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Title:
PIGMENT-BASED MICRONUTRIENT BIOSENSORS
Document Type and Number:
WIPO Patent Application WO/2016/205308
Kind Code:
A1
Abstract:
A whole-cell biosensor for a point-of-care assay using genetically engineered bacteria or fungi to report micronutrient, for example, zinc levels in blood samples is provided. One embodiment provides a micronutrient biosensor having a gene circuit expressing at least two different pigments in response to two different concentrations of the micronutrient, wherein expression of the pigment is visible to the naked eye and is indicative of a concentration of the micronutrient. Another embodiment provides the micronutrient biosensor expressing a third pigment in response to a different concentration of the micronutrient relative to the at least two pigments. Exemplary micronutrients include but are not limited to zinc, iron, vitamin D, vitamin B12, iodine, and folate. Representative pigments include but are not limited to violacein, lycopene, and beta-carotene.

Inventors:
STYCZYNSKI MARK PHILIP-WALTER (US)
WATSTEIN DANIEL M (US)
MCNERNEY MONICA (US)
Application Number:
PCT/US2016/037542
Publication Date:
December 22, 2016
Filing Date:
June 15, 2016
Export Citation:
Click for automatic bibliography generation   Help
Assignee:
GEORGIA TECH RES INST (US)
International Classes:
C12N15/74; C12N15/82; C12N15/87; G01N33/20; G01N33/48
Foreign References:
US20050114923A12005-05-26
US20140170700A12014-06-19
CN104419701A2015-03-18
Other References:
KIRTI ET AL.: "Colorful World of Microbes: Carotenoids and Their Applications.", ADV BIOL., vol. 2014, 2014, pages 1 - 13, XP055338687
DATABASE GenBank 5 August 2008 (2008-08-05), MISAWA: "Pantoea ananatis carotenoid biosynthesis gene cluster (crtE, crtX, crtY, crtl, crtB, crtZ), complete cds.", XP055338705, retrieved from ncbi Database accession no. D90087
DATABASE GenBank [O] 21 April 2008 (2008-04-21), SHETTY: "BioBrick cloning vector pSB3T5-152001, complete sequence.", XP055338698, retrieved from ncbi Database accession no. EU496104
JAJDA ET AL.: "Comparative efficacy of two standard methods for determination of iron and zinc in fruits, pulses and cereals.", J FOOD SCI TECHNOL., vol. 52, no. 2, February 2015 (2015-02-01), pages 1096 - 102, XP035448445
YOSHIDA ET AL.: "Novel Carotenoid-Based Biosensor for Simple Visual Detection of Arsenite: Characterization and Preliminary Evaluation for Environmental Application.", APPL ENVIRON MICROBIOL., vol. 74, no. 21, 2008, pages 6730 - 8, XP055338691
AHMETAGIC ET AL.: "Stable high level expression of the violacein indolocarbazole anti-tumour gene cluster and the Streptomyces lividans amyA gene in E. coli K12.", PLASMID., vol. 63, no. 2, 2010, pages 79 - 85, XP026878730
KANG ET AL.: "One step engineering of T7-expression strains for protein production: increasing the host-range of the T7-expression system.", PROTEIN EXPR PURIF., vol. 55, no. 2, 2007, pages 325 - 33, XP022238139
WATSTEIN ET AL.: "Precise metabolic engineering of carotenoid biosynthesis in Escherichia coli towards a low-cost biosensor.", METAB ENG., vol. 31, September 2015 (2015-09-01), pages 171 - 80, XP055338692
Attorney, Agent or Firm:
PABST, Patrea L. et al. (1545 Peachtree Street N.E., Suite 32, Atlanta GA, US)
Download PDF:
Claims:
We claim:

1. A micronutrient biosensor comprising a gene circuit expressing at least two different pigments in response to two different concentrations of the micronutrient, wherein expression of the at least two different pigments is visible to the naked eye and is indicative of a concentration of the micronutrient.

2. The micronutrient biosensor of claim 1 , further expressing a third pigment in response to a different concentration of the micronutrient relative to the at least two pigments.

3. The micronutrient biosensor of any one of claims 1 or 2, wherein the micronutrient is selected from the group consisting of zinc, iron, vitamin D, vitamin B12, iodine, and folate.

4. The micronutrient biosensor of any one of claims 1 or 2, wherein the pigment is selected from the group consisting of violacein, lycopene, and beta-carotene.

5. The micronutrient biosensor of claim 1 comprising at least 85, 90, 95, 99 or 100 % sequence identity to SEQ ID NO: 20.

6. The micronutrient biosensor of any one of claim 1 or 2, wherein the micronutrient biosensor is a bacterium or fungus genetically engineered to express the gene circuit.

7. A method for detecting micronutrients in a blood sample comprising: separating serum from the blood sample obtained from a subj ect, contacting the micronutrient biosensor of any one of claims 1 -6,

visually detecting expression of a pigment produced by the micronutrient biosensor, wherein the visual detection of the pigment is indicative of a concentration range of the micronutrient.

8. The method of claim 7, further comprising adding growth media to the micronutrient biosensor.

9. A kit comprising:

a container housing any one of the micronutrient biosensors of claims 1 -6, and instructions for using the micronutrient biosensor.

10. The kit of claim 9, wherein the micronutrient biosensors are lyophilized.

1 1. The kit of any one of claims 9-10, further comprising instruments for obtaining the blood sample.

12. The kit of any one of claims 9-11 , further comprising means for separating serum from the blood sample.

13. The kit of any one of claims 9-12, further comprising growth media for the micronutrient biosensor.

14. The micronutrient biosensor of any one of claims 1-6, further comprising SEQ ID NO: 17.

15. An engineered T7 system having at least 85, 90, 95, 99, or 100 percent sequence identity to SEQ ID NO: 17.

16. The kit of any one of claims 9-13, wherein the micronutrient biosensor further comprises the engineered T7 system of claim 15.

Description:
PIGMENT-BASED MICRONUTRIENT BIOSENSORS

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit of and priority to U.S. Provisional Patent Application No. 62/175,576 filed on June 15, 2015, and where permissible, is incorporated by reference in its entirety.

FIELD OF THE INVENTION

The invention is generally directed to genetically engineered biosensors and kits for determining serum levels of micronutrients.

BACKGROUND OF THE INVENTION

Micronutrients (vitamins and minerals) are critical components of human health and development, and micronutrient deficiencies are responsible for hundreds of thousands of deaths per year worldwide (Black et al, Lancet, 371 :243-260 (2008)). One of the most significant challenges in treating micronutrient deficiencies is in the identification of the populations most at risk for deficiencies or most in need of nutritional interventions (which are performed on populations, not individuals, and are prohibitively expensive for continuous application on a widescale (Bhutta et al, Lancet, 382: 452-477 (2013)). Zinc deficiency is estimated to be responsible for 116,000 annual deaths of children younger than five (Black et al, Lancet, 382: 427-451 (2013)) and contributes to growth stunting and increased incidence of pneumonia, diarrhea, and perhaps malaria (Fischer Walker and Black, Annu. Rev. Nutr., 24: 255-275 (2004)).

More precise assessment of global zinc deficiency pervasiveness and impacts is lacking due to the high cost and logistical difficulties of measuring zinc status on a regional population scale. In developing countries (which bear the overwhelming burden of nutritional deficiencies) and in the wake of disasters, on-site testing is hindered by the lack of availability of the sophisticated equipment required to measure serum zinc levels (Kelson and Shamberger, Clin. Chem, 24: 240-244 (1978)), and often by a lack of available electricity. Off-site analyses require continuous cold storage (logistically difficult) during international shipping, can take up to a week for results, and cost tens of dollars each, which is prohibitive for the required epidemiological-scale measurements.

Therefore, it is an object of the invention to provide assays for detecting or quantifying micronutrients in serum that require minimal or no equipment for measurement and interpretation.

It is another object of the invention to provide kits for assaying micronutrient levels in serum without using electricity.

SUMMARY OF THE INVENTION

A whole-cell biosensor for a point-of-care assay using genetically engineered bacteria or fungi to report micronutrient, for example, zinc levels in blood samples is provided. One embodiment provides a micronutrient biosensor having a gene circuit expressing at least two different pigments in response to two different concentrations of the micronutrient, wherein expression of the pigment is visible to the naked eye and is indicative of a concentration of the micronutrient. Another embodiment provides the micronutrient biosensor expressing a third pigment in response to a different concentration of the micronutrient relative to the at least two pigments. Exemplary micronutrients include but are not limited to zinc, iron, vitamin D, vitamin B12, iodine, and folate. Representative pigments include but are not limited to violacein, lycopene, and beta-carotene.

One embodiment provides micronutrient biosensor having at least 85, 90, 95, 99 or 100 % sequence identity to SEQ ID NO:20.

Another embodiment provides a method for detecting micronutrients in a blood sample by separating serum from the blood sample obtained from a subject, contacting the micronutrient biosensor with the serum, and visually detecting expression of a pigment produced by the micronutrient biosensor, wherein the visual detection of the pigment is indicative of a concentration range of the micronutrient. Growth media can optionally be added to the micronutrient biosensor. Still another embodiment provides a kit including a container housing the micronutrient biosensor and instructions for using the micronutrient biosensor. The kit can optionally include micronutrient biosensors that are lyophilized, instruments for obtaining the blood sample, means for separating serum from the blood sample and growth media for the micronutrient biosensor.

Another embodiment provides an engineered T7 system having at least 85, 90, 95, 99, or 100 percent sequence identity to SEQ ID NO: 17. The engineered T7 system can be used in combination with any of the disclosed micronutrient biosensors and kits.

A key innovation in this design is the use of a reporter that requires no equipment for readout. Previous technologies for sensing zinc-produced fluorescence or luminescence proportional to zinc concentrations . While such assays give the potential for quantitative results, they require specialized equipment to detect and quantify these readouts that limit application in low-resource areas. Instead, the disclosed biosensors use pigment production as a readout, removing the need for quantification. Pigment production is performed by heterologous genes expressed in E. coli under the control of micronutrient-responsive transcription factor/promoter systems.

The pigments used for an exemplary biosensor were the red and orange carotenoids lycopene and β-carotene and the purple pigment violacein, each of which have previously been the targets of metabolic engineering efforts whose results and insights have contributed to the engineering efforts described here. There has been extensive work on the engineered overproduction of lycopene and other isoprenoids, as they have significant industrial value as chemicals, and isoprenoids are a precursor to yet other extremely valuable chemicals, including taxol. Violacein has also been the subject of significant metabolic engineering efforts, both due to the novelty and complexity of the pathway that produces it, and for its potential value as an antimicrobial or antitumor drug. In these cases, the chief goal of the metabolic engineering efforts to date has been high titer production with the downstream goal of purification.

An effective bacterial-based, pigment-producing diagnostic required (1) sufficient pigment to be visible to the naked eye, (2) in a reasonable amount of time, and (3) with sufficient control over production of all pigments to enable discrete color states. The first two of these criteria directly relate to traditional metabolic engineering efforts, though with a greater emphasis on productivity to meet a minimum (visible) titer rather than maximizing titer. Meeting the third criterion was the most challenging for the final engineering implementation of the sensor, and was the focus of much effort .Two pigments (lycopene and β-carotene) from the linear carotenoid pathway were chosen with the expectation that it would facilitate the creation of discrete color states: each molecule of red lycopene could be stoichiometrically converted into orange β-carotene upon induction of the appropriate enzyme by the presence of sufficient micronutrient, leaving no residual red color in the orange state. This advantage is also a potential challenge, though, if that micronutrient-inducible promoter has substantial baseline uninduced transcription of that enzyme, which could prevent accumulation of lycopene.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 A shows a schematic of the workflow envisioned for the eventual final implementation of the diagnostic biosensor. Cells from general culture (bottom) are lyophilized for long-term storage. A blood sample (top) is centrifuged using, for example, an egg beater or salad spinner (Wong et al, 2008) to separate plasma, which is then added to the stored cells. These cells then produce pigment (right) based on the level of zinc in the plasma sample. Physiological concentrations of zinc are included for reference. Figure IB shows a schematic of the circuit design intended for

implementation of the initial biosensor. Transcriptional regulators ZntR and Zur are expressed on the same plasmid as the construct with violacein synthesis driven by the zinc-repressible PznuC promoter, lycopene synthesis driven by a weak promoter, and the gene catalyzing the transformation of lycopene to β-carotene driven by the zinc-inducible PzntA promoter. The bolded zinc annotation for PzntA indicates that activation by zinc happens at higher concentrations than for PznuC.

Figure 2A shows a schematic of the reporter construct used to simultaneously measure expression from PznuC and PzntA promoters. The transcription factors regulating expression from each promoter were included on the plasmid. The bolded zinc annotation for PzntA indicates that activation by zinc happens at higher concentrations than for PznuC. Figure 2B is a bar graph of eGFP Fluorescence (AU) or mRFP Fluorescence (AU) for PznuC-eGFP (left column of each pair) and PzntA-mRFP (right column of each pair) and shows expression from each promoter in arbitrary units specific to each fluorescent protein (RFP is right axis, GFP is left axis). There is significant induction of expression from PzntA even at low levels of added zinc and significant repression of expression from PznuC, suggesting that the baseline expression of crtY may be the cause of a lycopene-only state being difficult to capture. Error bars represent standard error of the mean.

Figure 3 A is bar graph of Fluorescence (AU) for the indicated constructs and shows fluorescence measurements of the library of regulatory element combinations. Inset illustrates fluorescence levels at a higher gain setting for library members with insufficient fluorescence to be measured accurately at the same gain setting as in the main plot. Construct names are the promoter name (R0011 and R0040 known strong promoters, J23116 and J23117 known weak promoters, and the PznuC and PzntA promoters), followed by an indicator of the ribosomal binding site part number (31-34), and then the degradation signal added to the protein sequence (no signal, weak DAS signal, or strong LAA signal). Error bars represent the standard error of the mean. Figure 3B is a bar graph of Fluorescence (AU) for R0011 (hatched), R0040 (grey) and pZnuC (black) for ribosomal binding sites B0034, B0031, B0032, B0033 and shows changing ribosomal binding sites for strong or weak promoters provides orders of magnitude reduction in expression. Error bars represent the standard error of the mean. Figure 3C shows flow cytometry plots of Frequency versus Fluorescence (AU) showing increasingly mixed populations for higher-strength (B0034) ribosomal binding sites (top) as opposed to weaker ribosomal binding sites (B0031 bottom), indicating that the bulk fluorescence measured for B0034 RBS under the strong promoters used here is an underestimate of the actual per- cell fluorescence for B0034 under unaltered promoters. Sequencing of plasmid DNA from cultures with bimodal populations yielded mixed traces, indicating mutation of the plasmids to reduce fluorescence production.

Figure 3D is a bar graph of Fluorescence for B0031 (hatched), B0032 (grey), and B0033 (black) for no tag, DAS tag, and LLA tag and shows degradation tags can also yield orders of magnitude reduction of protein levels; the strong LAA tag typically provides two or more orders of magnitude of decrease in fluorescence, while the weaker DAS tag is more variable in its reduction of fluorescence. Error bars represent standard error of the mean; only positive error is indicated due to the logarithmic scale of the plot because the lowest values are near the detection limits of the instrument and would include zero in the opposite direction.

Figure 4A shows a simplified version of the construct used to test only carotenoid production for a selection of constructs with varied regulatory sequences and vectors. Ribosomal binding sites and degradation tags are omitted for simplicity. Figure 4B is a line graph of concentration ^g/ml) of lycopene (■) or β-carotene (o) versus [Ζη 2+ ](μΜ) for B0033

1 AK3 and shows that changing only to a very weak ribosomal binding site is insufficient to produce a lycopene-only state, and generally insufficient to produce any lycopene at all. This suggests that multiple levels of control must be used. Figure 4C is a line graph of concentration ^g/ml) of lycopene (■) or β-carotene (o) versus [Ζη 2+ ](μΜ) for B0032 32 LAA 1 C3 and shows using a moderate ribosomal binding site and a strong degradation tag (LAA) enables the detectable production of lycopene but still does not enable a lycopene-dominated state. Figure 4D is a line graph of fraction of β-carotene versus [Ζη 2+ ](μΜ) for B0032 32 LAA - 1C3 (□), 3C5 (·), 6A1 (Δ), and Fosmid (♦) and shows changing the vector carrying the construct from panel C enables the presence of a lycopene-only state. For the lowest-copy plasmid (the fosmid), so little CrtY is accumulated even at full zinc induction that only a lycopene-dominated state can be observed. The low-copy 6A1 vector offers two distinct states. Figure 4E shows co-transforming with a plasmid containing the mevalonate pathway (pJBEI-MEV) to supplement production of lycopene alters the transition point between the lycopene and β-carotene states. At 50 μΜ Zn 2+ , the pJBEI-MEV-supplemented cells have still produced orders of magnitude more lycopene than β-carotene, while the non- supplemented strain is already transitioning to a β-carotene state. Of note is that the lycopene production at no supplemented zinc is three times higher in the supplemented strain, as expected.

Figures 5 A through 5D correspond to Figures 4B through 4E in showing that the behavior of the cells in minimal and rich medium (which includes an expected baseline level of zinc) is extremely similar. Error bars all represent the standard error of the mean. Measurements were all taken at 0, 50 and 50 μΜ Zn 2+ , though in Figure 5C an offset has been applied for visualization purposes. Figure 5 A is a line graph of concentration ^g/ml)of lycopene (■) or β-carotene (o) versus Zinc (μΜ) for B0033 1AK3 and shows changing only to a very weak ribosomal binding site is insufficient to produce a lycopene-only state, and generally insufficient to produce any lycopene at all. This suggests that multiple levels of control must be used. Figure 5B is a line graph of concentration ^g/ml)of lycopene (■) or β- carotene (o) versus Zinc (μΜ) for B0032 LAA 1C3 and shows using a moderate ribosomal binding site and a strong degradation tag (LAA) enables the detectable production of lycopene but still does not enable a lycopene- dominated state. Figure 5C is a line graph of fraction of β-carotene versus [Ζη 2+ ](μΜ) for B0032 32 LAA - 1C3 (V), 3C5 (·), 6A1 (Δ), and Fosmid (■) and shows changing the vector carrying the construct from Figure 5C enables the presence of a lycopene-only state. For the lowest-copy plasmid (the fosmid), so little CrtY is accumulated even at full zinc induction that only a lycopene-dominated state can be observed. The low-copy 6A1 vector offers two distinct states. Figure 5D is a line graph of fraction of β-carotene versus [Ζη 2+ ](μΜ) for B0032 32 LAA - 6A1 (o) and 6A1 + pJBEI-MEV and shows co-transforming with a plasmid containing the mevalonate pathway (pJBEI-MEV) to supplement production of lycopene alters the transition point between the lycopene and β-carotene states. At 50 μΜ Zn 2+ , the pJBEI- MEV-supplemented cells still have still produced much more lycopene than β-carotene, while the non-supplemented strain is already transitioning to a β- carotene state. Of note is that the lycopene production at no supplemented zinc is three times higher in the supplemented strain, as expected.

