to the toehold sequence "c" and its adjacent sequence "b". As illustrated, it is only in the presence of both triggers 1 and 2 that the switch can be opened.

In still other instances, the switch is opened in the presence of one trigger but not in the presence of another trigger, wherein the latter trigger may be regarded as a deactivating trigger. In that case, the system may be referred to as having an "ANDNOT gate" intending that the opening of the switch requires the presence of one trigger and the absence of another trigger (e.g., trigger A and not (deactivating) trigger B). In these situations, one trigger alone activates the repressed switch. However, the second "deactivating" trigger may be designed to hybridize to the first trigger, thereby interfering with its ability to bind to and unwind the switch. Thus, activation of the switch requires that the first trigger be expressed but not the second trigger. In that case, activation occurs if the first stimulus is present and if the second stimulus is absent. It will be clear that the stimuli that induce expression of the first and second stimuli should be different.

The invention contemplates that the various components of a riboregulator system, namely the switch and the trigger(s), are provided as expression constructs and that the transcription from such constructs is regulated by external stimuli such as environmental stimuli. Such stimuli include but are not limited to temperature (e.g., heat or cold), pH, water or humidity, small molecules, and the like. A list of such stimuli is provided herein.

Transcriptional control of these expression constructs can occur through the use of a promoter system that is responsive to the external stimulus. Such a promoter system may, for example, be bound by a repressive element such as a repressor in one condition and then not bound by the repressive element under another condition. The condition may act on the repressive element, and may for example degrade or initiate the degradation of the repressive element, or it may cause the dissociation of the repressive element from the promoter, for example. An example of such a promoter is shown in FIG. 2, albeit in the simple form of a promoter and generic output gene. The Figure illustrates the use of a cl-ts857 promoter system in which the repressor binds to the promoter and represses transcription of the output gene under cold temperature conditions. Even under such repression, some leakage from the locus is still observed. When the temperature is increased, the repressor is degraded and the output gene is transcribed. Temperature cut-offs are shown in Table 1.

Thus the invention contemplates a system comprising an expression construct that comprises the switch DNA, and one or more expression constructs that each comprise a trigger DNA. Importantly, the transcription of the switch DNA to form the switch (which is RNA in nature) and transcription of each of the triggers (which also are RNA in nature) may be regulated by the same stimulus. This is illustrated in FIG. 3 which provides the expression constructs for the switch and the two triggers of an AND gate (trigger 1 and trigger 2).

Because this is an AND gate, both triggers must be present in order to activate (in this case, de-repress) the switch. By designing the system so that the expression of all elements (i.e., the switch and the triggers) are controlled by the same external stimulus, the system will be better suited to reduce false positives. This is represented in FIG. 5, where it is shown that using a heat- sensitive promoter system, the concentration of switch RNA and trigger RNA is low under cold temperature conditions, thereby resulting in low leakage from the system.

The Figure further illustrates that under increased temperature high output of the output gene is expected and that such output should be roughly proportional to the concentration of the switch. FIGs. 7A and 7B provide additional data demonstrating the efficacy of a

temperature-controlled system comprising a toehold switch and a trigger which are both under temperature control. As shown in these Figures, the reporter protein encoded by the toehold switch is produced in a temperature-dependent manner.

FIG. 6 further illustrates a system in which components of the system, including the trigger(s), are designed to each respond to a different stimulus. For example, the system may be an OR gate in which any one of a number of triggers must be present to de-repress the switch. As illustrated, each of the triggers may itself by regulated by a different stimulus. Thus, as an example, the system may comprise a switch DNA that is transcribed in the presence of water or moisture, a first trigger that is transcribed in the presence of heat, and a second trigger that is transcribed in the presence of a particular small molecule. Alternatively, these triggers and switch may be designed to be an AND gate, in which case the switch would be activated only if the external stimuli of water/moisture, heat and the small molecule were present.

The output genes in the switches of the invention may be any variety of proteins. Of particular interest are enzymes or enzyme co-factors. The invention contemplates that the systems can then be used to induce production of an enzyme only under certain external conditions. Thus, the resultant enzymatic activity is itself induced only under certain external conditions. Examples of enzymes include cellulose or other paper-digesting enzyme, amylase or other starch-digesting enzyme, beta-glucosidases, exoglucanases, endoglucanases, proteases or peptidases that digest particular amino acid sequences, and the like. As another example, the protein may be a reporter protein such as GFP. In this latter instance, the reporter protein is used as an indicator of a particular condition.

The various riboregulator components, including the switch and the triggers, can be introduced into various cells including for example bacterial cells and may be integrated into the genome of such cells. The switch and triggers may be comprised of RNA in whole or in part. They may comprise naturally occurring nucleotides and/or non-naturally occurring nucleotides.