Figure 6 is a diagram of an exemplary T7 system to control lycopene expression. The engineered T7 system can be used in combination with the above constructs to repress pigment production during preculture to high cell density, and then released to allow pigment production in a few hours of assay time. This example can be modified to repress preculture production of violacein or other pigments to be produced by the cell, until the repression is released to allow rapid production of pigment during the assay.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

The use of the terms "a," "an," "the," and similar referents in the context of describing the presently claimed invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

Use of the term "about" is intended to describe values either above or below the stated value in a range of approx. +/- 10%; in other embodiments the values may range in value either above or below the stated value in a range of approx. +1- 5%; in other embodiments the values may range in value either above or below the stated value in a range of approx. +/- 2%; in other embodiments the values may range in value either above or below the stated value in a range of approx. +/- 1 %. The preceding ranges are intended to be made clear by context, and no further limitation is implied. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non- claimed element as essential to the practice of the invention.

The term "micronutrient" refers to dietary components, often referred to as vitamins and minerals, which although only required by the body in small amounts, are vital to development, disease prevention, and wellbeing. Representative micronutrients include but are not limited to zinc, iron, vitamin D, vitamin B 12, iodine, and folate.

A "transgenic organism" as used herein, is any organism, in which one or more of the cells of the organism contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art. The nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus. Suitable transgenic organisms include, but are not limited to, bacteria, cyanobacteria, fungi, plants and animals. The nucleic acids described herein can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation. Techniques for transferring DNA into such organisms are widely known and provided in references such as Sambrook, et al. (2000) Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1-3, Cold Spring Harbor Press, Plainview N.Y.

The term "construct" refers to a recombinant genetic molecule having one or more isolated polynucleotide sequences. Genetic constructs used for transgene expression in a host organism include in the 5 '-3 ' direction, a promoter sequence; a sequence encoding a gene of interest; and a termination sequence. The construct may also include selectable marker gene(s) and other regulatory elements for expression. The term "gene" refers to a DNA sequence that encodes through its template or messenger RNA a sequence of amino acids characteristic of a specific peptide, polypeptide, or protein. The term "gene" also refers to a DNA sequence that encodes an RNA product. The term gene as used herein with reference to genomic DNA includes intervening, non-coding regions as well as regulatory regions and can include 5 ' and 3 ' ends.

The term "endogenous" with regard to a nucleic acid refers to nucleic acids normally present in the host.

The term "heterologous" refers to elements occurring where they are not normally found. For example, a promoter may be linked to a heterologous nucleic acid sequence, e.g., a sequence that is not normally found operably linked to the promoter. When used herein to describe a promoter element, heterologous means a promoter element that differs from that normally found in the native promoter, either in sequence, species, or number. For example, a heterologous control element in a promoter sequence may be a control/ regulatory element of a different promoter added to enhance promoter control, or an additional control element of the same promoter. The term "heterologous" thus can also encompass "exogenous" and "non-native" elements.

The term "percent (%) sequence identity" is defined as the percentage of nucleotides or amino acids in a candidate sequence that are identical with the nucleotides or amino acids in a reference nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.

For purposes herein, the % sequence identity of a given nucleotides or amino acids sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given sequence C that has or comprises a certain % sequence identity to, with, or against a given sequence D) is calculated as follows:

100 times the fraction W/Z, where W is the number of nucleotides or amino acids scored as identical matches by the sequence alignment program in that program's alignment of C and D, and where Z is the total number of nucleotides or amino acids in D. It will be appreciated that where the length of sequence C is not equal to the length of sequence D, the % sequence identity of C to D will not equal the % sequence identity of D to C.

The term "stringent hybridization conditions" as used herein mean that hybridization will generally occur if there is at least 95% and preferably at least 97% sequence identity between the probe and the target sequence. Examples of stringent hybridization conditions are overnight incubation in a solution comprising 50% formamide, 5X SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5X Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared carrier DNA such as salmon sperm DNA, followed by washing the hybridization support in 0.1X SSC at approximately 65°C. Other hybridization and wash conditions are well known and are exemplified in Green and Sambrook.

(2012) Molecular Cloning: A Laboratory Manual 4rd. edition, Cold Spring Harbor Laboratory Press

As used herein, the term "low stringency" refers to conditions that permit a polynucleotide or polypeptide to bind to another substance with little or no sequence specificity.

The term "subject" refers to a mammal, preferably a human.

Unless otherwise indicated, the disclosure encompasses conventional techniques of molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Green and Sambrook. (2012) Molecular Cloning: A Laboratory Manual 4rd. edition, Cold Spring Harbor Laboratory Press; Current Protocols In Molecular Biology [(F. M. Ausubel, et al. eds., (1987)]; Coligan, Dunn, Ploegh, Speicher and Wingfeld, eds. (1995) Current Protocols in Protein Science (John Wiley & Sons, Inc.); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A

Practical Approach (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)].

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Lewin, Genes VII, published by Oxford University Press, 2000; Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Wiley-Interscience., 1999; and Robert A. Meyers (ed.), Molecular Biology and Biotechnology, a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995; Green and Sambrook. (2012) Molecular Cloning: A Laboratory Manual 4rd. edition, Cold Spring Harbor Laboratory Press.

The term "pigment biosynthesis pathway" refers to all of the genes required to express the desired pigment.

II. Micronutrient Biosensors

The design and engineering of a biosensor capable of responding to extracellular levels of a micronutrient, for example zinc, with potential application to blood test diagnostics are described. Specifically, a biosensor was designed to produce pigment, rather than fluorescence or related readouts, to facilitate equipment-free usage since the color of the readout can be assessed with the naked eye. Initial efforts yielded only a two-state sensor, despite the design of the construct to have three distinct regimes of pigment production. (A two-color sensor would still be useful, though having a third state provides increased micronutrient status resolution and thus more information for determining when and where to perform nutritional interventions.) This prompted the need for metabolic engineering of the construct, as baseline levels of the last gene in one pigment production pathway prevented the accumulation of an intermediate intended to represent a distinct readout state in the device.

The data described in the Examples advances in a number of different directions within the purview of developing metabolically engineered pigment-based biosensors. In one embodiment, two different heterologous pigment production pathways were integrated, and were able to shut one off completely so as to enable the visibility of the other. The use of natural zinc- responsive promoters and transcription factors provided one system

(Zur/PznuC) that was appropriate for one application, but another system (ZntR/PzntA), with a wide dynamic range and excessively high baseline expression when in an uninduced state, required significant engineering to be usable in the sensor. Engineering efforts were limited to components besides the promoter and regulator themselves, as mutating those would likely have been prone to loss of zinc specificity (Khan et al., 2002) (particularly true given the mechanism of action of ZntR). Without promoter engineering as a viable possibility, the focus was on post-transcriptional regulation to harness a very small portion of the dynamic range of the system and to sufficiently suppress expression to allow multiple color states within a single pathway. The system started with no switching capability whatsoever, but we were able to engineer in switching capability as well as demonstrate our ability to change the level at which the switch occurs. This is particularly relevant since one application is limited to the small fraction of ZntR's dynamic range that is physiologically relevant.

To sufficiently control the production of the most downstream enzyme in the carotenoid production pathway, CrtY, multiple levels of regulation were necessary. Combining the use of weaker ribosomal binding sites with protein degradation tags and changing the vector allowed for the establishment of inducible color change and tuning of the micronutrient concentration at which the color change occurred. Although the lower copy vectors were more effective at allowing a lycopene-dominated state, this phenomenon cannot be solely attributed to copy number since vector- specific differences in readthrough transcription affect the level of transcription of (promoterless) crtEBI and thus the balance of lycopene and crtY. Nonetheless, the data, along with intuition, suggest that copy number likely plays a role in this process; even if not, the fact that selection of vector has such a substantial effect on the metabolic phenotype of the cells is still an important observation. Fluorescence measurements were used on a library of regulatory combinations as the basis for rational exploration of the design space for production of carotenoids. It is possible that the reduction in fluorescence caused by the addition of degradation tags was not just due to increased degradation, but caused also by decreased fluorescence per molecule due to the tag changing the protein conformation. While this was not something controlled for and tempers the interpretations of tag effectiveness from the fluorescence data, the carotenoid production results are consistent with the interpretation of the fluorescence data on the importance of degradation tags for tuning the behavior of the cells.

While the use of native, rather than orthogonal, sensing (zinc- responsive) elements is not commonplace, it did not prevent successful metabolic engineering of the strain. While this aspect of the approach may have led to more non-linear impacts of the various regulatory changes implemented due to impacts on host cell behavior or impacts of host cell genomic expression, this disadvantage was outweighed by the many tools available for genetic manipulation in E. coli and the fact that the regulators used are well-characterized zinc-responsive elements, one of which was already known to respond within a physiologically relevant concentration range.

A. Gene Circuit Components

The disclosed micronutrient biosensors include an organism, preferably bacteria or fungi, genetically engineered to express a gene circuit that expresses a different pigment or different combination of pigments in response to different amounts of micronutrient, for example borderline levels of the micronutrient, low levels of the micronutrient, and normal levels of the micronutrient. Thus, the genetically engineered organism contains a gene circuit that includes one or more micronutrient responsive promoters, one or more transcription regulators, and one or more pigment biosynthesis pathways. The gene circuits may also contain translational regulation, such as riboswitches, and post-translational regulation such as protein degradation tags. 1. Micronutrient Responsive Promoters

The micronutrient responsive promoters can be selected based on the micronutrient to be detected. The micronutrient responsive promoters can induce expression or repress expression based on levels of micronutrients. Exemplary micronutrients to be detected include, but are not limited to zinc, iron, vitamin D, vitamin B12, iodine, and folate. Representative zinc response promoters include, but are not limited to PznuC and PzntA.

Micronutrient responsive promoters for iron include but are not limited to PfbpA from B. subtilis, and ryhBp in E. coli. The transcription of genes including but not limited to FolT from L. casei or other Firmicutes is known to be controlled by folate levels, indicating that the promoter of FolT is micronutrient responsive.

Other micronutrient responsive promoters are known in the art, based on the selected transcription regulator, and can also be used herein.

2. Transcriptional and Other Regulators

The micronutrient biosensors can also contain transcription regulators to induce or repress transcription of pigment biosynthesis pathways.

Transcription regulators that can be used for zinc biosensors include, but are not limited to ZntR and Zur. ZntR serves and an inducer in the presence of zinc and Zur acts as a repressor in the presence of zinc.

Other regulators that can be used in biosensors for other

micronutrients include Fur from ?. coli, B. subtilis, and other species for iron; the transcription factors that bind upstream of FolT from . casei or other Firmicutes for folate; and the riboswitch that regulates BtuB from E. coli for Vitamin B12.

3. Pigments

The micronutrient biosensors can express one or more pigments the have a color that can be visually detected with the naked eye. Exemplary pigments that can be express include, but are not limited to violacein, lycopene, and beta-carotene. Thus, the micronutrient biosensor can contain all of the genes necessary to express one or more pigments. Other pigments that can be used in the micronutrient biosensors include, but are not limited to zeaxanthin in the carotenoid pathway via crtZ from P. ananatis, deoxychromoviridans via deletion or modifications of vioCD from C.

violaceum, and melanin via melA from S. colwelliana.

B. Exemplary Construct Sequences

An exemplary gene circuit described below causes the conditional production of pigments depending on the concentration of zinc in the environment. The sequences are described individually but when combined form the exemplary construct. The two transcriptional regulators, ZntR and Zur, are constitutively expressed at defined levels. Lycopene, a red pigment, is constitutively produced. At low zinc concentrations, Zur repression of PznuC is alleviated, resulting in the expression of five genes in the violacein pathway and the production of the purple pigment violacein. In this state, both violacein and lycopene are produced; however, violacein predominates resulting in purple cells. At intermediate zinc concentrations, Zur is active as a repressor, and prevents expression of the violacein genes, resulting in cells that predominately contain lycopene. At high zinc concentrations, ZntR activates expression from PzntA producing CrtY, a gene catalyzing the reaction of lycopene to beta-carotene, an orange pigment. In this state, only beta-carotene is present and cells are orange.

CCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCG TTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCT CACCTTCGGGTGGGCCTTTCTGCGTTTATA (SEQ ID NO: l)

SEQ ID NO: 1 is a transcriptional terminator used to insulate the insert from transcription initiating in or reading through the vector.

TACTAGAGCTGATGGCTAGCTCAGTCCTAGGGATTATGCTAGC (SEQ ID NO:2)

SEQ ID NO:2 is a weak constitutive transcriptional promoter and assembly scar used to drive crtEBI

TACTAGAGGAGGTACTAGATGACGGTCTGCGCAAAAAAACACGTTCATC TCACTCGCGATGCTGCGGAGCAGTTACTGGCTGATATTGATCGACGCCTT GATCAGTTATTGCCCGTGGAGGGAGAACGGGATGTTGTGGGTGCCGCGA TGCGTGAAGGTGCGCTGGC ACCGGGAAAACGT ATTCGCCCC ATGTTGCT GTTGCTGACCGCCCGCGATCTGGGTTGCGCTGTCAGCCATGACGGATTA CTGGATTTGGCCTGTGCGGTGGAAATGGTCCACGCGGCTTCGCTGATCCT TGACGATATGCCCTGCATGGACGATGCGAAGCTGCGGCGCGGACGCCCT ACCATTCATTCTCATTACGGAGAGCATGTGGCAATACTGGCGGCGGTTG CCTTGCTGAGTAAAGCCTTTGGCGT AATTGCCGATGC AGATGGCCTC AC GCCGCTGGCAAAAAATCGGGCGGTTTCTGAACTGTCAAACGCCATCGGC ATGCAAGGATTGGTTCAGGGTCAGTTCAAGGATCTGTCTGAAGGGGATA AGCCGCGCAGCGCTGAAGCTATTTTGATGACGAATCACTTTAAAACCAG CACGCTGTTTTGTGCCTCCATGCAGATGGCCTCGATTGTTGCGAATGCCT CCAGCGAAGCGCGTGATTGCCTGCATCGTTTTTCACTTGATCTTGGTCAG GCATTTCAACTGCTGGACGATTTGACCGATGGCATGACCGACACCGGTA AGGATAGCAATCAGGACGCCGGTAAATCGACGCTGGTCAATCTGTTAGG CCCGAGGGCGGTTGAAGAACGTCTGAGACAACATCTTCAGCTTGCCAGT GAGCATCTCTCTGCGGCCTGCCAACACGGGCACGCCACTCAACATTTTA TTCAGGCCTGGTTTGACAAAAAACTCGCTGCCGTCAGTTAATAATACTA GAGCTCAAGGAGGTACTAGATGAATAATCCGTCGTTACTCAATCATGCG GTCGAAACGATGGCAGTTGGCTCGAAAAGTTTTGCGACAGCCTCAAAGT TATTTGATGCAAAAACCCGGCGCAGCGTACTGATGCTCTACGCCTGGTG CCGCCATTGTGACGATGTTATTGACGATCAGACGCTGGGCTTTCAGGCC CGGCAGCCTGCCTTACAAACGCCCGAACAACGTCTGATGCAACTTGAGA TGAAAACGCGCCAGGCCTATGCAGGATCGCAGATGCACGAACCGGCGTT TGCGGCTTTTCAGGAAGTGGCTATGGCTCATGATATCGCCCCGGCTTACG CGTTTGATCATCTGGAAGGCTTCGCCATGGATGTACGCGAAGCGCAATA CAGCCAACTGGATGATACGCTGCGCTATTGCTATCACGTTGCAGGCGTT GTCGGCTTGATGATGGCGCAAATCATGGGCGTGCGGGATAACGCCACGC TGGACCGCGCCTGTGACCTTGGGCTGGCATTTCAGTTGACCAATATTGCT CGCGATATTGTGGACGATGCGCATGCGGGCCGCTGTTATCTGCCGGCAA GCTGGCTGGAGCATGAAGGTCTGAACAAAGAGAATTATGCGGCACCTG AAAACCGTCAGGCGCTGAGCCGTATCGCCCGTCGTTTGGTGCAGGAAGC AGAACCTTACTATTTGTCTGCCACAGCCGGCCTGGCAGGGTTGCCCCTG CGTTCCGCCTGGGCAATCGCTACGGCGAAGCAGGTTTACCGGAAAATAG GTGTCAAAGTTGAACAGGCCGGTCAGCAAGCCTGGGATCAGCGGCAGTC AACGACCACGCCCGAAAAATTAACGCTGCTGCTGGCCGCCTCTGGTCAG GCCCTTACTTCCCGGATGCGGGCTCATCCTCCCCGCCCTGCGCATCTCTG GCAGCGCCCGCTCTAATAATACTAGAGCTCAAGGAGGTACTAGATGAAA CCAACT ACGGTAATTGGTGCAGGCTTCGGTGGCCTGGC ACTGGC AATTC GTCTACAAGCTGCGGGGATCCCCGTCTTACTGCTTGAACAACGTGATAA ACCCGGCGGTCGGGCTTATGTCTACGAGGATCAGGGGTTTACCTTTGAT GCAGGCCCGACGGTTATCACCGATCCCAGTGCCATTGAAGAACTGTTTG CACTGGCAGGAAAACAGTTAAAAGAGTATGTCGAACTGCTGCCGGTTAC GCCGTTTT ACCGCCTGTGTTGGGAGTC AGGGAAGGTCTTTAATT ACGATA ACGATCAAACCCGGCTCGAAGCGCAGATTCAGCAGTTTAATCCCCGCGA TGTCGAAGGTTATCGTCAGTTTCTGGACTATTCACGCGCGGTGTTTAAAG AAGGCTATCTAAAGCTCGGTACTGTCCCTTTTTTATCGTTCAGAGACATG CTTCGCGCCGCACCTCAACTGGCGAAACTGCAAGCATGGAGAAGCGTTT ACAGTAAGGTTGCCAGTTACATCGAAGATGAACATCTGCGCCAGGCGTT TTCTTTCCACTCGCTGTTGGTGGGCGGCAATCCCTTCGCCACCTCATCCA TTTATACGTTGATACACGCGCTGGAGCGTGAGTGGGGCGTCTGGTTTCC GCGTGGCGGCACCGGCGCATTAGTTCAGGGGATGATAAAGCTGTTTCAG GATCTGGGTGGCGAAGTCGTGTTAAACGCCAGAGTCAGCCATATGGAAA CGACAGGAAACAAGATTGAAGCCGTGCATTTAGAGGACGGTCGCAGGT TCCTGACGCAAGCCGTCGCGTCAAATGCAGATGTGGTTCATACCTATCG CGACCTGTTAAGCCAGCACCCTGCCGCGGTTAAGCAGTCCAACAAACTG CAAACTAAGCGCATGAGTAACTCTCTGTTTGTGCTCTATTTTGGTTTGAA TCACCATCATGATCAGCTCGCGCATCACACGGTTTGTTTCGGCCCGCGTT ACCGCGAGCTGATTGACGAAATTTTTAATCATGATGGCCTCGCAGAGGA CTTCTCACTTTATCTGCACGCGCCCTGTGTCACGGATTCGTCACTGGCGC CTGAAGGTTGCGGCAGTTACTATGTGTTGGCGCCGGTGCCGCATTTAGG CACCGCGAACCTCGACTGGACGGTTGAGGGGCCAAAACTACGCGACCGT ATTTTTGCGTACCTTGAGCAGCATTACATGCCTGGCTTACGGAGTCAGCT GGTCACGCACCGGATGTTTACGCCGTTTGATTTTCGCGACCAGCTTAATG CCTATCATGGCTCAGCCTTTTCTGTGGAGCCCGTTCTTACCCAGAGCGCC TGGTTTCGGCCGCATAACCGCGATAAAACCATTACTAATCTCTACCTGGT CGGCGCAGGCACGCATCCCGGCGCAGGCATTCCTGGCGTCATCGGCTCG GCAAAAGCGACAGCAGGTTTGATGCTGGAGGATCTGATATAATAA (SEQ ID NO:3)

SEQ ID NO:3 is the coding sequences for crtEBI, genes used to produce the carotenoid lycopene with ribosomal binding sites and assembly scars. T ACTAGAGCCAGGC ATC AAATAAAACGAAAGGCTC AGTCGAAAGACTG GGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTC ACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATA (SEQ ID NO:4)

SEQ ID NO:4 is a transcriptional terminator and assembly scar.