Similarly, the expression constructs that encode the switches and triggers may be comprised of DNA in whole or in part. They may comprise naturally occurring nucleotides and/or non- naturally occurring nucleotides.

Provided herein is a system comprising a host cell having, integrated or encoded into its genome, one or more riboregulator systems, each riboregulator system comprising (1) a switch RNA comprising (i) a single-stranded toehold domain, (ii) a fully or partially double- stranded stem domain comprising an initiation codon (AUG), (iii) a loop domain comprising a ribosome binding site (RBS), and (iv) a coding domain, and (2) one or more trigger RNA. The host cell may comprise expression constructs encoding the switch RNA and the trigger RNA(s) and such expression constructs may all comprise inducible promoters that respond to the same or different external stimulus. As used herein, an external stimulus is a stimulus that is applied to or that comes in contact with the promoter of the expression constructs and/or the cell in which such constructs exist. The external stimuli is typically not a stimulus that arises sua sponte in the cell without external interference. Preferably, it is a stimulus that originates outside the cell, such as an environmental stimulus (e.g., light, heat, pH, water or moisture, etc.). The stimulus will be present or exist within the cell but its genesis will be external. Examples of promoters that are induced by external stimuli and known in the art and include those provided in Table 1.

The host cell may be a prokaryotic cell such as but not limited to a bacterial cell. In certain embodiments, the host cell is an E. coli bacterium.

The invention contemplates that the components within a single riboregulator system may each be induced by a different external stimulus. The invention also contemplates that the components within a single riboregulator system may each be induced by the same external stimulus.

The invention generally provides nucleic acids, constructs, plasmids, host cells and methods for regulation of protein expression using external stimuli.

Various embodiments of the nucleic acids of the invention may be referred to herein as non-naturally occurring, artificial, engineered or synthetic. This means that the nucleic acid is not found naturally or in naturally occurring, unmanipulated, sources. A non-naturally occurring, artificial, engineered or synthetic nucleic acid may be similar in sequence to a naturally occurring nucleic acid but may contain at least one artificially created insertion, deletion, inversion, or substitution relative to the sequence found in its naturally occurring counterpart. A cell that contains an engineered nucleic acid may be referred to as an engineered cell. Cells into which riboregulator expression constructs are introduced are considered engineered cells, and such cells are not naturally occurring.

Various embodiments of the invention involve nucleic acid sequences that are complementary to each other. In some instances, the sequences are preferably fully complementary (i.e., 100% complementary). In other instances, however the sequences are only partially complementary. Partially complementary sequences may be at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% complementary. Sequences that are only partially complementary, when hybridized to each other, will comprise double- stranded regions and single- stranded regions. The single-stranded regions may be single mismatches, loops (where for instances a series of consecutive nucleotides on one strand are

unhybridized), bulges (where for instances a series of consecutive nucleotides on both strands, opposite to each other, are unhybridized). It will be appreciated that

complementarity may be determined with respect to the entire length of the two sequences or with respect to portions of the sequences. Nucleic acids and/or other moieties of the invention may be isolated. As used herein,

"isolated" means separate from at least some of the components with which it is usually associated whether it be from a naturally occurring source or made synthetically.

Nucleic acids and/or other moieties of the invention may be purified. As used herein, purified means separated from the majority of other compounds or entities. A compound or moiety may be partially purified or substantially purified. Purity may be denoted by a weight by weight measure and may be determined using a variety of analytical techniques such as but not limited to mass spectrometry, HPLC, etc. Nucleic acids generally refer to polymers comprising nucleotides or nucleotide analogs joined together through backbone linkages such as but not limited to phosphodiester bonds. Nucleic acids include deoxyribonucleic acids (DNA) and ribonucleic acids (RNA) such as messenger RNA (mRNA), transfer RNA (tRNA), etc. Nucleic acids may be single- stranded, double-stranded, and also tripled- stranded.

A naturally occurring nucleotide consists of a nucleoside, i.e., a nitrogenous base linked to a pentose sugar, and one or more phosphate groups which is usually esterified at the hydroxyl group attached to C-5 of the pentose sugar (indicated as 5') of the nucleoside. Such compounds are called nucleoside 5'-phosphates or 5'-nucleotides. In DNA the pentose sugar is deoxyribose, whereas in RNA the pentose sugar is ribose. The nitrogenous base can be a purine such as adenine or guanine (found in DNA and RNA), or a pyrimidine such as cytosine (found in DNA and RNA), thymine (found in DNA) or uracil (found in RNA). Thus, the major nucleotides of DNA are deoxyadenosine 5'-triphosphate (dATP), deoxyguanosine 5'-triphosphate (dGTP), deoxycytidine 5'-triphosphate (dCTP), and deoxythymidine 5'- triphosphate (dTTP). The major nucleotides of RNA are adenosine 5'-triphosphate (ATP), guanosine 5'-triphosphate (GTP), cytidine 5'-triphosphate (CTP) and uridine 5'-triphosphate (UTP). In general, stable base pairing interactions occur between adenine and thymine (AT), adenine and uracil (AU), and guanine and cytosine (GC). Thus adenine and thymidine, adenine and uracil, and guanine and cytosine (and the corresponding nucleosides and nucleotides) are referred to as being complementary to each other.