TACTAGAGTTGACAGCTAGCTCAGTCCTAGGGATTGTGCTAGC (SEQ ID NO:5)

SEQ ID NO: 5 is the medium strength constitutive promoter and assembly scar.

TACTAGAGAAAGAGGAGAAATACTAGATGTATCGCATTGGTGAGCTGGC AAAAATGGCGGAAGTAACACCCGACACGATTCGTTATTACGAAAAACA GCAGATGATGGAGCATGAAGTGCGTACTGAAGGTGGGTTTCGCCTATAT ACCGAAAGCGATCTCCAGCGATTGAAATTTATCCGCCATGCCAGACAAC TAGGTTTCAGTCTGGAGTCGATCCGCGAGTTGCTGTCGATCCGCATCGAT CCTGAACACCATACCTGTCAGGAGTCAAAAGGCATTGTGCAGGAAAGAT TGCAGGAAGTCGAAGCACGGATAGCCGAGTTGCAGAGTATGCAGCGTTC CTTGCAACGCCTTAACGATGCCTGTTGTGGGACTGCTCATAGCAGTGTTT ATTGTTCGATTCTTGAAGCTCTTGAACAAGGGGCGAGTGGCGTTAAGAG TGGTTGTTGA (SEQ ID NO:6)

SEQ ID NO: 6 is a strong ribosomal binding site, assembly scars, and coding sequence for ZntR, a zinc responsive transcriptional activator for PzntA. TACTAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGG CCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACA CTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATA (SEQ ID NO:7) SEQ ID NO: 7 is a transcriptional terminator and assembly scar.

TACTAGAGTTGACAGCTAGCTCAGTCCTAGGGATTGTGCTAGC (SEQ ID NO:8) SEQ ID NO: 8 is a medium strength constitutive promoter and assembly scar.

TACTAGAGTCACACAGGAAACCTACTAGATGGAAAAGACCACAACGCA

GGAGTTATTAGCGCAGGCTGAAAAAATCTGCGCGCAGCGTAATGTGCGC

CTGACCCCACAGCGCCTGGAAGTGTTGCGCCTGATGAGTCTCCAAGATG GCGCT ATC AGCGCTT ATGATCTGCTTGATTTACTGCGCGAAGCTGAACCG CAAGCCAAGCCGCCAACGGTTTATCGCGCGCTGGATTTTCTGCTTGAGC AAGGTTTTGTGCATAAGGTGGAATCCACCAACAGTTATGTGCTCTGTCAT CTGTTCGATCAGCCCACCCATACGTCAGCCATGTTTATTTGCGATCGCTG CGGCGCAGTGAAAGAAGAGTGTGCAGAAGGCGTGGAAGACATTATGCA TACGCTGGCGGCAAAAATGGGGTTTGCCCTGCGGCATAATGTGATTGAA GCACATGGGCTCTGTGCGGCATGTGTAGAAGTGGAAGCGTGTCGTCATC CTGAACAGTGCCAGCATGATCACTCTGTGCAGGTGAAAAAGAAACCGCG TTAA (SEQ ID NO:9) SEQ ID NO:9 is an intermediate strength ribosomal binding site, assembly scars, and coding sequence for Zur, a zinc responsive transcriptional repressor for PznuC.

TACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTG GGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTC ACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATA (SEQ ID NO: 10)

SEQ ID NO: 10 is a transcriptional terminator and assembly scar. TACTAGAGCTGTATCTCTGATAAAACTTGACTCTGGAGTCGACTCCAGA GTGTATCCTTCGGTTAAT (SEQ ID NO: 11)

SEQ ID NO: 11 is Pznta, a promoter sequence positively regulated by ZntR, and assembly scar.

TACTAGAGTCACACAGGACTACTAGATGCAACCGCATTATGATCTGATT CTCGTGGGGGCTGGACTCGCGAATGGCCTTATCGCCCTGCGTCTTCAGC AGCAGCAACCTGATATGCGTATTTTGCTTATCGACGCCGCACCCCAGGC GGGCGGGAATC ATACGTGGTC ATTTC ACC ACGATGATTTGACTGAGAGC CAACATCGTTGGATAGCTCCGCTGGTGGTTCATCACTGGCCCGACTATCA GGTACGCTTTCCCACACGCCGTCGTAAGCTGAACAGCGGCTACTTTTGTA TTACTTCTCAGCGTTTCGCTGAGGTTTTACAGCGACAGTTTGGCCCGCAC TTGTGGATGGATACCGCGGTCGCAGAGGTTAATGCGGAATCTGTTCGGT TGAAAAAGGGTC AGGTT ATCGGTGCCCGCGCGGTGATTGACGGGCGGG GTTATGCGGCAAATTCAGCACTGAGCGTGGGCTTCCAGGCGTTTATTGG CCAGGAATGGCGATTGAGCCACCCGCATGGTTTATCGTCTCCCATTATCA TGGATGCCACGGTCGATCAGCAAAATGGTTATCGCTTCGTGTACAGCCT GCCGCTCTCGCCGACCAGATTGTTAATTGAAGACACGCACTATATTGAT AATGCGACATTAGATCCTGAATGCGCGCGGCAAAATATTTGCGACTATG CCGCGCAACAGGGTTGGCAGCTTCAGACACTGCTGCGAGAAGAACAGG GCGCCTTACCCATTACTCTGTCGGGCAATGCCGACGCATTCTGGCAGCA GCGCCCCCTGGCCTGTAGTGGATTACGTGCCGGTCTGTTCCATCCTACCA CCGGCTATTCACTGCCGCTGGCGGTTGCCGTGGCCGACCGCCTGAGTGC ACTTGATGTCTTTACGTCGGCCTCAATTCACCATGCCATTACGCATTTTG CCCGCGAGCGCTGGCAGCAGCAGGGCTTTTTCCGCATGCTGAATCGCAT GCTGTTTTTAGCCGGACCCGCCGATTCACGCTGGCGGGTTATGCAGCGTT TTTATGGTTTACCTGAAGATTTAATTGCCCGTTTTTATGCGGGAAAACTC ACGCTGACCGATCGGCTACGTATTCTGAGCGGCAAGCCGCCTGTTCCGG TATTAGCAGCATTGCAAGCCATTATGACGACTCATCGTGCTGCTAACGA CGAAAACTACGCTCTGGCTGCTTAATAAT (SEQ ID NO: 12)

SEQ ID NO: 12 is a weak ribosomal binding site, coding sequence for carotenoid gene to catalyze reaction of lycopene into beta carotene, and assembly scars. TACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTG GGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTC ACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATA (SEQ ID NO: 13) SEQ ID N: 13 is a transcriptional terminator and assembly scar.

TACTAGAGAACATAATGCGACCAATAATCGTAATGAATATGAGAAGTGT GATATTATAACATTT (SEQ ID NO: 14) SEQ ID NO: 14 is PznuC, a promoter sequence negatively regulated by Zur.

TACTAGAGTTAAGGAGGTAAAAAAAATGAAACATTCTTCCGATATCTGC ATTGTTGGTGCTGGTATTTCTGGTTTGACGTGCGCAAGCCATCTGCTGGA CAGCCCGGCATGCCGTGGTCTGAGCCTGCGTATCTTTGACATGCAGCAA GAAGCCGGTGGCCGT ATCCGCAGC AAAATGCTGGATGGT AAGGC AAGC ATTGAACTGGGCGCAGGTCGCTACTCCCCTCAGTTGCACCCGCATTTCCA AAGCGCAATGCAGCACTATAGCCAAAAGAGCGAAGTCTATCCGTTCACC CAGTTGAAGTTCAAATCTCACGTGCAGCAAAAGCTGAAGCGCGCCATGA ATGAACTGTCCCCGCGTCTGAAAGAGCATGGTAAAGAGAGCTTTTTGCA GTTTGTCAGCCGTTATCAAGGTCACGATAGCGCGGTTGGTATGATCCGCT CTATGGGTTACGACGCACTGTTCCTGCCGGATATCAGCGCAGAAATGGC CTACGACATTGTGGGTAAGCACCCGGAGATCCAGAGCGTGACGGACAA CGACGCGAACCAATGGTTTGCAGCGGAAACGGGCTTTGCTGGTCTGATT CAGGGCATCAAGGCTAAGGTT AAGGC GGCAGGTGCGCGTTTTAGCCTGG GTTATCGTCTGCTGAGCGTCCGTACCGACGGTGACGGCTACCTGCTGCA ACTGGCAGGTGACGACGGCTGGAAACTGGAGCACCGTACCCGCCATCTG ATTCTGGCGATTCCGCCGAGCGCGATGGCGGGTTTGAATGTTGATTTTCC AGAAGCCTGGTCCGGTGCGCGCTATGGCAGCCTGCCGCTGTTTAAGGGC TTTCTGACGTACGGTGAGCCGTGGTGGTTGGACTACAAACTGGACGATC AGGTGCTGATTGTTGACAACCCGCTGCGCAAAATCTATTTCAAAGGCGA TAAGTACCTGTTCTTCTATACCGATAGCGAGATGGCGAATTACTGGCGC GGTTGTGTCGCGGAGGGCGAGGACGGTTACCTGGAGCAAATTCGCACCC ATTTGGCTAGCGCACTGGGTATCGTCCGTGAACGTATCCCGCAACCGCT GGCACACGTTCACAAGTATTGGGCGCACGGCGTTGAGTTTTGCCGTGAT TCTGATATTGACCACCCGAGCGCACTGTCTCATCGCGACAGCGGTATCA TCGCGTGCTCCGATGCGTACACGGAGCATTGTGGTTGGATGGAGGGCGG TCTGCTGAGCGCCCGTGAGGCAAGCCGTCTGCTGTTGCAGCGTATCGCC GCGTGATTAAGGAGGTAAAAAAAATGAGCATTCTGGATTTCCCGCGTAT CCACTTCCGTGGCTGGGCCCGTGTCAATGCGCCGACCGCGAACCGCGAT CCGCACGGCCACATCGATATGGCCAGCAATACCGTGGCGATGGCGGGTG AGCCGTTCGACCTGGCACGCCATCCTACGGAGTTCCACCGTCACCTGCG CTCCCTGGGTCCGCGCTTCGGCTTGGATGGTCGTGCTGACCCGGAAGGC CCGTTCAGCCTGGCCGAGGGCTACAACGCTGCCGGTAACAACCACTTTT CGTGGGAGAGCGCAACCGTTAGCCACGTGCAATGGGATGGCGGTGAGG CGGATCGTGGTGACGGTCTGGTCGGTGCTCGTTTGGC ACTGTGGGGTC A CTACAATGATTATCTGCGTACCACCTTCAATCGTGCTCGTTGGGTCGACA GCGACCCGACGCGCCGTGACGCTGCACAAATCTATGCGGGCCAATTCAC CATTAGCCCGGCTGGTGCCGGTCCGGGTACGCCGTGGCTGTTTACGGCA GACATTGATGATAGCCATGGTGCACGTTGGACGCGTGGCGGCCACATTG C AGAGCGTGGCGGCC ACTTCTTGGATGAAGAGTTTGGTCTGGC ACGCCT GTTTCAGTTCTCTGTGCCGAAAGATCACCCACATTTTCTGTTTCACCCGG GTCCGTTTGATTCCGAGGCCTGGCGTCGTCTGCAATTGGCTCTGGAGGAT GACGACGTTCTGGGTCTGACCGTGCAATATGCGTTGTTCAATATGAGCA CCCCGCCTCAGCCGAACAGCCCGGTTTTTCACGATATGGTCGGTGTTGTC GGTCTGTGGCGTCGTGGTGAACTGGCGAGCTACCCGGCTGGTCGTCTGC TGCGTCCGCGTCAACCGGGTCTGGGTGACCTGACCCTGCGCGTCAACGG TGGTCGCGTTGCGCTGAATTTGGCGTGTGCCATTCCGTTCAGCACTCGTG CCGCGCAGCCAAGCGCACCGGACCGCCTGACCCCGGACCTGGGTGCCA AACTGCCGCTGGGCGATCTGCTGCTGCGTGATGAGGACGGCGCACTGTT GGCACGTGTGCCGCAGGCTCTGTACCAAGACTATTGGACGAATCACGGT ATTGTGGACCTGCCGCTGCTGCGCGAACCGCGTGGTAGCTTGACCCTGA GCAGCGAACTGGCGGAGTGGCGTGAGCAAGACTGGGTCACCCAAAGCG ACGCGTCTAACCTGTACCTGGAGGCACCGGATCGCCGTCACGGTCGCTT TTTCCCTGAGAGCATCGCGCTGCGCAGCTACTTTCGCGGTGAAGCGCGT GCGCGTCCGGATATCCCGCATCGTATCGAGGGCATGGGCCTGGTCGGCG TCGAATCTCGTCAGGATGGCGACGCTGCGGAATGGCGTCTGACGGGTCT GCGTCCGGGTCCGGCACGCATTGTTCTGGACGATGGTGCCGAGGCGATC CCTCTGCGTGTTCTGCCTGACGATTGGGCGCTGGATGACGCGACCGTCG AAGAAGTGGATTACGCCTTTTTGTACCGCCACGTTATGGCGTATTACGA GCTGGTGTATCCATTCATGAGCGACAAGGTGTTTTCCCTGGCTGATCGTT GCAAATGTGAAACGTACGCACGTCTGATGTGGCAGATGTGTGATCCGCA GAACCGCAACAAGTCCTATTACATGCCGAGCACCCGCGAACTGTCGGCA CCGAAAGCTCGTTTGTTCTTGAAGTATCTGGCCCACGTGGAAGGCCAGG CACGCCTGCAAGCACCTCCGCCAGCGGGTCCGGCACGCATTGAATCTAA AGCCCAGTTGGCGGCAGAGCTGCGTAAAGCCGTCGACCTGGAGCTGTCT GTGATGCTGCAATACCTGTACGCGGCGTATAGCATTCCGAACTATGCAC AGGGCCAACAACGTGTTCGTGACGGTGCGTGGACCGCCGAGCAGCTGCA ACTGGCGTGCGGTAGCGGTGACCGTCGCCGTGATGGCGGTATTCGTGCA GCACTGCTGGAAATTGCTCATGAAGAAATGATTCATTACCTGGTCGTTA AC AACCTGCTGATGGCCCTGGGCGAGCCGTTCT ACGCGGGTGTCCCGCT GATGGGCGAAGCGGCACGTCAGGCGTTTGGCCTGGACACCGAGTTCGCT CTGGAACCGTTTAGCGAAAGCACGCTGGCACGTTTTGTTCGTCTGGAAT GGCCGCACTTTATCCCAGCACCGGGCAAATCCATCGCGGACTGCTATGC CGCCATTCGTCAGGCGTTTTTGGATCTGCCGGACTTGTTTGGTGGCGAGG C AGGTAAGCGTGGCGGTGAAC ACCACCTGTTCCTGAATGAGCTGACC AA CCGTGCGCATCCGGGTTATCAACTGGAAGTTTTCGATCGCGACTCGGCG CTGTTTGGTATTGCATTTGTGACCGATCAGGGCGAAGGTGGCGCTCTGG ACAGCCCGCACTACGAACATAGCCATTTTCAACGTCTGCGTGAAATGAG CGCGCGTATCATGGCTCAAAGCGCACCGTTCGAACCGGCGCTGCCGGCG TTGCGTAATCCGGTTCTGGATGAGAGCCCGGGTTGCCAACGTGTCGCAG ACGGTCGTGCGCGTGCGCTGATGGCATTGTACCAAGGCGTTTATGAGCT GATGTTTGCGATGATGGCGCAGCACTTCGCCGTGAAACCGCTGGGTAGC TTGCGTCGCAGCCGCCTGATGAACGCAGCAATCGATCTGATGACCGGTC TGTTGCGTCCGCTGAGCTGCGCGCTGATGAACCTGCCAAGCGGCATCGC CGGTCGCACGGCCGGTCCGCCGCTGCCGGGTCCGGTTGACACCCGTAGC TATGACGACTACGCGCTGGGCTGTCGCATGCTGGCACGCCGTTGCGAGC GTCTGCTGGAGCAGGCGAGCATGCTGGAACCGGGTTGGCTGCCGGATGC GCAGATGGAGCTGCTGGATTTCTATCGTCGCCAAATGCTGGACTTGGCG TGCGGCAAACTGAGCCGCGAGGCCTAAGGATCCTTAAGGAGGTAAAAA AAATGAAACGTGCGATTATCGTTGGTGGCGGCCTGGCGGGTGGCCTGAC CGCGATCTACCTGGCGAAGCGTGGCTACGAAGTGCACGTCGTGGAGAAG CGTGGTGATCCTCTGCGCGATCTGAGCTCTTACGTGGACGTTGTTAGCAG CCGTGCGATCGGCGTGAGCATGACCGTTCGTGGTATCAAGAGCGTTTTG GCTGCGGGCATTCCGCGTGCAGAGCTGGATGCGTGTGGCGAACCGATCG TGGCAATGGCTTTCTCCGTGGGTGGTCAGTATCGCATGCGCGAACTGAA GCCGTTGGAGGATTTCCGTCCGCTGAGCTTGAACCGTGCGGCGTTTCAA AAGCTGCTGAACAAATACGCGAACCTGGCAGGCGTTCGTTACTACTTTG AGCATAAGTGCCTGGATGTTGACCTGGATGGTAAGAGCGTGTTGATTCA GGGCAAAGATGGTCAGCCGCAGCGTCTGCAAGGTGACATGATTATCGGT GCGGATGGCGCCCACAGCGCCGTCCGTCAGGCGATGCAGAGCGGCCTG CGTCGTTTCGAGTTCCAGCAAACGTTCTTCCGCCATGGCTACAAAACCCT GGTTTTGCCGGACGCGCAAGCACTGGGTTACCGTAAAGACACGCTGTAC TTTTTCGGCATGGATTCCGGTGGCCTGTTCGCGGGTCGTGCGGCTACGAT CCCAGATGGTAGCGTCAGCATCGCCGTTTGCCTGCCGTACTCGGGTAGC CCTTCCCTGACGACC ACCGACGAACCGACGATGCGTGCGTTCTTCGATC GTTACTTCGGTGGCCTGCCGCGTGACGCGCGTGACGAAATGCTGCGTCA GTTTCTGGCGAAGCCGAGCAACGACCTGATTAACGTGCGCTCTAGCACC TTTCACTATAAGGGTAATGTGCTGTTGCTGGGTGATGCTGCGCATGCGAC TGCGCCGTTCCTGGGTCAGGGTATGAACATGGCGCTGGAGGACGCCCGC ACGTTTGTCGAGCTGCTGGACCGCC ACC AGGGCGACCAAGAC AAAGCCT TTCCGGAGTTCACGGAGCTGCGCAAAGTCCAGGCAGACGCAATGCAAG ACATGGCTCGCGCCAACTATGACGTTTTGAGCTGCTCGAACCCGATCTTT TTCATGCGTGCGCGTTACACGCGTTACATGCATTCCAAGTTTCCGGGCCT GTATCCGCCGGATATGGCCGAGAAACTGTACTTTACGAGCGAGCCGTAC GATCGTCTGCAACAAATCCAGCGTAAACAGAATGTTTGGTACAAGATTG GTCGCGTGAATTGAAGATCTTTAAGGAGGTAAAAAAAATGAAGATTCTG GTCATTGGTGCTGGTCCAGCTGGTCTGGTTTTCGCATCCCAACTGAAGCA GGCACGCCCTTTGTGGGCCATTGACATCGTGGAGAAGAATGACGAGCAA GAAGTGCTGGGCTGGGGTGTCGTGCTGCCTGGCCGTCCGGGTCAGCACC CGGCGAACCCGCTGTCCTATCTGGATGCACCGGAGCGTCTGAATCCGCA ATTTCTGGAGGACTTCAAACTGGTGCATCATAATGAGCCGTCCTTGATGT CCACGGGCGTTTTGTTGTGCGGCGTGGAGCGTCGCGGTCTGGTTCACGC GCTGCGCGATAAGTGCCGCAGCCAAGGCATTGCTATTCGTTTCGAAAGC CCGTTGCTGGAACACGGTGAGCTGCCGCTGGCGGACTATGATCTGGTGG TCCTGGCTAATGGTGTTAATCACAAAACCGCGCATTTCACCGAGGCTCT GGTCCCGCAGGTGGACTACGGCCGCAATAAGTACATTTGGTATGGCACT AGCCAGCTGTTCGATCAGATGAATCTGGTTTTTCGTACCCATGGTAAAG ATATCTTTATCGCGCATGCCTATAAGTATAGCGATACCATGAGCACGTTC ATTGTCGAATGTAGCGAAGAGACTTACGCACGCGCACGCCTGGGCGAAA TGTCCGAAGAGGCGAGCGCAGAATACGTTGCTAAGGTGTTCCAGGCCGA GCTGGGTGGTCACGGCCTGGTGAGCCAGCCGGGTCTGGGTTGGCGTAAC TTCATGACGTTGTCTCATGACCGTTGTCATGATGGTAAGTTGGTTCTGCT GGGTGACGCGCTGCAAAGCGGTCACTTTAGCATCGGCCACGGCACCACG ATGGCCGTGGTGGTGGCGCAGCTGCTGGTTAAAGCGCTGTGTACCGAAG ATGGTGTGCCTGCCGCGCTGAAACGTTTCGAAGAGCGTGCCCTGCCGCT GGTGCAGTTGTTCCGTGGCCACGCAGACAACAGCCGCGTTTGGTTCGAA ACCGTCGAAGAGCGCATGCACCTGTCCTCGGCGGAATTTGTGCAAAGCT TCGACGCACGCCGCAAAAGCCTGCCGCCGATGCCGGAAGCACTGGCGC AGAATCTGCGTTATGCTTTGCAGCGCTGATGATCATTAAGGAGGTAAAA AAAATGGAGAACCGTGAGCC ACC ACTGTTGCC AGCCCGTTGGAGCAGCG CCTATGTCTCTTATTGGAGCCCGATGCTGCCGGATGACCAGCTGACCAG CGGCTATTGCTGGTTCGACTATGAACGTGACATCTGTCGTATTGACGGCC TGTTCAATCCGTGGAGCGAGCGTGATACTGGTTATCGCCTGTGGATGTC GGAGGTTGGTAATGCGGCCAGCGGCCGTACCTGGAAACAAAAAGTCGC CTATGGTCGTGAGCGTACCGCCCTGGGTGAACAGCTGTGTGAGCGTCCG CTGGATGATGAGACTGGCCCTTTTGCCGAATTGTTCCTGCCACGCGATGT CCTGCGCCGTCTGGGTGCCCGTCACATTGGCCGTCGCGTGGTTCTGGGTC GCGAAGCGGACGGTTGGCGTTACCAGCGCCCAGGTAAAGGTCCGAGCA CCCTGTACCTGGATGCGGCGAGCGGCACTCCACTGCGCATGGTCACCGG CGATGAAGCGTCGCGTGCAAGCCTGCGTGATTTTCCGAATGTGAGCGAG GCGGAGATCCCGGACGCGGTTTTCGCGGCCAAGCGCTAA (SEQ ID NO: 15)

SEQ ID NO: 15 is coding sequences, assembly scars, and ribosomal binding sites for vioABCDE for production of violacein.

TACTAGTAGCGGCCGCTGCAGGAGTCACTAAGGGTTAGTTAGTTAGATT AGCAGAAAGTCAAAAGCCTCCGACCGGAGGCTTTTGACTAAAACTTCCC TTGGGGTTATCATTGGGGCTCACTCAAAGGCGGTAATCAGATAAAAAAA ATCCTTAGCTTTCGCTAAGGATGATTTCTGCTAGAGATGGAATAGACTG GATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCG GCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTC GCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGT AGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAG ACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCA GACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTA ATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAA ATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCTTAATAAG ATGATCTTCTTGAGATCGTTTTGGTCTGCGCGTAATCTCTTGCTCTGAAA ACGAAAAAACCGCCTTGCAGGGCGGTTTTTCGAAGGTTCTCTGAGCTAC CAACTCTTTGAACCGAGGTAACTGGCTTGGAGGAGCGCAGTCACCAAAA CTTGTCCTTTCAGTTTAGCCTTAACCGGCGCATGACTTCAAGACTAACTC CTCTAAATCAATTACCAGTGGCTGCTGCCAGTGGTGCTTTTGCATGTCTT TCCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGG ACTGAACGGGGGGTTCGTGC AT AC AGTCC AGCTTGGAGCGAACTGCCT A CCCGGAACTGAGTGTCAGGCGTGGAATGAGACAAACGCGGCCATAACA GCGGAATGACACCGGTAAACCGAAAGGCAGGAACAGGAGAGCGCACGA GGGAGCCGCCAGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTT CGCCACCACTGATTTGAGCGTCAGATTTCGTGATGCTTGTCAGGGGGGC GGAGCCTATGGAAAAACGGCTTTGCCGCGGCCCTCTC ACTTCCCTGTTA AGTATCTTCCTGGCATCTTCCAGGAAATCTCCGCCCCGTTCGTAAGCCAT TTCCGCTCGCCGCAGTCGAACGACCGAGCGTAGCGAGTCAGTGAGCGAG GAAGCGGAATATATCCTGTATCACATATTCTGCTGACGCACCGGTGCAG CCTTTTTTCTCCTGCCACATGAAGCACTTCACTGACACCCTCATCAGTGC CAACATAGTAAGCCAGTATACACTCCGCTAGCGCTGAGGTCTGCCTCGT GAAGAAGGTGTTGCTGACTCATACCAGGCCTGAATCGCCCCATCATCCA GCCAGAAAGTGAGGGAGCCACGGTTGATGAGAGCTTTGTTGTAGGTGGA CCAGTTGGTGATTTTGAACTTTTGCTTTGCCACGGAACGGTCTGCGTTGT CGGGAAGATGCGTGATCTGATCCTTCAACTCAGCAAAAGTTCGATTTAT TCAACAAAGCCACGTTGTGTCTCAAAATCTCTGATGTTACATTGCACAA GATAAAAATATATCATCATGAACAATAAAACTGTCTGCTTACATAAACA GTAATACAAGGGGTGTTTACTAGAGGAGATTCTCATGTTTGACAGCTTA TCATCGATAAGCTTTAATGCGGTAGTTTATCACAGTTAAATTGCTAACGC AGTCAGGCACCGTGTATGAAATCTAACAATGCGCTCATCGTCATTCTCG GCACCGTCACCCTGGACGCTGTAGGCATAGGCTTGGTTATGCCGGTACT GCCGGGCCTCTTGCGGGATATCGTCCATTCCGACAGTATTGCCAGTCACT ATGGCGTGCTGCTTGCGCTCTATGCGTTGATGCAATTTCTTTGCGCACCC GTTCTCGGAGCCCTGTCCGACCGCTTTGGCCGCCGTCCAGTCCTGCTCGC TTCGCTCCTTGGAGCCACTATCGACTACGCGATCATGGCGACCACACCC GTCCTGTGGATTCTCTACGCCGGACGCATCGTGGCGGGCATCACGGGTG CCACAGGTGCGGTTGCTGGTGCCTATATCGCCGACATCACCGATGGGGA AGATCGGGCTCGCCACTTCGGGCTCATGAGCGCTTGTTTCGGCGTGGGT ATGGTGGCAGGCCCCGTGGCCGGGGGACTGTTGGGTGCCATCTCCTTGC ATGCACCATTCCTTGCGGCGGCGGTGCTCAACGGCCTCAACCTCCTCCTG GGCTGCTTCCTTATGCAGGAATCGCATAAGGGAGAGCGCCGTCCGATGC CCTTGCGTGCCTTCAATCCAGTCAGCTCCTTCCGGTGGGCGCGGGGCAT GACTATCGTCGCCGCACTTATGACTGTTTTCTTTATCATGCAACTCGTAG GACAGGTTCCGGCAGCGCTCTGGGTCATTTTCGGCGAGGACCGCTTTCG CTGGAGCGCGACGATGATCGGCCTGTCGCTTGCGGTATTCGGAATCTTG C ACGCCCTCGCTC AAGCCTTCGTC ACGGGCCCCGCC ACC AAACGTTTCG GCGAGAAGCAGGCCATTATCGCGGGCATGGCGGCCGACGCGCTGGGCT ACGTCTTGCTGGCGTTCGCGACGCGCGGCTGGATGGCCTTCCCCATTATG ATTCTTCTCGCTTCCGGCGGCATCGGTATGCCCGCGTTGCAGGCCATGCT GTCCCGCCAAGTAGATGACGACCATCAGGGACAGCTTCAAGGGTCGCTC GCGGCTCTTACC AGCCTC ACTTCGATC ATTGGACCGCTGATCGTCACGGC GATTTATGCCGCCTCGGCGAGCACATGGAACGGGTTGGCATGGATTGTA GGTGCCGCCCTTTACCTTGTCTGCCTCCCCGCGTTGCGTCGCGGTGCATG GAGCCGGGCCACCTCGACCTAATAATACTAGCTCCGGCAAAAAAACGG GCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTT CGCGTTTGCCACCTGACGTCTAAGAAAAGGAATATTCAGCAATTTGCCC GTGCCGAAGAAAGGCCCACCCGTGAAGGTGAGCCAGTGAGTTGATTGCT ACGTAATTAGTTAGTTAGCCCTTAGTGACTCGAATTCGCGGCCGCTTCTA GAG (SEQ ID NO: 16) SEQ ID NO: 16 is the sequence for pSB3T5, a medium copy vector. The copy number of the vector is important for proper operation of the circuit.