In general, one end of a nucleic acid has a 5 '-hydroxyl group and the other end of the nucleic acid has a 3 '-hydroxyl group. As a result, the nucleic acid has polarity. The position or location of a sequence or moiety or domain in a nucleic acid may be denoted as being upstream or 5' of a particular marker, intending that it is between the marker and the 5' end of the nucleic acid. Similarly, the position or location of a sequence or moiety or domain in a nucleic acid may be denoted as being downstream or 3' of a particular marker, intending that it is between the marker and the 3' end of the nucleic acid.

Nucleic acids may comprise nucleotide analogs including non-naturally occurring nucleotide analogs. Such analogs include nucleoside analogs (e.g., 2-aminoadenosine, 2- thiothymidine, inosine, 3 -methyl adenosine, C5-propynylcytidine, C5-propynyluridine, C5- bromouridine, C5-fluorouridine, C5-iodouridine, C5-methylcytidine, 7-deazaadenosine, 7- deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, and 2-thiocytidine), chemically modified bases, biologically modified bases (e.g., methylated bases), intercalated bases, modified sugars (e.g., 2'-fluororibose, ribose, 2 '-deoxyribose, arabinose, and hexose), or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite linkages).

The nucleic acids of the invention, including the switch RNA and trigger RNA, or the DNA-based expression vectors that encode these RNAs, may be provided or present in a larger nucleic acid. The larger nucleic acid may comprise a nucleotide sequence that is transcribed to produce the switch RNA and trigger RNA of the invention. For convenience, the invention may refer to the larger nucleic acid as comprising the switch RNA and/or trigger RNA although it is to be understood that in practice this intends that the larger nucleic acid comprises a sequence that encodes the switch RNA and/or trigger RNA. Such encoding sequences may be operably linked to other sequences in the larger nucleic acid such as but not limited to origins of replication. As used herein, "operably linked" refers to a relationship between two nucleic acid sequences wherein the production or expression of one of the nucleic acid sequences is controlled by, regulated by, modulated by, etc., the other nucleic acid sequence. For example, the transcription of a nucleic acid sequence is directed by an operably linked promoter sequence; post-transcriptional processing of a nucleic acid is directed by an operably linked processing sequence; the translation of a nucleic acid sequence is directed by an operably linked translational regulatory sequence; the transport or localization of a nucleic acid or polypeptide is directed by an operably linked transport or localization sequence; and the post-translational processing of a polypeptide is directed by an operably linked processing sequence. Preferably a nucleic acid sequence that is operably linked to a second nucleic acid sequence is covalently linked, either directly or indirectly, to such a sequence, although any effective association is acceptable.

As used herein, a regulatory sequence or element intends a region of nucleic acid sequence that directs, enhances, or inhibits the expression (e.g., transcription, translation, processing, etc.) of sequence(s) with which it is operatively linked. The term includes promoters, enhancers and other transcriptional and/or translational control elements. The switch RNA and trigger RNA moieties of the invention may be considered to be regulatory sequences or elements to the extent they control translation of a gene of interest that is operably linked to the switch RNA. The invention contemplates that the switch RNA and trigger RNA of the invention may direct constitutive or inducible protein expression.

Inducible protein expression may be controlled in a temporal or developmental manner.

As described in detail herein, the translation of the operably linked protein is induced by the presence of one or more external stimuli (or alternatively by the presence of one or more external stimuli and the absence of one or more stimuli, as the case may be). This inducible translation is accomplished by controlling the transcription of the switch or trigger(s) or deactivator moieties in a manner that is dependent on the presence or absence of one or more external stimuli. Thus, it may be the case that one element in the system is constitutively expressed, but more typically all the elements in the system will be inducibly expressed as a function of the presence or absence or one or more external stimuli, including some combination of external stimuli. The term vector or construct, as the terms are used interchangeably, refers to a nucleic acid capable of mediating entry of, e.g., transferring, transporting, etc., a second nucleic acid molecule into a cell. The transferred nucleic acid is generally linked to, e.g., inserted into, the vector nucleic acid. A vector may include sequences that direct autonomous replication, or may include sequences sufficient to allow integration into host cell DNA. Useful vectors include, for example, plasmids (typically DNA molecules although RNA plasmids are also known), cosmids, and viral vectors.

Reporter proteins may be used to visualize activation of the switch RNA, and thus may also be used as a readout of the presence of one or more external stimuli and/or absence of one or more stimuli and/or a combination of the presence and absence of one or more stimuli.