TACTAGTAGCGGCCGCTGCAGGAGTCACTAAGGGTTAGTTAGTTAGATT AGCAGAAAGTCAAAAGCCTCCGACCGGAGGCTTTTGACTAAAACTTCCC TTGGGGTTATCATTGGGGCTCACTCAAAGGCGGTAATCAGATAAAAAAA ATCCTTAGCTTTCGCTAAGGATGATTTCTGCTAGAGATGGAATAGACTG GATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCG GCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTC GCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGT AGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAG ACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCA GACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTA ATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAA ATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCTTAATAAG ATGATCTTCTTGAGATCGTTTTGGTCTGCGCGTAATCTCTTGCTCTGAAA ACGAAAAAACCGCCTTGCAGGGCGGTTTTTCGAAGGTTCTCTGAGCTAC CAACTCTTTGAACCGAGGTAACTGGCTTGGAGGAGCGCAGTCACCAAAA CTTGTCCTTTCAGTTTAGCCTTAACCGGCGCATGACTTCAAGACTAACTC CTCTAAATCAATTACCAGTGGCTGCTGCCAGTGGTGCTTTTGCATGTCTT TCCGGGTTGGACTC AAGACGATAGTTACCGGATAAGGCGCAGCGGTCGG ACTGAACGGGGGGTTCGTGCATACAGTCCAGCTTGGAGCGAACTGCCTA CCCGGAACTGAGTGTCAGGCGTGGAATGAGACAAACGCGGCCATAACA GCGGAATGACACCGGTAAACCGAAAGGCAGGAACAGGAGAGCGCACG AGGGAGCCGCCAGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTT TCGCC ACC ACTGATTTGAGCGTC AGATTTCGTGATGCTTGTC AGGGGGG CGGAGCCTATGGAAAAACGGCTTTGCCGCGGCCCTCTCACTTCCCTGTT AAGTATCTTCCTGGCATCTTCCAGGAAATCTCCGCCCCGTTCGTAAGCCA TTTCCGCTCGCCGCAGTCGAACGACCGAGCGTAGCGAGTCAGTGAGCGA GGAAGCGGAATATATCCTGTATCACATATTCTGCTGACGCACCGGTGCA GCCTTTTTTCTCCTGCCACATGAAGCACTTCACTGACACCCTCATCAGTG CCAACATAGTAAGCCAGTATACACTCCGCTAGCGCTGAGGTCTGCCTCG TGAAGAAGGTGTTGCTGACTCATACCAGGCCTGAATCGCCCCATCATCC AGCCAGAAAGTGAGGGAGCCACGGTTGATGAGAGCTTTGTTGTAGGTGG ACCAGTTGGTGATTTTGAACTTTTGCTTTGCCACGGAACGGTCTGCGTTG TCGGGAAGATGCGTGATCTGATCCTTCAACTCAGCAAAAGTTCGATTTA TTCAACAAAGCCACGTTGTGTCTCAAAATCTCTGATGTTACATTGCACAA GATAAAAATATATCATCATGAACAATAAAACTGTCTGCTTACATAAACA GTAATACAAGGGGTGTTTACTAGAGGAGATTCTCATGTTTGACAGCTTA TCATCGATAAGCTTTAATGCGGTAGTTTATCACAGTTAAATTGCTAACGC AGTCAGGCACCGTGTATGAAATCTAACAATGCGCTCATCGTCATTCTCG GCACCGTCACCCTGGACGCTGTAGGCATAGGCTTGGTTATGCCGGTACT GCCGGGCCTCTTGCGGGATATCGTCCATTCCGACAGTATTGCCAGTCACT ATGGCGTGCTGCTTGCGCTCTATGCGTTGATGCAATTTCTTTGCGCACCC GTTCTCGGAGCCCTGTCCGACCGCTTTGGCCGCCGTCCAGTCCTGCTCGC TTCGCTCCTTGGAGCCACTATCGACTACGCGATCATGGCGACCACACCC GTCCTGTGGATTCTCTACGCCGGACGCATCGTGGCGGGCATCACGGGTG CCACAGGTGCGGTTGCTGGTGCCTATATCGCCGACATCACCGATGGGGA AGATCGGGCTCGCCACTTCGGGCTCATGAGCGCTTGTTTCGGCGTGGGT ATGGTGGCAGGCCCCGTGGCCGGGGGACTGTTGGGTGCCATCTCCTTGC ATGCACCATTCCTTGCGGCGGCGGTGCTCAACGGCCTCAACCTCCTCCTG GGCTGCTTCCTTATGCAGGAATCGCATAAGGGAGAGCGCCGTCCGATGC CCTTGCGTGCCTTCAATCCAGTCAGCTCCTTCCGGTGGGCGCGGGGCAT GACTATCGTCGCCGCACTTATGACTGTTTTCTTTATCATGCAACTCGTAG GACAGGTTCCGGCAGCGCTCTGGGTCATTTTCGGCGAGGACCGCTTTCG CTGGAGCGCGACGATGATCGGCCTGTCGCTTGCGGTATTCGGAATCTTG CACGCCCTCGCTCAAGCCTTCGTCACGGGCCCCGCCACCAAACGTTTCG GCGAGAAGCAGGCCATTATCGCGGGCATGGCGGCCGACGCGCTGGGCT ACGTCTTGCTGGCGTTCGCGACGCGCGGCTGGATGGCCTTCCCCATTATG ATTCTTCTCGCTTCCGGCGGCATCGGTATGCCCGCGTTGCAGGCCATGCT GTCCCGCC AAGTAGATGACGACC ATC AGGGAC AGCTTC AAGGGTCGCTC GCGGCTCTTACCAGCCTCACTTCGATCATTGGACCGCTGATCGTCACGGC GATTTATGCCGCCTCGGCGAGCACATGGAACGGGTTGGCATGGATTGTA GGTGCCGCCCTTTACCTTGTCTGCCTCCCCGCGTTGCGTCGCGGTGCATG GAGCCGGGCCACCTCGACCTAATAATACTAGCTCCGGCAAAAAAACGG GCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTT CGCGTTTGCCACCTGACGTCTAAGAAAAGGAATATTCAGCAATTTGCCC GTGCCGAAGAAAGGCCCACCCGTGAAGGTGAGCCAGTGAGTTGATTGCT ACGTAATTAGTTAGTTAGCCCTTAGTGACTCGAATTCGCGGCCGCTTCTA GAGTAACACCGTGCGTGTTGACTATTTTACCTCTGGCGGTGATAATGGTT GCTACTAGTCACACAGGACTACTAGATGAAACCAGTAACGTTATACGAT GTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGA ACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGG CGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGC GGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTG CACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAAC TGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAG CCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCT GATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCT GCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACC CATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTG GAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCC CATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATAT CTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGG AGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCA TCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGC AATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCG GTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGT TAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGA CCGCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTG TTGCCCGTCTCACTGGTGAAAAGAAAAACC ACCCTGGCGCCC AATACGC AAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACG ACAGGTTTCCCGATACTAGCCAGGCATCAAATAAAACGAAAGGCTCAGT CGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTC TACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATAT ACTAGAATTGTGAGCGGAT AAC AACTGATGGTGTGAGCGGAT AACAAG ATTATGAGCACATACTAGTCACACAGGACTACTAGATGAACACGATTAA CATCGCTAAGAACGACTTCTCTGACATCGAACTGGCTGCTATCCCGTTCA ACACTCTGGCTGACCATTACGGTGAGCGTTTAGCTCGCGAACAGTTGGC CCTTGAGCATGAGTCTTACGAGATGGGTGAAGCACGCTTCCGCAAGATG TTTGAGCGTCAACTTAAAGCTGGTGAGGTTGCGGATAACGCTGCCGCCA AGCCTCTCATCACTACCCTACTCCCTAAGATGATTGCACGCATCAACGA CTGGTTTGAGGAAGTGAAAGCTAAGCGCGGCAAGCGCCCGACAGCCTTC CAGTTCCTGCAAGAAATCAAGCCGGAAGCCGTAGCGTACATCACCATTA AGACCACTCTGGCTTGCCTAACCAGTGCTGACAATACAACCGTTCAGGC TGTAGCAAGCGCAATCGGTCGGGCCATTGAGGACGAGGCTCGCTTCGGT CGTATCCGTGACCTTGAAGCTAAGCACTTCAAGAAAAACGTTGAGGAAC AACTCAACAAGCGCGTAGGGCACGTCTACAAGAAAGCATTTATGCAAGT TGTCGAGGCTGACATGCTCTCTAAGGGTCTACTCGGTGGCGAGGCGTGG TCTTCGTGGCATAAGGAAGACTCTATTCATGTAGGAGTACGCTGCATCG AGATGCTCATTGAGTCAACCGGAATGGTTAGCTTACACCGCCAAAATGC TGGCGTAGTAGGTCAAGACTCTGAGACTATCGAACTCGCACCTGAATAC GCTGAGGCTATCGCAACCCGTGCAGGTGCGCTGGCTGGCATCTCTCCGA TGTTCCAACCTTGCGTAGTTCCTCCTAAGCCGTGGACTGGCATTACTGGT GGTGGCTATTGGGCTAACGGTCGTCGTCCTCTGGCGCTGGTGCGTACTC ACAGTAAGAAAGCACTGATGCGCTACGAAGACGTTTACATGCCTGAGGT GTACAAAGCGATTAACATTGCGCAAAACACCGCATGGAAAATCAACAA GAAAGTCCTAGCGGTCGCCAACGTAATCACCAAGTGGAAGCATTGTCCG GTCGAGGACATCCCTGCGATTGAGCGTGAAGAACTCCCGATGAAACCGG AAGACATCGACATGAATCCTGAGGCTCTCACCGCGTGGAAACGTGCTGC CGCTGCTGTGTACCGCAAGGACAAGGCTCGCAAGTCTCGCCGTATCAGC CTTGAGTTCATGCTTGAGCAAGCCAATAAGTTTGCTAACCATAAGGCCA TCTGGTTCCCTTACAACATGGACTGGCGCGGTCGTGTTTACGCTGTGTCA ATGTTCAACCCGCAAGGTAACGATATGACCAAAGGACTGCTTACGCTGG CGAAAGGTAAACCAATCGGTAAGGAAGGTTACTACTGGCTGAAAATCC ACGGTGC AAACTGTGCGGGTGTCGAT AAGGTTCCGTTCCCTGAGCGC AT CAAGTTCATTGAGGAAAACCACGAGAACATCATGGCTTGCGCTAAGTCT CCACTGGAGAACACTTGGTGGGCTGAGCAAGATTCTCCGTTCTGCTTCCT TGCGTTCTGCTTTGAGTACGCTGGGGTACAGCACCACGGCCTGAGCTAT AACTGCTCCCTTCCGCTGGCGTTTGACGGGTCTTGCTCTGGCATCCAGCA CTTCTCCGCGATGCTCCGAGATGAGGT AGGTGGTCGCGCGGTT AACTTG CTTCCTAGTGAAACCGTTCAGGACATCTACGGGATTGTTGCTAAGAAAG TCAACGAGATTCTACAAGCAGACGCAATCAATGGGACCGATAACGAAG TAGTTACCGTGACCGATGAGAACACTGGTGAAATCTCTGAGAAAGTCAA GCTGGGCACTAAGGCACTGGCTGGTCAATGGCTGGCTTACGGTGTTACT CGCAGTGTGACTAAGCGTTCAGTCATGACGCTGGCTTACGGGTCCAAAG AGTTCGGCTTCCGTCAACAAGTGCTGGAAGATACCATTCAGCCAGCTAT TGATTCCGGCAAGGGTCTGATGTTCACTCAGCCGAATCAGGCTGCTGGA TACATGGCTAAGCTGATTTGGGAATCTGTGAGCGTGACGGTGGTAGCTG CGGTTGAAGCAATGAACTGGCTTAAGTCTGCTGCTAAGCTGCTGGCTGC TGAGGTCAAAGATAAGAAGACTGGAGAGATTCTTCGCAAGCGTTGCGCT GTGCATTGGGTAACTCCTGATGGTTTCCCTGTGTGGCAGGAATACAAGA AGCCTATTCAGACGCGCTTGAACCTGATGTTCCTCGGTCAGTTCCGCTTA CAGCCTACCATTAACACCAACAAAGATAGCGAGATTGATGCACACAAA CAGGAGTCTGGTATCGCTCCTAACTTTGTACACAGCCAAGACGGTAGCC ACCTTCGTAAGACTGTAGTGTGGGCACACGAGAAGTACGGAATCGAATC TTTTGCACTGATTCACGACTCCTTCGGTACCATTCCGGCTGACGCTGCGA ACCTGTTCAAAGCAGTGCGCGAAACTATGGTTGACACATATGAGTCTTG TGATGTACTGGCTGATTTCTACGACCAGTTCGCTGACCAGTTGCACGAGT CTCAATTGGACAAAATGCCAGCACTTCCGGCTAAAGGTAACTTGAACCT CCGTGACATCTTAGAGTCGGACTTCGCGTTCGCGTAATAATACTAGCCA GGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTT TATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCAC CTTCGGGTGGGCCTTTCTGCGTTTATATACTAGTAATACGACTCACTATA GGAATTGTGAGCGGATAACAAGATTATAATTGTGAGCGCTCACAATTTA CTAGTCACACAGGAAAGTACTAGATGACGGTCTGCGCAAAAAAACACG TTCATCTCACTCGCGATGCTGCGGAGCAGTTACTGGCTGATATTGATCGA CGCCTTGATCAGTTATTGCCCGTGGAGGGAGAACGGGATGTTGTGGGTG CCGCGATGCGTGAAGGTGCGCTGGCACCGGGAAAACGTATTCGCCCCAT GTTGCTGTTGCTGACCGCCCGCGATCTGGGTTGCGCTGTCAGCCATGAC GGATT ACTGGATTTGGCCTGTGCGGTGGAAATGGTCC ACGCGGCTTCGC TGATCCTTGACGATATGCCCTGCATGGACGATGCGAAGCTGCGGCGCGG ACGCCCTACCATTCATTCTCATTACGGAGAGCATGTGGCAATACTGGCG GCGGTTGCCTTGCTGAGTAAAGCCTTTGGCGTAATTGCCGATGCAGATG GCCTCACGCCGCTGGCAAAAAATCGGGCGGTTTCTGAACTGTCAAACGC C ATCGGC ATGC AAGGATTGGTTCAGGGTC AGTTCAAGGATCTGTCTGAA GGGGATAAGCCGCGCAGCGCTGAAGCTATTTTGATGACGAATCACTTTA AAACCAGCACGCTGTTTTGTGCCTCCATGCAGATGGCCTCGATTGTTGCG AATGCCTCCAGCGAAGCGCGTGATTGCCTGCATCGTTTTTCACTTGATCT TGGTCAGGCATTTCAACTGCTGGACGATTTGACCGATGGCATGACCGAC ACCGGTAAGGATAGCAATCAGGACGCCGGTAAATCGACGCTGGTCAAT CTGTTAGGCCCGAGGGCGGTTGAAGAACGTCTGAGACAACATCTTCAGC TTGCCAGTGAGCATCTCTCTGCGGCCTGCCAACACGGGCACGCCACTCA ACATTTTATTCAGGCCTGGTTTGACAAAAAACTCGCTGCCGTCAGTTAAT ACTAGTCACACAGGAAAGTACTAGATGAATAATCCGTCGTTACTCAATC ATGCGGTCGAAACGATGGCAGTTGGCTCGAAAAGTTTTGCGACAGCCTC AAAGTTATTTGATGCAAAAACCCGGCGCAGCGTACTGATGCTCTACGCC TGGTGCCGCCATTGTGACGATGTTATTGACGATCAGACGCTGGGCTTTCA GGCCCGGCAGCCTGCCTTACAAACGCCCGAACAACGTCTGATGCAACTT GAGATGAAAACGCGCCAGGCCTATGCAGGATCGCAGATGCACGAACCG GCGTTTGCGGCTTTTCAGGAAGTGGCTATGGCTCATGATATCGCCCCGG CTTACGCGTTTGATCATCTGGAAGGCTTCGCCATGGATGTACGCGAAGC GCAATACAGCCAACTGGATGATACGCTGCGCTATTGCTATCACGTTGCA GGCGTTGTCGGCTTGATGATGGCGCAAATCATGGGCGTGCGGGATAACG CCACGCTGGACCGCGCCTGTGACCTTGGGCTGGCATTTCAGTTGACCAA TATTGCTCGCGATATTGTGGACGATGCGCATGCGGGCCGCTGTTATCTGC CGGCAAGCTGGCTGGAGCATGAAGGTCTGAACAAAGAGAATTATGCGG CACCTGAAAACCGTCAGGCGCTGAGCCGTATCGCCCGTCGTTTGGTGCA GGAAGCAGAACCTTACTATTTGTCTGCCACAGCCGGCCTGGCAGGGTTG CCCCTGCGTTCCGCCTGGGCAATCGCTACGGCGAAGCAGGTTTACCGGA AAATAGGTGTCAAAGTTGAACAGGCCGGTCAGCAAGCCTGGGATCAGC GGCAGTCAACGACCACGCCCGAAAAATTAACGCTGCTGCTGGCCGCCTC TGGTCAGGCCCTTACTTCCCGGATGCGGGCTCATCCTCCCCGCCCTGCGC ATCTCTGGCAGCGCCCGCTCTAATACTAGTCACACAGGAAAGTACTAGA TGAAACCAACTACGGTAATTGGTGCAGGCTTCGGTGGCCTGGCACTGGC AATTCGTCTAC AAGCTGCGGGGATCCCCGTCTTACTGCTTGAAC AACGT GATAAACCCGGCGGTCGGGCTTATGTCTACGAGGATCAGGGGTTTACCT TTGATGCAGGCCCGACGGTTATCACCGATCCCAGTGCCATTGAAGAACT GTTTGCACTGGCAGGAAAACAGTTAAAAGAGTATGTCGAACTGCTGCCG GTTACGCCGTTTTACCGCCTGTGTTGGGAGTCAGGGAAGGTCTTTAATTA CGATAACGATCAAACCCGGCTCGAAGCGCAGATTC AGC AGTTT AATCCC CGCGATGTCGAAGGTTATCGTCAGTTTCTGGACTATTCACGCGCGGTGTT TAAAGAAGGCTATCTAAAGCTCGGTACTGTCCCTTTTTTATCGTTCAGAG ACATGCTTCGCGCCGCACCTCAACTGGCGAAACTGCAAGCATGGAGAAG CGTTTACAGTAAGGTTGCCAGTTACATCGAAGATGAACATCTGCGCCAG GCGTTTTCTTTCCACTCGCTGTTGGTGGGCGGCAATCCCTTCGCCACCTC ATCCATTTATACGTTGATACACGCGCTGGAGCGTGAGTGGGGCGTCTGG TTTCCGCGTGGCGGCACCGGCGCATTAGTTCAGGGGATGATAAAGCTGT TTCAGGATCTGGGTGGCGAAGTCGTGTTAAACGCCAGAGTCAGCCATAT GGAAACGACAGGAAACAAGATTGAAGCCGTGCATTTAGAGGACGGTCG CAGGTTCCTGACGCAAGCCGTCGCGTCAAATGCAGATGTGGTTCATACC TATCGCGACCTGTTAAGCCAGCACCCTGCCGCGGTTAAGCAGTCCAACA AACTGCAAACTAAGCGCATGAGTAACTCTCTGTTTGTGCTCTATTTTGGT TTGAATCACCATCATGATCAGCTCGCGCATCACACGGTTTGTTTCGGCCC GCGTTACCGCGAGCTGATTGACGAAATTTTTAATCATGATGGCCTCGCA GAGGACTTCTCACTTTATCTGCACGCGCCCTGTGTCACGGATTCGTCACT GGCGCCTGAAGGTTGCGGCAGTTACTATGTGTTGGCGCCGGTGCCGCAT TTAGGCACCGCGAACCTCGACTGGACGGTTGAGGGGCCAAAACTACGC GACCGTATTTTTGCGTACCTTGAGCAGCATTACATGCCTGGCTTACGGAG TCAGCTGGTCACGCACCGGATGTTTACGCCGTTTGATTTTCGCGACCAGC TTAATGCCTATCATGGCTCAGCCTTTTCTGTGGAGCCCGTTCTTACCCAG AGCGCCTGGTTTCGGCCGCATAACCGCGATAAAACCATTACTAATCTCT ACCTGGTCGGCGCAGGCACGCATCCCGGCGCAGGCATTCCTGGCGTCAT CGGCTCGGCAAAAGCGACAGCAGGTTTGATGCTGGAGGATCTGATATAA TAA (SEQ ID NO: 17) SEQ ID NO: 17 is an engineered T7 system to control lycopene expression (Figure 6). The engineered T7 system can be used in combination with the above constructs to repress pigment production during preculture to high cell density, and then released to allow pigment production in a few hours. CC AGGC ATC AAATAAAACGAAAGGCTC AGTCGAAAGACTGGGCC TTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTC ACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATATACTAGA GCTGATGGCTAGCTCAGTCCTAGGGATTATGCTAGCTACTAGAGG AGGTACTAGATGACGGTCTGCGCAAAAAAACACGTTCATCTCACT CGCGATGCTGCGGAGCAGTTACTGGCTGATATTGATCGACGCCTT GATCAGTTATTGCCCGTGGAGGGAGAACGGGATGTTGTGGGTGCC GCGATGCGTGAAGGTGCGCTGGCACCGGGAAAACGTATTCGCCCC ATGTTGCTGTTGCTGACCGCCCGCGATCTGGGTTGCGCTGTCAGCC ATGACGGATTACTGGATTTGGCCTGTGCGGTGGAAATGGTCCACG CGGCTTCGCTGATCCTTGACGATATGCCCTGCATGGACGATGCGA AGCTGCGGCGCGGACGCCCTACCATTCATTCTCATTACGGAGAGC ATGTGGCAATACTGGCGGCGGTTGCCTTGCTGAGTAAAGCCTTTG GCGTAATTGCCGATGCAGATGGCCTCACGCCGCTGGCAAAAAATC GGGCGGTTTCTGAACTGTCAAACGCCATCGGCATGCAAGGATTGG TTCAGGGTCAGTTCAAGGATCTGTCTGAAGGGGATAAGCCGCGCA GCGCTGAAGCTATTTTGATGACGAATCACTTTAAAACCAGCACGC TGTTTTGTGCCTCCATGCAGATGGCCTCGATTGTTGCGAATGCCTC CAGCGAAGCGCGTGATTGCCTGCATCGTTTTTCACTTGATCTTGGT CAGGCATTTCAACTGCTGGACGATTTGACCGATGGCATGACCGAC ACCGGTAAGGATAGCAATCAGGACGCCGGTAAATCGACGCTGGT CAATCTGTTAGGCCCGAGGGCGGTTGAAGAACGTCTGAGACAAC ATCTTCAGCTTGCCAGTGAGCATCTCTCTGCGGCCTGCCAACACG GGCACGCCACTCAACATTTTATTCAGGCCTGGTTTGACAAAAAAC TCGCTGCCGTCAGTTAATAATACTAGAGCTCAAGGAGGTACTAGA TGAATAATCCGTCGTTACTCAATCATGCGGTCGAAACGATGGCAG TTGGCTCGAAAAGTTTTGCGACAGCCTCAAAGTTATTTGATGCAA AAACCCGGCGCAGCGTACTGATGCTCTACGCCTGGTGCCGCCATT GTGACGATGTTATTGACGATCAGACGCTGGGCTTTCAGGCCCGGC AGCCTGCCTTACAAACGCCCGAACAACGTCTGATGCAACTTGAGA TGAAAACGCGCCAGGCCTATGCAGGATCGCAGATGCACGAACCG GCGTTTGCGGCTTTTCAGGAAGTGGCTATGGCTCATGATATCGCC CCGGCTTACGCGTTTGATCATCTGGAAGGCTTCGCCATGGATGTA CGCGAAGCGCAATACAGCCAACTGGATGATACGCTGCGCTATTGC TATCACGTTGCAGGCGTTGTCGGCTTGATGATGGCGCAAATCATG GGCGTGCGGGATAACGCCACGCTGGACCGCGCCTGTGACCTTGGG CTGGCATTTCAGTTGACCAATATTGCTCGCGATATTGTGGACGAT GCGC ATGCGGGCCGCTGTTATCTGCCGGCAAGCTGGCTGGAGCAT GAAGGTCTGAACAAAGAGAATTATGCGGCACCTGAAAACCGTCA GGCGCTGAGCCGTATCGCCCGTCGTTTGGTGCAGGAAGCAGAACC TTACTATTTGTCTGCCACAGCCGGCCTGGCAGGGTTGCCCCTGCGT TCCGCCTGGGCAATCGCTACGGCGAAGCAGGTTTACCGGAAAATA GGTGTCAAAGTTGAACAGGCCGGTCAGCAAGCCTGGGATCAGCG GCAGTCAACGACCACGCCCGAAAAATTAACGCTGCTGCTGGCCGC CTCTGGTCAGGCCCTTACTTCCCGGATGCGGGCTCATCCTCCCCGC CCTGCGCATCTCTGGCAGCGCCCGCTCTAATAATACTAGAGCTCA AGGAGGTACTAGATGAAACCAACTACGGTAATTGGTGCAGGCTTC GGTGGCCTGGCACTGGCAATTCGTCTACAAGCTGCGGGGATCCCC GTCTTACTGCTTGAACAACGTGATAAACCCGGCGGTCGGGCTTAT GTCTACGAGGATCAGGGGTTTACCTTTGATGCAGGCCCGACGGTT ATCACCGATCCCAGTGCCATTGAAGAACTGTTTGCACTGGCAGGA AAACAGTTAAAAGAGTATGTCGAACTGCTGCCGGTTACGCCGTTT TACCGCCTGTGTTGGGAGTCAGGGAAGGTCTTTAATTACGATAAC GATCAAACCCGGCTCGAAGCGCAGATTCAGCAGTTTAATCCCCGC GATGTCGAAGGTTATCGTCAGTTTCTGGACTATTCACGCGCGGTG TTTAAAGAAGGCTATCTAAAGCTCGGTACTGTCCCTTTTTTATCGT TCAGAGACATGCTTCGCGCCGCACCTCAACTGGCGAAACTGCAAG CATGGAGAAGCGTTTACAGTAAGGTTGCCAGTTACATCGAAGATG AACATCTGCGCCAGGCGTTTTCTTTCCACTCGCTGTTGGTGGGCGG CAATCCCTTCGCCACCTCATCCATTTATACGTTGATACACGCGCTG GAGCGTGAGTGGGGCGTCTGGTTTCCGCGTGGCGGCACCGGCGCA TTAGTTCAGGGGATGATAAAGCTGTTTCAGGATCTGGGTGGCGAA GTCGTGTTAAACGCCAGAGTCAGCCATATGGAAACGACAGGAAA CAAGATTGAAGCCGTGCATTTAGAGGACGGTCGCAGGTTCCTGAC GCAAGCCGTCGCGTCAAATGCAGATGTGGTTCATACCTATCGCGA CCTGTTAAGCCAGCACCCTGCCGCGGTTAAGCAGTCCAACAAACT GCAAACTAAGCGCATGAGTAACTCTCTGTTTGTGCTCTATTTTGGT TTGAATCACCATCATGATCAGCTCGCGCATCACACGGTTTGTTTCG GCCCGCGTTACCGCGAGCTGATTGACGAAATTTTTAATCATGATG GCCTCGCAGAGGACTTCTCACTTTATCTGCACGCGCCCTGTGTCAC GGATTCGTCACTGGCGCCTGAAGGTTGCGGCAGTTACTATGTGTT GGCGCCGGTGCCGCATTTAGGCACCGCGAACCTCGACTGGACGGT TGAGGGGCCAAAACTACGCGACCGTATTTTTGCGTACCTTGAGCA GCATTACATGCCTGGCTTACGGAGTCAGCTGGTCACGCACCGGAT GTTTACGCCGTTTGATTTTCGCGACCAGCTTAATGCCTATCATGGC TCAGCCTTTTCTGTGGAGCCCGTTCTTACCCAGAGCGCCTGGTTTC GGCCGCATAACCGCGATAAAACCATTACTAATCTCTACCTGGTCG GCGCAGGCACGCATCCCGGCGCAGGCATTCCTGGCGTCATCGGCT CGGCAAAAGCGACAGCAGGTTTGATGCTGGAGGATCTGATATAAT AATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAA AGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTC TACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTT ATATACTAGAGTTGACAGCTAGCTCAGTCCTAGGGATTGTGCTAG CTACTAGAGAAAGAGGAGAAATACTAGATGTATCGCATTGGTGA GCTGGCAAAAATGGCGGAAGTAACACCCGACACGATTCGTTATTA CGAAAAACAGCAGATGATGGAGCATGAAGTGCGTACTGAAGGTG GGTTTC GC CT AT AT AC CGAAAGC GATCTCC AGCGATTGAAATTT A TCCGCCATGCCAGACAACTAGGTTTCAGTCTGGAGTCGATCCGCG AGTTGCTGTCGATCCGCATCGATCCTGAACACCATACCTGTCAGG AGTCAAAAGGCATTGTGCAGGAAAGATTGCAGGAAGTCGAAGCA CGGATAGCCGAGTTGCAGAGTATGCAGCGTTCCTTGCAACGCCTT AACGATGCCTGTTGTGGGACTGCTCATAGCAGTGTTTATTGTTCGA TTCTTGAAGCTCTTGAACAAGGGGCGAGTGGCGTTAAGAGTGGTT GTTGATACTAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGA AAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCT CTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTT TATATACTAGAGTTGAC AGCTAGCTC AGTCCTAGGGATTGTGCTA GCTACTAGAGTCACACAGGAAACCTACTAGATGGAAAAGACCAC AACGCAGGAGTTATTAGCGCAGGCTGAAAAAATCTGCGCGCAGC GTAATGTGCGCCTGACCCCACAGCGCCTGGAAGTGTTGCGCCTGA TGAGTCTCCAAGATGGCGCTATCAGCGCTTATGATCTGCTTGATTT ACTGCGCGAAGCTGAACCGCAAGCC AAGCCGCC AACGGTTTATCG CGCGCTGGATTTTCTGCTTGAGCAAGGTTTTGTGCATAAGGTGGA ATCCACCAACAGTTATGTGCTCTGTCATCTGTTCGATCAGCCCACC CAT AC GTC AGCC ATGTTT ATTTGC GATC GCTGCGGC GC AGTGAAA GAAGAGTGTGCAGAAGGCGTGGAAGACATTATGCATACGCTGGC GGCAAAAATGGGGTTTGCCCTGCGGCATAATGTGATTGAAGCACA TGGGCTCTGTGCGGCATGTGTAGAAGTGGAAGCGTGTCGTCATCC TGAACAGTGCCAGCATGATCACTCTGTGCAGGTGAAAAAGAAAC CGCGTTAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCA GTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAAC GCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCT GCGTTTATATACTAGAGCTGTATCTCTGATAAAACTTGACTCTGGA GTCGACTCCAGAGTGTATCCTTCGGTTAATTACTAGAGTCACACA GGACTACTAGATGCAACCGCATTATGATCTGATTCTCGTGGGGGC TGGACTCGCGAATGGCCTTATCGCCCTGCGTCTTCAGCAGCAGCA ACCTGATATGCGTATTTTGCTTATCGACGCCGCACCCCAGGCGGG CGGGAATCATACGTGGTCATTTCACCACGATGATTTGACTGAGAG CCAACATCGTTGGATAGCTCCGCTGGTGGTTCATCACTGGCCCGA CTATCAGGTACGCTTTCCCACACGCCGTCGTAAGCTGAACAGCGG CTACTTTTGTATTACTTCTCAGCGTTTCGCTGAGGTTTTACAGCGA CAGTTTGGCCCGCACTTGTGGATGGATACCGCGGTCGCAGAGGTT AATGCGGAATCTGTTCGGTTGAAAAAGGGTCAGGTTATCGGTGCC CGCGCGGTGATTGACGGGCGGGGTTATGCGGCAAATTCAGCACTG AGCGTGGGCTTCCAGGCGTTTATTGGCCAGGAATGGCGATTGAGC CACCCGCATGGTTTATCGTCTCCCATTATCATGGATGCCACGGTCG ATCAGCAAAATGGTTATCGCTTCGTGTACAGCCTGCCGCTCTCGC CGACCAGATTGTTAATTGAAGACACGCACTATATTGATAATGCGA C ATT AGATC CTGAATGC GCGC GGC AAAATATTTGC GACT ATGC CG CGCAACAGGGTTGGCAGCTTCAGACACTGCTGCGAGAAGAACAG GGCGCCTTACCCATTACTCTGTCGGGCAATGCCGACGCATTCTGG CAGCAGCGCCCCCTGGCCTGTAGTGGATTACGTGCCGGTCTGTTC CATCCTACCACCGGCTATTCACTGCCGCTGGCGGTTGCCGTGGCC GACCGCCTGAGTGCACTTGATGTCTTTACGTCGGCCTCAATTC ACC ATGCCATTACGCATTTTGCCCGCGAGCGCTGGCAGCAGCAGGGCT TTTTCCGCATGCTGAATCGCATGCTGTTTTTAGCCGGACCCGCCGA TTCACGCTGGCGGGTTATGCAGCGTTTTTATGGTTTACCTGAAGAT TTAATTGCCCGTTTTTATGCGGGAAAACTCACGCTGACCGATCGG CTACGTATTCTGAGCGGCAAGCCGCCTGTTCCGGTATTAGCAGCA TTGCAAGCCATTATGACGACTCATCGTGCTGCTAACGACGAAAAC TACGCTCTGGCTGCTTAATAATACTAGAGCCAGGCATCAAATAAA ACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTG TTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCG GGTGGGCCTTTCTGCGTTTATATACTAGAGAACATAATGCGACCA ATAATCGTAATGAATATGAGAAGTGTGATATTATAACATTTTACT AGAGTTAAGGAGGTAAAAAAAATGAAACATTCTTCCGATATCTGC ATTGTTGGTGCTGGTATTTCTGGTTTGACGTGCGCAAGCCATCTGC TGGACAGCCCGGCATGCCGTGGTCTGAGCCTGCGTATCTTTGACA TGCAGCAAGAAGCCGGTGGCCGTATCCGCAGCAAAATGCTGGAT GGTAAGGCAAGCATTGAACTGGGCGCAGGTCGCTACTCCCCTCAG TTGCACCCGCATTTCCAAAGCGCAATGCAGCACTATAGCCAAAAG AGCGAAGTCTATCCGTTCACCCAGTTGAAGTTCAAATCTCACGTG CAGCAAAAGCTGAAGCGCGCCATGAATGAACTGTCCCCGCGTCTG AAAGAGCATGGTAAAGAGAGCTTTTTGCAGTTTGTCAGCCGTTAT CAAGGTCACGATAGCGCGGTTGGTATGATCCGCTCTATGGGTTAC GAC GC ACTGTTC CTGCC GGATATC AGC GC AGAAATGGCCT ACGAC ATTGTGGGTAAGCACCCGGAGATCCAGAGCGTGACGGACAACGA CGCGAACCAATGGTTTGCAGCGGAAACGGGCTTTGCTGGTCTGAT TCAGGGCATCAAGGCTAAGGTTAAGGCGGCAGGTGCGCGTTTTAG CCTGGGTTATCGTCTGCTGAGCGTCCGTACCGACGGTGACGGCTA CCTGCTGC AACTGGC AGGTGACGACGGCTGGAAACTGGAGC ACC GTACCCGCCATCTGATTCTGGCGATTCCGCCGAGCGCGATGGCGG GTTTGAATGTTGATTTTCCAGAAGCCTGGTCCGGTGCGCGCTATG GCAGCCTGCCGCTGTTTAAGGGCTTTCTGACGTACGGTGAGCCGT GGTGGTTGGACTACAAACTGGACGATCAGGTGCTGATTGTTGACA ACCCGCTGCGCAAAATCTATTTCAAAGGCGATAAGTACCTGTTCT TCTATACCGATAGCGAGATGGCGAATTACTGGCGCGGTTGTGTCG CGGAGGGC GAGGAC GGTTAC CTGGAGC AAATTC GC ACC C ATTTGG CTAGCGCACTGGGTATCGTCCGTGAACGTATCCCGCAACCGCTGG CACACGTTCACAAGTATTGGGCGCACGGCGTTGAGTTTTGCCGTG ATTCTGATATTGACCACCCGAGCGCACTGTCTCATCGCGACAGCG GTATCATCGCGTGCTCCGATGCGTACACGGAGCATTGTGGTTGGA TGGAGGGCGGTCTGCTGAGCGCCCGTGAGGCAAGCCGTCTGCTGT TGCAGCGTATCGCCGCGTGATTAAGGAGGTAAAAAAAATGAGCA TTCTGGATTTCCCGCGTATCCACTTCCGTGGCTGGGCCCGTGTCAA TGCGCCGACCGCGAACCGCGATCCGCACGGCCACATCGATATGGC CAGCAATACCGTGGCGATGGCGGGTGAGCCGTTCGACCTGGCACG CCATCCTACGGAGTTCCACCGTCACCTGCGCTCCCTGGGTCCGCG CTTCGGCTTGGATGGTCGTGCTGACCCGGAAGGCCCGTTCAGCCT GGCCGAGGGCTACAACGCTGCCGGTAACAACCACTTTTCGTGGGA GAGCGCAACCGTTAGCCACGTGCAATGGGATGGCGGTGAGGCGG ATCGTGGTGACGGTCTGGTCGGTGCTCGTTTGGCACTGTGGGGTC ACTACAATGATTATCTGCGTACCACCTTCAATCGTGCTCGTTGGGT CGACAGCGACCCGACGCGCCGTGACGCTGCACAAATCTATGCGG GCCAATTCACCATTAGCCCGGCTGGTGCCGGTCCGGGTACGCCGT GGCTGTTTACGGCAGACATTGATGATAGCCATGGTGCACGTTGGA CGCGTGGCGGCCACATTGCAGAGCGTGGCGGCCACTTCTTGGATG AAGAGTTTGGTCTGGCACGCCTGTTTCAGTTCTCTGTGCCGAAAG ATCACCCACATTTTCTGTTTCACCCGGGTCCGTTTGATTCCGAGGC CTGGCGTCGTCTGCAATTGGCTCTGGAGGATGACGACGTTCTGGG TCTGACCGTGCAATATGCGTTGTTCAATATGAGCACCCCGCCTCA GC CGAAC AGC CC GGTTTTTC AC GATATGGTC GGTGTTGTCGGTCT GTGGCGTCGTGGTGAACTGGCGAGCTACCCGGCTGGTCGTCTGCT GCGTCCGCGTCAACCGGGTCTGGGTGACCTGACCCTGCGCGTCAA CGGTGGTCGCGTTGCGCTGAATTTGGCGTGTGCCATTCCGTTCAGC ACTCGTGCCGCGCAGCCAAGCGCACCGGACCGCCTGACCCCGGAC CTGGGTGCCAAACTGCCGCTGGGCGATCTGCTGCTGCGTGATGAG GACGGCGCACTGTTGGCACGTGTGCCGC AGGCTCTGTACCAAGAC TATTGGACGAATCACGGTATTGTGGACCTGCCGCTGCTGCGCGAA CCGCGTGGTAGCTTGACCCTGAGCAGCGAACTGGCGGAGTGGCGT GAGCAAGACTGGGTCACCCAAAGCGACGCGTCTAACCTGTACCTG GAGGCACCGGATCGCCGTCACGGTCGCTTTTTCCCTGAGAGCATC GCGCTGCGCAGCTACTTTCGCGGTGAAGCGCGTGCGCGTCCGGAT ATCCCGCATCGTATCGAGGGCATGGGCCTGGTCGGCGTCGAATCT CGTCAGGATGGCGACGCTGCGGAATGGCGTCTGACGGGTCTGCGT CCGGGTCCGGCACGCATTGTTCTGGACGATGGTGCCGAGGCGATC CCTCTGCGTGTTCTGCCTGACGATTGGGCGCTGGATGACGCGACC GTCGAAGAAGTGGATTACGCCTTTTTGTACCGCCACGTTATGGCG TATTACGAGCTGGTGTATCCATTCATGAGCGACAAGGTGTTTTCCC TGGCTGATCGTTGCAAATGTGAAACGTACGCACGTCTGATGTGGC AGATGTGTGATCCGCAGAACCGCAACAAGTCCTATTACATGCCGA GCACCCGCGAACTGTCGGCACCGAAAGCTCGTTTGTTCTTGAAGT ATCTGGCCCACGTGGAAGGCCAGGCACGCCTGCAAGCACCTCCGC C AGC GGGTC CGGC AC GC ATTGAATCT AAAGCC C AGTTGGCGGC AG AGCTGCGTAAAGCCGTCGACCTGGAGCTGTCTGTGATGCTGCAAT ACCTGTACGCGGCGTATAGCATTCCGAACTATGCACAGGGCCAAC AACGTGTTCGTGACGGTGCGTGGACCGCCGAGCAGCTGCAACTGG CGTGCGGTAGCGGTGACCGTCGCCGTGATGGCGGTATTCGTGCAG CACTGCTGGAAATTGCTCATGAAGAAATGATTCATTACCTGGTCG TTAACAACCTGCTGATGGCCCTGGGCGAGCCGTTCTACGCGGGTG TCCCGCTGATGGGCGAAGCGGCACGTCAGGCGTTTGGCCTGGACA CCGAGTTCGCTCTGGAACCGTTTAGCGAAAGCACGCTGGCACGTT TTGTTCGTCTGGAATGGCCGCACTTTATCCCAGCACCGGGCAAAT CCATCGCGGACTGCTATGCCGCCATTCGTCAGGCGTTTTTGGATCT GCCGGACTTGTTTGGTGGCGAGGCAGGTAAGCGTGGCGGTGAAC ACCACCTGTTCCTGAATGAGCTGACCAACCGTGCGCATCCGGGTT ATCAACTGGAAGTTTTCGATCGCGACTCGGCGCTGTTTGGTATTGC ATTTGTGACCGATCAGGGCGAAGGTGGCGCTCTGGACAGCCCGCA CTACGAACATAGCCATTTTCAACGTCTGCGTGAAATGAGCGCGCG TATC ATGGCTCAAAGCGCACCGTTCGAACCGGCGCTGCCGGCGTT GCGTAATCCGGTTCTGGATGAGAGCCCGGGTTGCCAACGTGTCGC AGACGGTCGTGCGCGTGCGCTGATGGCATTGTACCAAGGCGTTTA TGAGCTGATGTTTGCGATGATGGCGCAGCACTTCGCCGTGAAACC GCTGGGTAGCTTGCGTCGCAGCCGCCTGATGAACGCAGCAATCGA TCTGATGACCGGTCTGTTGCGTCCGCTGAGCTGCGCGCTGATGAA CCTGCCAAGCGGCATCGCCGGTCGCACGGCCGGTCCGCCGCTGCC GGGTCCGGTTGACACCCGTAGCTATGACGACTACGCGCTGGGCTG TCGCATGCTGGCACGCCGTTGCGAGCGTCTGCTGGAGCAGGCGAG CATGCTGGAACCGGGTTGGCTGCCGGATGCGCAGATGGAGCTGCT GGATTTCTATCGTCGCCAAATGCTGGACTTGGCGTGCGGCAAACT GAGCCGCGAGGCCTAAGGATCCTTAAGGAGGTAAAAAAAATGAA ACGTGCGATTATCGTTGGTGGCGGCCTGGCGGGTGGCCTGACCGC GATCTACCTGGCGAAGCGTGGCTACGAAGTGCACGTCGTGGAGA AGCGTGGTGATCCTCTGCGCGATCTGAGCTCTTACGTGGACGTTG TTAGCAGCCGTGCGATCGGCGTGAGCATGACCGTTCGTGGTATCA AGAGCGTTTTGGCTGCGGGCATTCCGCGTGCAGAGCTGGATGCGT GTGGCGAACCGATCGTGGCAATGGCTTTCTCCGTGGGTGGTCAGT ATCGCATGCGCGAACTGAAGCCGTTGGAGGATTTCCGTCCGCTGA GCTTGAACCGTGCGGCGTTTCAAAAGCTGCTGAACAAATACGCGA ACCTGGCAGGCGTTCGTTACTACTTTGAGCATAAGTGCCTGGATG TTGACCTGGATGGTAAGAGCGTGTTGATTCAGGGCAAAGATGGTC AGCCGCAGCGTCTGCAAGGTGACATGATTATCGGTGCGGATGGCG CCCACAGCGCCGTCCGTCAGGCGATGCAGAGCGGCCTGCGTCGTT TCGAGTTCCAGCAAACGTTCTTCCGCCATGGCTACAAAACCCTGG TTTTGCCGGACGCGCAAGCACTGGGTTACCGTAAAGACACGCTGT ACTTTTTCGGCATGGATTCCGGTGGCCTGTTCGCGGGTCGTGCGGC TACGATCCCAGATGGTAGCGTC AGCATCGCCGTTTGCCTGCCGTA CTCGGGTAGCCCTTCCCTGACGACCACCGACGAACCGACGATGCG TGCGTTCTTC GATCGTTACTTC GGTGGC CTGCC GC GTGAC GCGC GT GACGAAATGCTGCGTCAGTTTCTGGCGAAGCCGAGCAACGACCTG ATTAACGTGCGCTCTAGCACCTTTCACTATAAGGGTAATGTGCTGT TGCTGGGTGATGCTGCGCATGCGACTGCGCCGTTCCTGGGTCAGG GTATGAACATGGCGCTGGAGGACGCCCGCACGTTTGTCGAGCTGC TGGACCGCCACCAGGGCGACCAAGACAAAGCCTTTCCGGAGTTCA CGGAGCTGCGCAAAGTCCAGGCAGACGCAATGCAAGACATGGCT CGCGCCAACTATGACGTTTTGAGCTGCTCGAACCCGATCTTTTTCA TGCGTGCGCGTTACACGCGTTACATGCATTCCAAGTTTCCGGGCCT GTATCCGCCGGATATGGCCGAGAAACTGTACTTTACGAGCGAGCC GTACGATCGTCTGCAACAAATCCAGCGTAAACAGAATGTTTGGTA CAAGATTGGTCGCGTGAATTGAAGATCTTTAAGGAGGTAAAAAA AATGAAGATTCTGGTCATTGGTGCTGGTCCAGCTGGTCTGGTTTTC GCATCCCAACTGAAGCAGGCACGCCCTTTGTGGGCCATTGACATC GTGGAGAAGAATGACGAGCAAGAAGTGCTGGGCTGGGGTGTCGT GCTGCCTGGCCGTCCGGGTCAGCACCCGGCGAACCCGCTGTCCTA TCTGGATGCACCGGAGCGTCTGAATCCGCAATTTCTGGAGGACTT CAAACTGGTGCATCATAATGAGCCGTCCTTGATGTCCACGGGCGT TTTGTTGTGCGGCGTGGAGCGTCGCGGTCTGGTTCACGCGCTGCG CGATAAGTGCCGCAGCCAAGGCATTGCTATTCGTTTCGAAAGCCC GTTGCTGGAACACGGTGAGCTGCCGCTGGCGGACTATGATCTGGT GGTCCTGGCTAATGGTGTTAATCACAAAACCGCGCATTTCACCGA GGCTCTGGTCCCGCAGGTGGACTACGGCCGCAATAAGTACATTTG GTATGGCACTAGCCAGCTGTTCGATCAGATGAATCTGGTTTTTCGT ACCCATGGTAAAGATATCTTTATCGCGCATGCCTATAAGTATAGC GATACCATGAGCACGTTCATTGTCGAATGTAGCGAAGAGACTTAC GCACGCGCACGCCTGGGCGAAATGTCCGAAGAGGCGAGCGCAGA ATACGTTGCTAAGGTGTTCCAGGCCGAGCTGGGTGGTCACGGCCT GGTGAGCCAGCCGGGTCTGGGTTGGCGTAACTTCATGACGTTGTC TCATGACCGTTGTCATGATGGTAAGTTGGTTCTGCTGGGTGACGC GCTGC AAAGCGGTC ACTTTAGC ATCGGCC ACGGC ACC ACGATGGC CGTGGTGGTGGCGCAGCTGCTGGTTAAAGCGCTGTGTACCGAAGA TGGTGTGCCTGCCGCGCTGAAACGTTTCGAAGAGCGTGCCCTGCC GCTGGTGCAGTTGTTCCGTGGCCACGCAGACAACAGCCGCGTTTG GTTCGAAACCGTCGAAGAGCGCATGCACCTGTCCTCGGCGGAATT TGTGCAAAGCTTCGACGCACGCCGCAAAAGCCTGCCGCCGATGCC GGAAGCACTGGCGCAGAATCTGCGTTATGCTTTGCAGCGCTGATG ATCATTAAGGAGGTAAAAAAAATGGAGAACCGTGAGCCACCACT GTTGCCAGCCCGTTGGAGCAGCGCCTATGTCTCTTATTGGAGCCC GATGCTGCCGGATGACCAGCTGACCAGCGGCTATTGCTGGTTCGA CTATGAACGTGACATCTGTCGTATTGACGGCCTGTTCAATCCGTG GAGCGAGCGTGATACTGGTTATCGCCTGTGGATGTCGGAGGTTGG TAATGCGGCCAGCGGCCGTACCTGGAAACAAAAAGTCGCCTATG GTCGTGAGCGTACCGCCCTGGGTGAACAGCTGTGTGAGCGTCCGC TGGATGATGAGACTGGCCCTTTTGCCGAATTGTTCCTGCCACGCG ATGTCCTGCGCCGTCTGGGTGCCCGTCACATTGGCCGTCGCGTGG TTCTGGGTCGCGAAGCGGACGGTTGGCGTTACCAGCGCCCAGGTA AAGGTCCGAGCACCCTGTACCTGGATGCGGCGAGCGGCACTCCAC TGCGCATGGTCACCGGCGATGAAGCGTCGCGTGCAAGCCTGCGTG ATTTTCCGAATGTGAGCGAGGCGGAGATCCCGGACGCGGTTTTCG CGGCCAAGCGCTAATACTAGTAGCGGCCGCTGCAGGAGTCACTAA GGGTTAGTTAGTTAGATTAGCAGAAAGTCAAAAGCCTCCGACCGG AGGCTTTTGACTAAAACTTCCCTTGGGGTTATCATTGGGGCTCACT CAAAGGCGGTAATCAGATAAAAAAAATCCTTAGCTTTCGCTAAGG ATGATTTCTGCTAGAGATGGAATAGACTGGATGGAGGCGGATAA AGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTT ATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATC ATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTT ATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAG ACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACT GTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTT CATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATC TCATGACC AAAATCCCTTAACGTGAGTTTTCGTTCC ACTGAGCGTC AGACCCCTTAATAAGATGATCTTCTTGAGATCGTTTTGGTCTGCGC GTAATCTCTTGCTCTGAAAACGAAAAAACCGCCTTGCAGGGCGGT TTTTCGAAGGTTCTCTGAGCTACCAACTCTTTGAACCGAGGTAACT GGCTTGGAGGAGCGCAGTCACCAAAACTTGTCCTTTCAGTTTAGC CTTAACCGGCGCATGACTTC AAGACTAACTCCTCTAAATCAATTA CCAGTGGCTGCTGCCAGTGGTGCTTTTGCATGTCTTTCCGGGTTGG ACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGACTGA ACGGGGGGTTCGTGCATACAGTCCAGCTTGGAGCGAACTGCCTAC CCGGAACTGAGTGTCAGGCGTGGAATGAGACAAACGCGGCCATA ACAGCGGAATGACACCGGTAAACCGAAAGGCAGGAACAGGAGA GCGCACGAGGGAGCCGCCAGGGGAAACGCCTGGTATCTTTATAGT CCTGTCGGGTTTCGCCACCACTGATTTGAGCGTCAGATTTCGTGAT GCTTGTCAGGGGGGCGGAGCCTATGGAAAAACGGCTTTGCCGCG GCCCTCTCACTTCCCTGTTAAGTATCTTCCTGGCATCTTCCAGGAA ATCTCCGCCCCGTTCGTAAGCCATTTCCGCTCGCCGCAGTCGAAC GACCGAGCGTAGCGAGTCAGTGAGCGAGGAAGCGGAATATATCC TGTATCACATATTCTGCTGACGCACCGGTGCAGCCTTTTTTCTCCT GCCACATGAAGCACTTCACTGACACCCTCATCAGTGCCAACATAG TAAGCCAGTATACACTCCGCTAGCGCTGAGGTCTGCCTCGTGAAG AAGGTGTTGCTGACTCATACCAGGCCTGAATCGCCCCATCATCCA GCCAGAAAGTGAGGGAGCCACGGTTGATGAGAGCTTTGTTGTAG GTGGACCAGTTGGTGATTTTGAACTTTTGCTTTGCCACGGAACGGT CTGCGTTGTCGGGAAGATGCGTGATCTGATCCTTCAACTCAGCAA AAGTTCGATTTATTCAACAAAGCCACGTTGTGTCTCAAAATCTCTG ATGTTACATTGCACAAGATAAAAATATATCATCATGAACAATAAA ACTGTCTGCTTACATAAACAGTAATACAAGGGGTGTTTACTAGAG GAGATTCTCATGTTTGACAGCTTATCATCGATAAGCTTTAATGCGG TAGTTTATCACAGTTAAATTGCTAACGCAGTCAGGCACCGTGTAT GAAATCTAACAATGCGCTCATCGTCATTCTCGGCACCGTCACCCT GGACGCTGTAGGCATAGGCTTGGTTATGCCGGTACTGCCGGGCCT CTTGCGGGATATCGTCCATTCCGACAGTATTGCCAGTCACTATGG CGTGCTGCTTGCGCTCTATGCGTTGATGC AATTTCTTTGCGCACCC GTTCTCGGAGCCCTGTCCGACCGCTTTGGCCGCCGTCCAGTCCTGC TCGCTTCGCTCCTTGGAGCCACTATCGACTACGCGATCATGGCGA CCACACCCGTCCTGTGGATTCTCTACGCCGGACGCATCGTGGCGG GCATCACGGGTGCCACAGGTGCGGTTGCTGGTGCCTATATCGCCG AC ATC ACCGATGGGGAAGATCGGGCTCGCC ACTTCGGGCTC ATGA GCGCTTGTTTCGGCGTGGGTATGGTGGCAGGCCCCGTGGCCGGGG GACTGTTGGGTGCCATCTCCTTGCATGCACCATTCCTTGCGGCGGC GGTGCTCAACGGCCTCAACCTCCTCCTGGGCTGCTTCCTTATGCAG GAATCGCATAAGGGAGAGCGCCGTCCGATGCCCTTGCGTGCCTTC AATCCAGTCAGCTCCTTCCGGTGGGCGCGGGGCATGACTATCGTC GCCGCACTTATGACTGTTTTCTTTATCATGCAACTCGTAGGACAGG TTCCGGCAGCGCTCTGGGTCATTTTCGGCGAGGACCGCTTTCGCTG GAGCGCGACGATGATCGGCCTGTCGCTTGCGGTATTCGGAATCTT GCACGCCCTCGCTCAAGCCTTCGTCACGGGCCCCGCCACCAAACG TTTCGGCGAGAAGCAGGCCATTATCGCGGGCATGGCGGCCGACGC GCTGGGCTACGTCTTGCTGGCGTTCGCGACGCGCGGCTGGATGGC CTTCCCCATTATGATTCTTCTCGCTTCCGGCGGCATCGGTATGCCC GCGTTGCAGGCCATGCTGTCCCGCCAAGTAGATGACGACCATCAG GGACAGCTTCAAGGGTCGCTCGCGGCTCTTACCAGCCTCACTTCG ATCATTGGACCGCTGATCGTCACGGCGATTTATGCCGCCTCGGCG AGCACATGGAACGGGTTGGCATGGATTGTAGGTGCCGCCCTTTAC CTTGTCTGCCTCCCCGCGTTGCGTCGCGGTGCATGGAGCCGGGCC ACCTCGACCTAATAATACTAGCTCCGGCAAAAAAACGGGCAAGG

TGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCG

CGTTTGCCACCTGACGTCTAAGAAAAGGAATATTCAGCAATTTGC

CCGTGCCGAAGAAAGGCCCACCCGTGAAGGTGAGCCAGTGAGTT

GATTGCTACGTAATTAGTTAGTTAGCCCTTAGTGACTCGAATTCGC

GGCCGCTTCTAGAG (SEQ ID NO:20)

SEQ ID NO: 20 is the nucleic acid sequence encoding one embodiment of the micronutrient biosensor for detecting levels of zinc in a sample.

C. Kits

Kits containing the disclosed micronutrient biosensors are also provided. The kits come as a container enclosing the genetically engineered bacteria or fungi serving as the micronutrient biosensor and instructions for its use. The kit can optionally contain equipment for drawing a blood sample and growth media to add to the micronutrient biosensors. The micronutrient biosensors are typically lyophilized and in a separate container, for example a tube or other vessel that can receive the blood sample or blood sample component such as serum. The kit can also contain an egg beater centrifuge to separate components of the blood sample.

III. Methods of Using the Compositions

One embodiment provides a method for detecting micronutrient levels in a subject by separating plasma from a blood sample, contacting the engineered cells with the serum, and allowing the cells to produce different pigments representative of corresponding to normal, borderline, and low levels of zinc respectively. In one embodiment, the plasma is separated using an egg beater centrifuge.

Examples

Example 1: Zinc

Materials and Methods

T4 DNA ligase, T5 exonuclease, Taq ligase, Phusion polymerase, Q5 polymerase, and restriction endonucleases were purchased from New England Biolabs (Ipswich, MA, USA). QIAprep Spin Miniprep Kits, QIAquick Gel Extraction Kits, and QIAquick PCR Purification Kits were purchased from QIAGEN (Valencia, CA, USA). Lycopene (98%) was purchased from Cayman Chemical (Ann Arbor, MI, USA), β-carotene (97%) was purchased from TCI America (Portland, OR, USA). SC-Ura amino acid supplement was purchased from Sunrise Science (San Diego, CA, USA).

Cloning and construct assembly

Most construct assembly was carried out via restriction endonuclease digestion of components and subsequent ligation and transformation following the BioBricks idempotent standard assembly. Constructs in pCClFOS were first assembled as above in BioBrick-compliant vectors. These were then digested with Notl and ligated into pCClFOS.

The initial attempted multi-state pigment-producing construct was assembled via Gibson assembly (Gibson et al, 2009). This method was also used to add ssrA tags to eGFP and CrtY and to remove illegal restriction sites for BioBrick standard assembly from coding sequences for eGFP, zur, and tetR.

Cell culture

For eGFP library expression, freshly transformed DH10B colonies were inoculated in triplicate into 5 mL LB media and were grown at 37 °C for 24 h. 125 of each culture was aliquotted into 96-well plates and measured on a Biotek Synergy H4 plate reader for OD600 and fluorescence at 488 nm excitation and 509 nm emission. Samples were also analyzed on a BD Accuri C6 flow cytometer.

For zinc carotenoid experiments, each strain was inoculated from freshly transformed colonies in triplicate into LB and minimal media at the following concentrations of supplemented ZnSCv 0, 50, 500, 1000 μΜ (LB) and 0, 5, 50, 500 μΜ (Minimal). LB cultures were incubated at 37 °C for 24 h.

To allow zinc titration, a modified minimal media based on M9 but lacking inorganic phosphate was developed containing the following: 2.5 g/L beta-glycerophosphate pentahydrate, 1.64 g/L KC1, 4.5 g/L NaCl, 1 g/L NFLC1, 3.9 g/L MES, 2 mM MgS0 4 , 0.1 mM CaCl 2 , 0.01% thiamine, 0.6% glycerol, and 1.92 g/L SC-Ura amino acid supplement. pH was adjusted to 7.4. The following antibiotics were used as needed for selection:

carbenicillin (100 μg/mL), chloramphenicol (34 μg/mL), and kanamycin (30 μg/mL). Minimal media cultures were incubated at 37 °C for 48 h.

Gradient agar plates were made by allowing supplemented media to gel on inclined petri dishes, forming wedges. The plates were then laid flat and filled in with unsupplemented media. LB gradient plates were made with three sections: supplemented 10 μΜ TP EN, LB alone, and LB with 250 μΜ ZnSCv Minimal gradient plates were made with two sections: plain minimal medium and 250 μΜ ZnSCv TPEN was used to simulate zinc deprivation in rich media. Plates were incubated at 37 °C for 24 and 48 h for LB and minimal agar plates, respectively. Color for streaks on LB gradient plates took approximately 24 h to develop, except for the final fosmid construct and the final pJBEI-supplemented strain with the pSB6Al construct, which took approximately 14-16 h to develop color.

Carotenoid extraction

1 mL of bacterial culture was pelleted at 16,000 rcf for 7 min. Cell pellets were resuspended in 50 μΐ. of ultrapure water. Carotenoids were extracted with 1 mL of acetone at 55 °C for 20 min with continuous shaking in a Grant Thermoshaker at 1400 rpm. Cellular debris was pelleted at 16,000 rcf and the supernatant was removed for analysis. All carotenoid extraction was carried out in low light conditions.

HPLC analysis

All HPLC analysis was conducted on a Shimadzu LC-20AD Liquid Chromatograph using a Shimadzu C18 4.6 mmx50 mm column with a 5 μπι particle size and a SPD-20A UV-vis detector. The instrument was run with a flow rate of 0.6 mL/min and a solvent ratio of 90: 10 acetonitrile:THF.

Retention times and intensities were compared to analytical standards spiked into control extractions from DH10B cells and calibration curves to convert peak areas to analyte concentrations were constructed. Plasmids and regulatory sequences

A list of all vectors used is provided in Table 1, along with their expected copy number. ZntR, Zur, and PznuC were amplified from DH10B genomic DNA. pJBEI-6409 (Alonso-Gutierrez et al., 2013) was obtained from Addgene (Cambridge, MA, USA) and is referred to here as pJBEI- MEV. PzntA sequence was synthesized by Genewiz (South Plainfield, NJ, USA). eGFP was provided by Julie Champion, and pCClFOS was provided by Brian Hammer. All other parts, including pSB-series plasmids (Shetty et al, 2008), ribosomal binding sites, promoters, and pigment biosynthesis genes were obtained from the Registry of Standard Biological Parts and were sequence-confirmed before use.

Table 1.

Vectors and source plasmids used in this work.

The pSB series of plasmids were originally developed as standardized vectors for BioBricks and were obtained from the Registry of Standard Biological Parts. To comply with BioBricks standards, the pSB3 series was constructed with a pi 5 A origin and regulatory region without illegal restriction sites. pJBEI-6409 also uses the pl5A origin and regulatory region and is known to function as a medium copy plasmid. A direct comparison of these regions of the plasmids with the genbank annotation of the pl5A origin and regulatory region of pACYC184, a known medium copy pi 5 A origin plasmid, shows an 83 base truncation and single base deletion in pSB3C5 relative to the reference sequence. Original construction of pSB3C5 and other pSB series plasmids is described in Shetty et al., J. Biol. Eng., 2:5 (2008).

Strain naming conventions

Fluorescence library members were named by concatenating the name or Parts Registry part number of the promoter, the last two digits of the Parts Registry part number for the ribosomal binding site, and then potentially three letters indicating whether a strong (LAA) or weak (DAS) ssrA protein degradation tag was added (AANDENYALAA (SEQ ID NO: 18) or AANDENYADAS SEQ ID NO: 19)). Carotenoid test parts were named by concatenating the Parts Registry part number of the RBS in front of crtY, an indicator of whether there was a degradation tag on crtY (LAA, DAS, or nothing), and the last three characters of the name of the vector the construct was cloned into (1C3, 3C5, 6A1, or FOS). The presence of +J indicates the carotenoid construct was co-transformed with pJBEI-MEV.

Results

Design of a zinc-responsive, pigment-producing, bacterial biosensor The overall workflow of an exemplary biosensor is illustrated in Figure 1 A. Sensor cells can be lyophilized or otherwise preserved for long-term storage. A blood sample is separated in the field via a low-cost, equipment-free approach such as the egg beater centrifuge (Wong et al, Lab Chip, 8: :2032- 2037 (2008)) The resulting plasma is then be added to the preserved cells (potentially with additional, zinc-free defined medium), and the cells produce different pigments in response to the levels of zinc in the physiologically relevant range of 8-15 μΜ (Hess et al., Food Nutr. Bull, 28: S403-S429 (2007)) corresponding to normal, borderline, and low levels of zinc.