Reporter proteins suitable for this purpose include but are not limited to fluorescent or chemiluminescent reporters (e.g., GFP variants, luciferase, e.g., luciferase derived from the firefly (Photinus pyralis) or the sea pansy (Renilla reniformis) and mutants thereof), enzymatic reporters (e.g., β-galactosidase, alkaline phosphatase, DHFR, CAT), etc. The eGFPs are a class of proteins that has various substitutions (e.g., Thr, Ala, Gly) of the serine at position 65 (Ser65). The blue fluorescent proteins (BFP) have a mutation at position 66 (Tyr to His mutation) which alters emission and excitation properties. This Y66H mutation in BFP causes the spectra to be blue-shifted compared to the wtGFP. Cyan fluorescent proteins (CFP) have a Y66W mutation with excitation and emission spectra wavelengths between those of BFP and eGFP. Sapphire is a mutant with the suppressed excitation peak at 495 nM but still retaining an excitation peak at 395 and the emission peak at 511 nM. Yellow FP (YFP) mutants have an aromatic amino acid (e.g. Phe, Tyr, etc.) at position 203 and have red-shifted emission and excitation spectra.

It is to be understood that although various embodiments of the invention are described in the context of RNA, the nucleic acids of the invention can be RNA or DNA. In general, RNA and DNA can be produced using in vitro systems, within cells, or by chemical synthesis using methods known in the art. It will be appreciated that insertion of switch RNA elements upstream of an open reading frame (ORF) can be accomplished by modifying a nucleic acid comprising the ORF. The invention provides DNA templates for transcription of a switch RNA or trigger RNA. The invention also provides expression constructs, including plasmids, comprising such DNA templates. In certain embodiments, the invention provides a construct comprising the template for transcription of a switch RNA or a trigger RNA operably linked to a promoter.

In certain embodiments, the invention provides a DNA construct comprising (i) a template for transcription of a switch RNA; and (ii) an inducible promoter located upstream of the template. In certain embodiments, a construct or plasmid of the invention includes a restriction site downstream of the 3' end of the portion of the construct that serves as a template for the switch RNA, to allow insertion of an ORF of choice. The construct may include part or all of a polylinker or multiple cloning site downstream of the portion that serves as a template for the switch RNA. The construct may also include an ORF downstream of the switch RNA.

In certain embodiments, the invention provides a DNA construct comprising (i) a template for transcription of a trigger RNA; and (ii) an inducible promoter located upstream of the template.

The invention further provides a DNA construct comprising: (i) a template for transcription of a switch RNA; (ii) an inducible promoter located upstream of the template for transcription of the switch RNA; (iii) a template for transcription of a trigger RNA; and (iv) an inducible promoter located upstream of the template for transcription of the trigger RNA. The inducible promoters may be the same or different.

The constructs may be or may be incorporated into plasmids, e.g., plasmids capable of replicating in bacteria. In certain embodiments, the plasmid is a high copy number plasmid (e.g., a pUC-based or pBR322-based plasmid), while in other embodiments, the plasmid is a low or medium copy number plasmid, as these terms are understood and known in the art. The plasmid may include any of a variety of origins of replication, which may provide different copy numbers. For example, any of the following may be used (copy numbers are listed in parenthesis): ColEl (50-70 (high)), pl5A (20-30 (medium)), pSClOl (10-12 (low)), pSOOl* (< 4 (lowest). It may be desirable to use plasmids with different copy numbers for transcription of the switch RNA and the trigger RNA in order to alter their relative amounts in a cell or system. In addition, in certain embodiments a tunable copy number plasmid is employed.

The invention further provides viruses and cells comprising the nucleic acids, constructs (such as DNA constructs), and plasmids described above. In various embodiments, the cell is a prokaryotic cell. In various embodiments, the cell is a eukaryotic cell (e.g., a fungal cell, mammalian cell, insect cell, plant cell, etc.). The nucleic acids or constructs may be integrated into a viral genome using recombinant nucleic acid technology, and infectious virus particles comprising the nucleic acid molecules and/or templates for their transcription can be produced. The nucleic acid molecules, DNA constructs, plasmids, or viruses may be introduced into cells using any of a variety of methods known in the art, e.g., electroporation, calcium-phosphate mediated transfection, viral infection, etc.

As discussed herein, the nucleic acid constructs can be integrated into the genome of a cell. Such cells may be present in vitro (e.g., in culture) or in vivo (e.g., in an organism). The cells may be eukaryotic or prokaryotic cells, including but not limited to mammalian cells and bacterial cells. An example of a bacterial cell is an E. coli bacterium. An example of a mammalian cell is a human cell or a mouse cell. The invention further provides transgenic plants and non-human transgenic animals comprising the nucleic acids, DNA constructs, and/or plasmids of the invention. Methods for generating such transgenic organisms are known in the art. The invention further provides a variety of kits. For example, the invention provides a kit comprising a plasmid, wherein a first plasmid comprises (i) a template for transcription of a switch RNA, and (ii) an inducible promoter located upstream of the template for transcription of the switch RNA element, and optionally a second plasmid that comprises (i) a template for transcription of a cognate (complementary) trigger RNA element, and (ii) an inducible promoter located upstream of the template for transcription of the trigger RNA element. The promoters may be the same or, preferably, different, intending for example that the promoters may be induced by the same external stimulus or a different external stimulus. One or more of the promoters may be inducible, and one but not all of the promoters may be constitutive. The plasmids may have the same or different copy numbers. The invention further provides a kit comprising a single plasmid that comprises a template for transcription of a switch RNA element and an inducible promoter located upstream of the template for transcription of the switch RNA element and further comprises a template for transcription of a cognate trigger RNA element and an inducible promoter located upstream of the template for transcription of the cognate trigger RNA element. In certain embodiments, the plasmids comprise one or more restriction sites upstream or downstream of the template for transcription of the switch RNA element. If downstream, the restriction sites may be used for insertion of an open reading frame of choice. The kits may further include one or more of the following components: (i) one or more inducers; (ii) host cells (e.g., prokaryotic or eukaryotic host cells); (iii) one or more buffers; (iv) one or more enzymes, e.g., a restriction enzyme; (v) nucleic acid isolation and/or purification reagents; (vi) a control plasmid lacking a switch RNA or trigger RNA sequence; (vii) a control plasmid containing a switch RNA or trigger RNA sequence or both; (viii) sequencing primers; (ix) instructions for use. The control plasmids may comprise a reporter sequence.

The riboregulators of the invention in some instances comprise a consensus prokaryotic RBS. However, in various embodiments of the invention any of a variety of alternative sequences may be used as the RBS. The sequences of a large number of bacterial ribosome binding sites have been determined, and the important features of these sequences are known. Preferred RBS sequences for high level translation contain a G-rich region at positions -6 to -11 with respect to the AUG and typically contain an A at position -3.

Exemplary RBS sequences for use in the present invention include, but are not limited to, AGAGGAGA (or subsequences of this sequence, e.g., subsequences at least 6 nucleotides in length, such as AGGAGG). Shorter sequences are also acceptable, e.g., AGGA, AGGGAG, GAGGAG, etc. Numerous synthetic ribosome binding sites have been created, and their translation initiation activity has been tested. In various embodiments any naturally occurring RBS may be used in the switch RNA constructs. The activity of any candidate sequence to function as an RBS may be tested using any suitable method. For example, expression may be measured as described in Example 1 of published PCT application WO 2004/046321, or as described in reference 53 of that published PCT application, e.g., by measuring the activity of a reporter protein encoded by an mRNA that contains the candidate RBS appropriately positioned upstream of the AUG. Preferably an RBS sequence for use in the invention supports translation at a level of at least 10% of the level at which the consensus RBS supports translation (e.g., as measured by the activity of a reporter protein). For example, if the candidate RBS is inserted into a control plasmid in place of the consensus RBS, the measured fluorescence will be at least 10% of that measured using the consensus RBS. In certain embodiments, an RBS that supports translation at a level of at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more relative to the level at which the consensus RBS supports translation is used. In certain embodiments of the invention an RBS that supports translation at higher levels than the consensus RBS is used. Further general teachings relating to riboregulators are found in published PCT applications WO 2004/046321, WO 2014/074648 and WO 2016/011089, the entire contents of each of which are incorporated by reference herein.

The invention contemplates use of the external stimuli sensitive riboregulator systems described herein in a variety of applications. For example, the riboregulator system may comprise a reporter protein, such that presence of one or more external stimuli, a combination of external stimuli, and/or a combination of the presence of one or more certain external stimuli and absence of one or more certain external stimuli can be detected via the production of the reporter protein. Presence of the reporter protein can be determined visually, for example if the reporter protein emits a signal such as a fluorescent signal. Presence of the reporter protein may also be determined using an existing assay with which the riboregulator system is associated.