One embodiment provdes a circuit using heterologous pigment production pathways driven by zinc-responsive transcription factor/promoter pairs present in E. coli ( Figure IB). The first heterologous pigment production pathway is the purple violacein biosynthesis pathway, which uses the vioABCDE operon (originally derived from Chromobacterium violaceum) to produce violacein from endogenous tryptophan. The second pigment production pathway is carotenoid biosynthesis, which uses the crtE, crtB, and crtl genes to produce red lycopene starting from endogenous FPP, and uses crtY to produce orange β-carotene from lycopene; these genes were originally derived from Pantoea ananatis. Expression of these pigment production pathways was driven by two endogenous E. coli zinc-responsive promoter/transcription factor pairs, PznuC/Zur and PzntA/ZntR, that control expression of zinc importer and exporter genes, respectively. (Zur is a zinc- activated transcriptional repressor and ZntR is a zinc-activated

transcriptional activator.) Previous work ( Yamamoto and Ishihama, 2005) suggested that Zur is able to fully repress expression from PznuC at approximately 10 μΜ zinc, while induction of transcription from PzntA should begin at least by 100 μΜ zinc (preliminary results suggested induction by 10 μΜ) with full induction not until approximately 1.1 mM (Brocklehurst et al, Mol. Microbiol, 31 : 893-902 (1999)).

These pieces were then combined together such that the circuit would provide discrete color states of purple, red, and orange depending on the zinc concentration of the sample. PznuC was used to drive expression of vioABCDE and PzntA was used to drive expression of crtY; zntR and zur were expressed from the same plasmid using their genomic promoters, while crtEBI was expressed from a mutated ρλ-based, extremely weak promoter.

The original goal of our first construct for Figure IB was to express crtEBI under the control of R0040, a ρλ-based promoter with two tetO operator sites for TetR-mediated repression. (This was to be followed by a B0034 RBS.) No constructs with intact promoters were isolated; plasmid sequencing of isolated constructs indicated intact ribosomal binding sites and mutated promoters, with at least one tetO sequence, along with intervening promoter sequence, removed. This resulted in a weak, mutated version of the original promoter. Given that the medium used for cloning was a rich medium with significant levels of zinc, this meant that the default state for the sensor should be the production of lycopene. Given this constraint, it is not surprising based on previous literature that there was heavy selection against high production of lycopene, as high expression of crtEBI is toxic. Thus, low expression of crtEBI was desirable for the construct.

At low zinc concentrations (below 10 μΜ), transcription from PzntA would be low and transcription from PznuC would be induced, resulting in violacein biosynthesis (which is so dark as to visibly overwhelm minor lycopene production). At borderline levels of zinc (approximately 10 μΜ), Zur would become activated and repress expression from PznuC. This would repress expression of the violacein biosynthesis operon, but expression of crtY to produce β-carotene would not be sufficiently induced, resulting in the production of only the red pigment lycopene. At normal levels of zinc (over 10 μΜ), violacein production would still be repressed, but ZntR would then be activated and crtY expression would increase, facilitating the

transformation of lycopene to β-carotene and yielding only orange pigment.

Upon implementation, the designed circuit only exhibited two distinct color states

The circuit design was engineered on a high copy number plasmid and expressed in E. coli. The entire construct was sequence confirmed.

The behavior of the implemented construct was observed as a streak on a zinc gradient plate (data not shown). There is a clear, fairly sharp transition between two color states, purple (low zinc) and orange (high zinc), but no observable red (intermediate zinc) state. This behavior provides a number of key insights. First, the fact that crtEBI is very weakly and constitutively expressed is not a problem, as the dark color produced by violacein overwhelms any potential background signal of carotenoid production. Next, the repression of violacein production at higher zinc concentrations is sufficient to allow for the carotenoid production to be visible without any interference from leakiness of the PznuC promoter. Finally, the basal uninduced expression of crtY from PzntA at low and intermediate zinc levels is too great to allow precise control over the pathway, as lycopene is converted to β-carotene even at zinc levels where crtY should not be strongly induced. Absence of a lycopene-only state is due to overlap in expression regimes

A fluorescence-based construct was implemented to assess the induction and repression of PzntA and PznuC, respectively. Figure 2A shows the structure of this construct: RFP expression is driven by the zinc-inducible PzntA promoter, while GFP expression is driven by the zinc-repressible PznuC promoter, with the transcription factors for each promoter (ZntR and Zur, respectively) constitutively expressed from the same high-copy plasmid. Figure 2B shows the quantitative results of GFP and RFP expression from the promoters. It is thus clear that while PzntA and PznuC can be significantly induced and repressed, respectively, there is some overlap in their expression regimes, whether due to their response curves to zinc or due to baseline leaky expression. It is worth noting that most of the repression from PznuC occurs over the low concentrations of zinc, yet there is clearly a regime where violacein is not visible. Since violacein is much easier to detect visually than the carotenoids, this suggests that even though there is measurable fluorescence (and thus measurable leakiness) for this test construct, the production of violacein corresponding to that level of fluorescence would not be visually detectable. Combined with knowledge that the maximum repression of PznuC has been shown to happen at the bottom end of the PzntA induction range, this supports the idea that the reason why no lycopene-only state is detectable is that crtY expression is too great at low zinc levels, rather than vioABCDE expression being too great at higher zinc levels. The expression of mRFP from PznuC and PzntA on parallel regulated constructs suggests that visibly there is likely to be a significant region of non-overlap between the repression of PznuC and the induction of PzntA (data not shown).

A library of fluorescence constructs enables characterization of post- transcriptional control

Modification of ribosomal binding sites and tagging of proteins for degradation were identified as the simplest approaches to decrease the level of CrtY at low levels of zinc. To do this most efficiently, the impact of multiple regulatory elements within our specific host strain was characterized using an eGFP reporter. The strength of multiple potentially constitutive promoters (which could drive expression of the zinc-responsive transcription factors) and the strength of our zinc-responsive promoters (without coexpressed regulators) were also characterized. This

characterization was intended to facilitate more rational and focused selection of regulatory elements for the final sensor construct rather than performing combinatorial syntheses that would be challenging based on product toxicities; the results of the characterization are presented in Figure 3.

Taken together, manipulating promoters and post-transcriptional regulatory sequences yielded over five orders of magnitude of variation of fluorescence (Figure 3A). Each level alone (transcriptional and post- transcriptional) offered at least three orders of magnitude of dynamic range, with low-fluorescence measurements being confounded by limits of detectability even at high plate reader gain settings.

Measurements of promoter strength across multiple sets of post- transcriptional regulatory sequences provided information for later selection of promoters (i.e., for zntR and zur) and provided an estimate of the strength of the PznuC and PzntA promoters. Unsurprisingly, the ρλ-based promoters (R0040 and R0011) were the strongest. Known weaker promoters (J23117 and J23116) performed roughly as expected, though the difference between these two promoters was greater than previously reported. The expression levels of these promoters were used to characterize some of the

transcriptional behavior of the two inducible promoters. Since these constructs had no supplementary regulator expression (i.e., Zur or ZntR) in order to be consistent with the rest of the library, the expression from PznuC is indicative of essentially unrepressed transcription, while expression from PzntA is indicative of essentially uninduced transcription. It was found that baseline expression from PzntA was stronger than at least one known weak constitutive promoter, J23117. This suggests that the underlying reason for the lycopene state being difficult to stabilize may be that the baseline transcription from PzntA is sufficiently high to result in significant production of CrtY.

Changing ribosomal binding sites (RBSs) also enabled many orders of magnitude of reduction for overall gene expression. Figure 3B

demonstrates the impact of using four different ribosomal binding sites taken from the Registry of Standard Biological Parts: B0031-B0034. The patterns of RBS strength as measured here were qualitatively consistent with previously measured values, with a few exceptions. First, B0031 was found to be slightly stronger than B0032 rather than vice versa; this was consistent across all promoter strengths. Second, the expression from B0034 was found not to be much stronger than from B0031, whereas previous reports have shown an order of magnitude difference between these two RBS options. Flow cytometry for library members with strong promoters (Figure 3C) showed that the reason for the unexpected low levels of fluorescence from B0034 is a bimodal population of low-expressing and high-expressing cells. This population split was ultimately due to mutation of promoters in library members with the B0034 RBS due to physiological stress from the high levels of protein expression: while measurements of all library members were done from fresh transformants from sequence-verified DNA, plasmid sequencing for B0034 library transformants exhibiting these bimodal populations returned multiple competing signals indicating that the dual operator sites in R0040 and R0011 had combined to heavily mutate the promoter.

Figure 3D shows that LAA and DAS protein degradation tags can significantly curtail the net expression of a gene and that the LAA tag can do so by three orders of magnitude. C-terminus of eGFP was tagged with one of two ssrA tags, the strong AANDENYALAA (SEQ ID NO: 18) tag or the weaker AANDENYADAS (SEQ ID NO: 19) tag. These tags induce SspB- mediated binding to the ClpX and ClpA proteases, which increases a protein's degradation rate (McGinness et al, Mol. Cell, 22: 701-707 (2006)). The magnitude of the effect of the LAA tag is as much as or greater than the impact of using the lowest-efficiency ribosomal binding site. Of note is the interaction between expression level and the efficiency of repression via tagging for protein degradation. The LAA tag effectively reduces protein levels even for highly-expressed proteins, whereas the DAS tag is most effective for lowly-expressed proteins (giving a reduction of up to 10-fold; for example, B0033 RBS or weak promoters) compared to highly-expressed proteins (with reductions typically closer to 2-fold; for example, B0031 RBS with a strong promoter). This suggests that overall gene dosage may play a role in the effectiveness of post-transcriptional regulation in some cases, though the mechanism by which this would occur is unclear.

Carotenoid production can be controlled post-transcriptionally Using this library of regulatory element combinations and the insights gleaned from them, 13 targeted carotenoid-expressing constructs were then assembled to identify those that could produce two distinct states: one dominated by lycopene and one dominated by β-carotene, which was not observed in our initial attempts. The test constructs consisted of (Figure 4A) crtY being driven by the PzntA promoter and zntR driven by a weak constitutive promoter. crtEBI was cloned in without an explicit promoter (discussed in greater detail in Section 4 and in Supplementary information S3) so as to yield very low transcription, consistent with previous work where crtEBI had an inducible promoter that was never induced since its expression is detrimental to cell growth ( Yoon et al., Biotechnol. Bioeng., 94: 1025-1032 (2006)). Regulatory elements of crtY were varied to control conversion of lycopene to β-carotene.

To manipulate levels of CrtY, we selected variants from the fluorescence characterization presented in Figure 3, as well as additional methods for manipulation of expression levels. The RBS of crtY was varied and the strong (LAA) degradation tag was added to crtY. The strong B0034 RBS was not explored further since preliminary pilot experiments indicated it would need such substantial reduction in expression to allow a lycopene- dominated state that RBS variation would likely be necessary. Another level of control was added over the amount of gene expression via variation of vectors, employing high, medium, low, and essentially-single copy vectors. This would titrate not only the total amount of CrtY in the cell from leaky or mildly -induced expression, but also the expression of crtEBI, which also may cause toxicity and could prevent greater accumulation of carotenoids.

Changing only the RBS of crtY was insufficient to yield a distinct lycopene state. Replacing the original B0034 RBS with the B0033 RBS yielded no visible lycopene in an uninduced state (and essentially no measurable lycopene), but a substantial amount of β-carotene ( Figure 4B), despite the B0033 RBS being two to three orders of magnitude weaker than the B0034 RBS used in the original construct. (Again, this supports the earlier assessment that the reason why no lycopene-only state could be detected was due to too much crtY expression at low zinc rather than too much vioABCDE expression at high zinc.) Even combining a lower-strength (B0032) RBS with a strong (LAA) degradation tag, which should yield four or more orders of magnitude less CrtY, was not sufficient to allow lycopene to persist in an uninduced state ( Figure 4C).

When the vector was varied, substantial amounts of lycopene were produced (Figure 4D). Medium and low copy number plasmids yielded substantial lycopene at low zinc that is repressed at high zinc, and a fosmid yielded such substantial production of lycopene that CrtY could not be induced enough to consume it (yielding a single state in the pathway dominated only by lycopene instead of by β-carotene). The most promising combinations appear to be the low-copy plasmid with a degradation tag and a moderate RBS, yielding a lycopene-dominated state and a β-carotene dominated state, though with less switch-like behavior than expected.

Similar behaviors were observed in minimal medium cultures

(Figures 5A-5D). Figures 5A through 5D in this figure correspond to Figures 4B through 4E, showing that the behavior of the cells in minimal and rich medium (which includes an expected baseline level of zinc) is extremely similar. Error bars all represent the standard error of the mean.

Measurements were all taken at 0, 50, 500 and 1000 μΜ Zn 2+ , though in Figure 5C an offset has been applied for visualization purposes. Figure 5A shows that changing only to a very weak ribosomal binding site is insufficient to produce a lycopene-only state, and generally insufficient to produce any lycopene at all. This suggests that multiple levels of control must be used. Figure 5B shows using a moderate ribosomal binding site and a strong degradation tag (LAA) enables the detectable production of lycopene but still does not enable a lycopene-dominated state. Figure 5C shows changing the vector carrying the construct from Figure 5C enables the presence of a lycopene-only state. For the lowest-copy plasmid (the fosmid), so little CrtY is accumulated even at full zinc induction that only a lycopene- dominated state can be observed. The low-copy 6A1 vector offers two distinct states. Figure 5D shows co-transforming with a plasmid containing the mevalonate pathway (pJBEI-MEV) to supplement production of lycopene alters the transition point between the lycopene and β-carotene states. At 50 μΜ Zn2+, the pJBEI-MEV-supplemented cells still have still produced much more lycopene than β-carotene, while the non-supplemented strain is already transitioning to a β-carotene state. Of note is that the lycopene production at no supplemented zinc is three times higher in the supplemented strain, as expected.

Worth noting is that in many constructs, the lycopene and/or β- carotene levels decrease at the 1 mM zinc condition. In previous literature, the full induction level of PzntA was found to be at 1.1 mM zinc, and E. coli has been shown to be capable of normal growth in an excess of 1 mM extracellular zinc. Additional metabolic stress of the cells from substantial transcription, translation, and pigment synthesis, combined with the toxicity of carotenoid operon genes and potential interference with zinc homeostasis due to overexpression of native regulators, increases the sensitivity of the engineered cells to zinc. Fortunately, this level of zinc is nowhere near that which would be relevant to the ultimate application of this biosensor in a blood micronutrient diagnostic (Hess et al, 2007).

Enhancing lycopene production affects sensor response

Another alternative to manipulate the apparent response of PzntA- driven expression is to stimulate the overall production of lycopene in the cells. The root cause of a β-carotene-only state is that there is sufficient CrtY to enzymatically act on all of the lycopene that is present. Lycopene production, or more generally the production of carotenoids and isoprenoids, has long been a target for metabolic engineering studies, with significant advances made in terms of overproducing background strains and identification of multiple independent ways to increase carotenoid titers. One well-known approach is the introduction of a mevalonate pathway to supplement the production of precursors for the carotenoid and isoprenoid pathways (Yoon et al, 2006).

By co-transforming engineered carotenoid constructs with a plasmid containing the mevalonate pathway (pJBEI-MEV), the response of the carotenoid-producing sensor was substantially affected (Figure 4E). A moderate RBS combined with a strong degradation tag and a low-copy plasmid initially yielded a system offering a crossover in production from lycopene to β-carotene. At approximately 50 μΜ of zinc, the levels of lycopene and β-carotene were of the same order of magnitude. However, by supplementing with pJBEI-MEV, production of lycopene was increased threefold, and at 50 μΜ lycopene levels remained much higher than β- carotene levels. This effectively changed the "switching point" between lycopene and β-carotene states in the supplemented cells. This behavior was observed both in rich medium and in minimal medium. While in this case it moved the switching point perhaps to a level beyond physiological relevance, application of this approach with other constructs allows for another level of manipulation to engineer the precise response desired.

Engineered distinct red and orange system states

A transition between red and orange states is now possible after substantial pathway engineering. In order for both states to be possible for the same cells, multiple components needed to be precisely controlled in combination. With a moderate RBS, a strong degradation tag, and the low- copy plasmid, levels of CrtY were limited at low zinc levels (preventing reaction of lycopene) but were adequately inducible to allow a switch to β- carotene at higher zinc levels. Selection of a different plasmid in this configuration would have different results: for example, on the the fosmid, CrtY levels are limited so much that the β-carotene state can never be reached (though for other configurations, for example without LAA tags, the fosmid did allow for accumulation of CrtY and production of β-carotene). Supplementation of lycopene production via the mevalonate pathway enabled further tuning of the zinc levels at which that transition occurred. Taken together, these strategies enabled the ultimate within-pathway state- switching goal of the biosensor.

The sensitivity of metabolic pathways to low levels of enzymatic expression - a key aspect of what one could call "precision metabolic engineering" - is not something that has been widely explored in the literature. Nonetheless, it is an extremely important aspect of harnessing metabolism for biosensor applications while minimizing the transcriptional and metabolic burden on cells (and thus using the same pathway to produce multiple different visible outputs). Pathway engineering was used to allow a lycopene-only intermediate state to persist, but it was also found that tuning the carotenoid pathway could strongly affect sensor response. Adjusting both upstream and downstream pathway expression controlled the zinc concentration at which pigment production switched, allowing more control over what concentrations would map to "intermediate zinc" and "high zinc" outputs.

Overall, the most difficult aspect of the engineering task was compensating for leaky expression of enzymes downstream of the first pigment indicator. This suggests that (when possible) the selection of regulatable promoters with the lowest levels of leakiness may outweigh other important factors (e.g., whether it is inducible or repressible, or the expected response threshold) when designing future pigment-based sensors. However, for natural promoters this will often not be possible, necessitating the precision engineering efforts that we have described. Ultimately, multiple levels of control were necessary to achieve the desired metabolite production given the limitations in inducible regulation and intermediate or product toxicity; we expect that such extensive control will typically be necessary in regulating the response of sensors using metabolite reporters. Perhaps the most interesting lesson for future strain design was that controlling the levels of precursors for the heterologous biosynthetic pathways can affect the switch point of the circuit. This provides a powerful, modular, orthogonal approach to basic pathway engineering of heterologous pigment production pathways, and could be considered an earlier option in strain design if (as in the mevalonate pathway) there are well-established methods to adjust precursor availability to optimize pigment switching around input levels of interest from arbitrary sensors.

The approaches identified and lessons learned could also be generalizable to other "precision" applications where extremely selective production of specific molecules is critical, including the design of multifunctional (potentially portable) microbial cell factories that can produce different products in response to different environmental conditions. A key result of this work is that significant, combinatorial regulation was necessary in order to limit the immediate production of β-carotene in our construct; while it was unsurprising that pigments from two different heterologous pathways could be combined via a switch for a visible output, the level of control necessary to prevent the reaction of all lycopene to β- carotene was unexpected. While the level of control necessary would ultimately be dependent upon enzyme turnover number rate, knowledge that such levels would need to be so precisely regulated for pathway control will likely play a key role in other precision metabolic engineering applications in the future.

While in the foregoing specification this invention has been described in relation to certain embodiments thereof, and many details have been put forth for the purpose of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein can be varied considerably without departing from the basic principles of the invention.

All references cited herein are incorporated by reference in their entirety. The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof and, accordingly, reference should be made to the appended claims, rather than to the foregoing specification, as indicating the scope of the invention.