In other embodiments, the riboregulator systems described herein may be used to trigger translation of the encoded protein so as to induce its activity at particular times, including remotely. In some embodiments, the riboregulator system is used to deploy a particular protein, including an enzyme, under certain environment conditions. The protein may then act on another moiety, including for example a packaging material or a housing material such as paper or a cellulose-based product, or starch, or a protein hydrogel, and the like. In this way, a packaging material or housing may be degraded upon exposure to a particular environmental condition. This has implications for clean technologies as well as other areas. Table 1. Promoter systems regulated by external stimuli

Type Environmental Exemplary Result and Source

Factor Conditions

BclXL repressor temperature-heat Heat-Induced Fibrillation www.ncbi.nlm.nih.gov/pmc/arti cles/PMC3706518/

HsfB l and repressor temperature-heat expression of Heat- www.ncbi.nlm.nih.gov/pmc/arti HsfB2b Inducible Hsfs cles/PMC3252156/

RheA repressor temperature-heat HSP18 heat shock www.ncbi.nlm.nih.gov/pubmed response in Streptomyces /l 0716740

albus

MAF2 repressor temperature-cold repress flowering www.ncbi.nlm.nih.gov/pmc/arti cles/PMC4425511/ trp repressor pH (low) gel retardation www.ncbi.nlm.nih.gov/pubmed

/3277190

LitR repressor temperature Biofilm Formation and www.ncbi.nlm.nih.gov/pmc/arti

Colony Morphology cles/PMC4136084/

NADPH transcripti pH Regulate the DNA pubs.acs.org/doi/abs/10.1021/bi on factor Binding Activity of 5008518

Neuronal PAS Domain

Protein 2 (NPAS2)

LexA repressor pH autodigestion without arizona.openrepository.com/ari

RecA zona/handle/10150/ 184488 lac promoter temperature three temperature- www.ncbi.nlm.nih.gov/pubmed sensitive mutant strains /225641

for RNA polymerase beta

or beta' subunits

lacUV5 promoter IPTG homepage.univie.ac.at/nikos.pi notsis/webPP/genetoprotein/clo _vector/promoters .html tac (hybrid) promoter IPTG homepage.univie.ac.at/nikos.pi notsis/webPP/genetoprotein/clo _vector/promoters .html trc (hybrid) promoter IPTG homepage.univie.ac.at/nikos.pi notsis/webPP/genetoprotein/clo _vector/promoters .html

P_syn (synthetic) promoter IPTG homepage.univie.ac.at/nikos.pi notsis/webPP/genetoprotein/clo _vector/promoters .html trp promoter tryptophan tryptophan starvation homepage.univie.ac.at/nikos.pi notsis/webPP/genetoprotein/clo _vector/promoters .html araBAD promoter L-arabinose homepage.univie.ac.at/nikos.pi notsis/webPP/genetoprotein/clo _vector/promoters .html ipp^a promoter IPTG, lactose homepage.univie.ac.at/nikos.pi notsis/webPP/genetoprotein/clo _vector/promoters .html lpp-lac (hybrid) promoter IPTG homepage.univie.ac.at/nikos.pi notsis/webPP/genetoprotein/clo _vector/promoters .html phoA promoter phosphate phosphate starvation homepage.univie.ac.at/nikos.pi notsis/webPP/genetoprotein/clo _vector/promoters .html recA promoter nalidixic acid homepage.univie.ac.at/nikos.pi notsis/webPP/genetoprotein/clo _vector/promoters .html proU promoter osmolarity homepage.univie.ac.at/nikos.pi notsis/webPP/genetoprotein/clo _vector/promoters .html cst-1 promoter glucose glucose starvation homepage.univie.ac.at/nikos.pi notsis/webPP/genetoprotein/clo _vector/promoters .html tetA promoter tetracyclin homepage.univie.ac.at/nikos.pi notsis/webPP/genetoprotein/clo _vector/promoters .html cadA promoter pH homepage.univie.ac.at/nikos.pi notsis/webPP/genetoprotein/clo _vector/promoters .html nar promoter anaerobic homepage.univie.ac.at/nikos.pi conditions notsis/webPP/genetoprotein/clo

_vector/promoters .html

PL promoter temperature-heat (shift to 42°C) homepage.univie.ac.at/nikos.pi notsis/webPP/genetoprotein/clo _vector/promoters .html cspA promoter temperature-cold (shift to below 20°C) homepage.univie.ac.at/nikos.pi notsis/webPP/genetoprotein/clo _vector/promoters .html

T7 promoter temperature homepage.univie.ac.at/nikos.pi notsis/webPP/genetoprotein/clo _vector/promoters .html

T7-lac operator promoter IPTG homepage.univie.ac.at/nikos.pi notsis/webPP/genetoprotein/clo _vector/promoters .html

T3-lac operator promoter IPTG homepage.univie.ac.at/nikos.pi notsis/webPP/genetoprotein/clo _vector/promoters .html

T5-lac operator promoter IPTG homepage.univie.ac.at/nikos.pi notsis/webPP/genetoprotein/clo _vector/promoters .html

T4 gene 32 promoter T4 infection homepage.univie.ac.at/nikos.pi notsis/webPP/genetoprotein/clo _vector/promoters .html nprM-lac promoter IPTG homepage.univie.ac.at/nikos.pi operator notsis/webPP/genetoprotein/clo

_vector/promoters .html

VHb promoter oxygen homepage.univie.ac.at/nikos.pi notsis/webPP/genetoprotein/clo _vector/promoters .html

Metallothionein promoter copper sulfate or wolfson.huji.ac.il/expression/ve cadmium ctor/Promoters.html#promoters chloride

EXAMPLES

Example 1. Temperature-controlled toehold switch and trigger. E. coli strain DH5a (endAl recAl gyrA96 thi-1 glnV44 reiki hsdR17(r_K- m_K ⁺) λ ) was used. Plasmids were constructed using PCR and Gibson assembly. The synthetic DNA strands purchased from Integrated DNA Technologies were amplified via PCR to form double-stranded DNAs. The resulting DNAs were then inserted into plasmid backbones using 30-bp homology domains via Gibson assembly. All plasmids were cloned in the E. coli

DH5a strain and validated through DNA sequencing. Backbones for the plasmids were taken from the commercial vectors pET15b, pCOLADuet, and pCDFDuet (EMD Millipore).

GFPmut3b-ASV was used as the reporter for the gate plasmids. This GFP is GFPmut3b with an ASV degradation tag. Key sequences of elements used in the plasmids are provided in Table 2.

Three plasmids were used for the experiment. The switch plasmid having a toehold switch under the control of lambda pR promoter followed by GFPmut3b-ASV reporter has a medium copy ColA origin with kanamycin resistance. The switch plasmid also encoded lac repressor under a constitutive promoter. The trigger plasmid having a toehold trigger under the control lambda pR promoter has a high copy ColEl origin with ampicillin resistance. The lambda repressor plasmid having thermo-labile lambda repressor cI2 under the control of lac promoter has a high copy CDF origin with spectinomycin resistance.

Temperature sensor circuits were tested using cells transformed with switch plasmid, trigger plasmid, and lambda repressor plasmid induced with different amounts of IPTG, to control the amount of lambda repressor cI2, and at different temperatures. Control cells with plasmid that constitutively expresses GFPmut3b-ASV and plasmid that does not encode GFP were included as positive and negative controls respectively.

E. coli DH5a cells were grown overnight in 96-well plates with shaking at 250 rpm and at 30°C were induced with 0.1 mM IPTG for high expression of lambda repressor and with appropriate antibiotics: ampicillin (50 μg mL^-1), spectinomycin (25 μg mL^-1), and kanamycin (30 μg mL^"1). Overnight cultured were then diluted by 100-fold into fresh LB media with antibiotics and 0.1 mM IPTG and returned to shaking (250 rpm, 30°C). After 80 minutes, cells were returned to the shaker (250 rpm) at three different temperatures (30°C, 33°C, 37°C) and measured after 6 hrs.

Flow cytometry measurements were performed using a BD LSRFortessa cell analyzer with a high-throughput sampler. Prior to sampling, cells were diluted by a factor of -40 into phosphate-buffered saline. Cells were detected using a forward scatter (FSC) trigger and at least 20,000 cells were recorded for each measurement. Cell populations were gated according to their FSC and side scatter (SSC) distributions and the GFP fluorescence levels of these gated cells were used to measure circuit output. GFP fluorescence histograms yielded unimodal population distributions and the mode fluorescence was recorded from at least two biological replicates. Cellular autofluorescence was not subtracted. The flow cytometry measurement showed that the control cell populations had little temperature dependence on GFP expression or cellular autofluorescence. On the other hand, the temperature sensor circuit showed fluorescence level close to cellular autofluorescence from the 30°C and 33°C culture, and induced GFP expression about 10 fold above background at 37°C.

Table 2. Sequence elements

Toehold switch 1 GATTGAATATGATAGAAGTTTAGTAGTAGACAATAGAACAGAGGAGA

TATTGATGACTACTAAACTA ( SEQ ID NO : 1 )

Toehold trigger 1 GATACACATAGAATCATGTGTATAACACTACTAAACTTCTATCATAT

TCAATCAC (SEQ ID NO : 2 )

pR promoter TGAGCTAACACCGTGCGTGTTGACAATTTTACCTCTGGCGGTGATAA

TGGTTGCA (SEQ ID NO : 3 )

pLac-Ol promoter ATAAATGTGAGCGGATAACATTGACATTGTGAGCGGATAACAAGATA

CTGAGCAC (SEQ ID NO : 4 )

Lambda repressor atgagcacaaaaaagaaaccattaacacaagagcagcttgaggacgc cI2 acgtcgccttaaagcaatttatgaaaaaaagaaaaatgaacttggct tatcccaggaatctgtcgcagacaagatggggatggggcagtcaggc gttggtgctttatttaatggcatcaatgcattaaatgcttataacgc cgcattgcttgcaaaaattctcaaagttagcgttgaagaatttagcc cttcaatcgccagagaaatctacgagatgtatgaagcggttagtatg cagccgtcacttagaagtgagtatgagtaccctgttttttctcatgt tcaggcagggatgttctcacctgagcttagaacctttaccaaaggtg atgcggagagatgggtaagcacaaccaaaaaagccagtgattctgca ttctggcttgaggttgaaggtaattccatgaccgcaccaacaggctc caagccaagctttcctgacggaatgttaattctcgttgaccctgagc aggctgttgagccaggtgatttctgcatagccagacttgggggtgat gagtttaccttcaagaaactgatcagggatagcggtcaggtgttttt acaaccactaaacccacagtacccaatgatcccatgcaatgagagtt gttccgttgtgggggaagttatcgctagtcagtggcctgaagagacg tttggctga (SEQ ID NO : 5 )

GFPmut3b-ASV atgcgtaaaggagaagaacttttcactggagttgtcccaattcttgt tgaattagatggtgatgttaatgggcacaaattttctgtcagtggag agggtgaaggtgatgcaacatacggaaaacttacccttaaatttatt tgcactactggaaaactacctgttccgtggccaacacttgtcactac tttcggttatggtgttcaatgctttgcgagatacccagatcacatga aacagcatgactttttcaagagtgccatgcccgaaggttacgtacag gaaagaactatatttttcaaagatgacgggaactacaagacacgtgc tgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaa aaggtattgattttaaagaagatggaaacattcttggacacaaattg gaatacaactataactcacacaatgtatacatcatggcagacaaaca aaagaatggaatcaaagttaacttcaaaattagacacaacattgaag atggaagcgttcaactagcagaccattatcaacaaaatactccgatt ggcgatggccctgtccttttaccagacaaccattacctgtccacaca atctgccctttcgaaagatcccaacgaaaagagagaccacatggtcc ttcttgagtttgtaaccgctgctgggattacacatggcatggatgaa ctatacaaaaggcctgcagcaaacgacgaaaactacgctgcatcagt ttaataa (SEQ ID NO : 6 )

EQUIVALENTS

While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

The indefinite articles "a" and "an," as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

The phrase "and/or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and/or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and/or" clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to "A and/or B", when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e. "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and/or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another

embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

In the claims, as well as in the specification above, all transitional phrases such as

"comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of and "consisting essentially of shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

Claims

What is claimed is: CLAIMS

1. A system comprising

a host cell comprising an expression construct that encodes a switch RNA and one or more expression constructs that encode a trigger RNA, wherein a switch RNA comprises

(i) a single- stranded toehold domain,

(ii) a fully or partially double- stranded stem domain comprising an initiation codon,

(iii) a loop domain comprising a ribosome binding site, and

(iv) a coding domain, and

wherein the one or more trigger RNA(s) alone or combined hybridize to the single- stranded toehold domain of a switch RNA, and

wherein expression of the switch RNA and the trigger RNA is induced in the presence of one or more external stimulus, or in the presence of one or more external stimuli and absence of one or more external stimuli, or in a combination of presence and absence of two or more external stimuli.

2. A method of inducing translation of a protein by an external stimulus, comprising providing a host cell comprising an expression construct that encodes a switch RNA and one or more expression constructs that encode a trigger RNA, wherein a switch RNA comprises

(i) a single- stranded toehold domain,

(ii) a fully or partially double- stranded stem domain comprising an initiation codon,

(iii) a loop domain comprising a ribosome binding site, and

(iv) a coding domain, and

wherein the one or more trigger RNA(s) alone or combined hybridize to the single- stranded toehold domain of a switch RNA, and

wherein expression of the switch RNA and the trigger RNA is induced in the presence of one or more external stimulus, or in the presence of one or more external stimuli and absence of one or more external stimuli, or in a combination of presence and absence of two or more external stimuli, and inducing translation of the coding domain by the presence of the external stimulus.

3. The system of claim 1 or the method of claim 2, wherein the coding domain encodes an enzyme or an enzyme cofactor.

4. The system of claim 1 or the method of claim 2, wherein the coding domain encodes a reporter protein.

5. The system or method of any of the foregoing claims, wherein the external stimulus is light, heat, water or moisture, pH, or one or more small molecules.

6. The system or method of any of the foregoing claims, wherein the host cell is provided in a housing.

7. The system or method of any of the foregoing claims, wherein the host cell is provided in a housing and the coding domain encodes an enzyme that degrades the housing.

8. The system or method of claim 7, wherein the housing consists of paper, starch, cellulose and/or a hydrogel.

9. The system or method of claim 8, wherein the enzyme is a cellulose, an amylase, a proteases, a peptidase, a beta-glucosidase, an exoglucanase, or an endoglucanase.

10. The system or method of any one of the foregoing claims, wherein the expression constructs comprise inducible promoters selected from the group consisting of cl-ts857, cspA, cadA, Lacl, TetR and AraC sensitive promoters.