WO2004076685A2 - Fungal biosensor and assay - Google Patents

Abstract

A fungal biosensor comprising luminescent or fluorescent filamentous fungi is provided. The luminescence/fluorescence of the fungi varies depending upon whether the predetermined test substance is present or absent. Typically the gene encoding the luminescent or fluorescent protein is genetically engineered to be under the control of an inducible promoter or enhancer sensitive to the test substance. Suitable fungi include Neurospora Sp., and Aspergillus Sp. Exemplary luminescent proteins include the Gaussia luciferase and the phosphoprotein obelin. An assay using the biosensor is also described.

Description

FUNGAL BIOSENSOR AND ASSAY

The present invention relates to a biosensor and assay using luminescent or fluorescent filamentous fungi for the detection of component substances within a sample by measuring the light output from the filamentous fungi . The present invention also relates to genetically engineered filamentous fungi able to luminesce or fluoresce.

It is known that certain bacteria are able to emit light without excitation (ie. they glow in the dark) and such organisms are said to be luminescent or bio-luminescent. Such luminescent bacteria are often of marine origin and examples include Vibrio harveyi , Vibrio fischeri , Photobacterium phosphoreum and Photobacterium leiognathi . The luminescence of bacteria is known to be sensitive to a wide variety of toxic substances such as pesticides or heavy metals and this sensitivity has made them suitable for use in methods for the detection of the presence of toxic substances in substrates such as soil, effluent, marine environments etc. Prokaryotic biosensors founded on luminescent bacteria are commercially available, for example MICROTOX, which is based on the bacterium Vibrio fisheri NRRL B- 11177. However, such biosensors are either insensitive or only poorly sensitive to eukaryotic- specific molecules. Moreover these biosensors operate only within a narrow pH range . In the case of MICROTOX the luciferase within Vibrio fisheri is dependent on ATP for luminescence, which limits the strength of luminescence available. Thus, the presence of toxic compounds, inhibition of cellular activity or decreased rate of respiration results in a corresponding decrease in the rate of luminescence.

WO-A-0G,'14267 proposes a new eukaryotic biosensor derived from Saccharomyces cerevisiae genetically engineered to express a light emitting protein, for instaπc luciferase. However, such bio-assays based upon yeast, such as Saccharomyces cerevisiae, require the luminescent organism to be maintained in a liquid suspension form which is a significant disadvantage. To be accurate, an actively growing culture must be prepared for each assay and this requirement severely limits the possibility of conducting the assay outside a laboratory ' environment. In addition, the assay is non-specific,- it sirrply suggests that a molecule is toxic, but does rot imply the mechanism of toxicity. The Saccha romyces biosensor is based on firefly luciferase, which is also dependent upon ATP, and similarly to MICROTOX, inhibition of respiration corresponds to decrease in bioluminescence.

The present invention is concerned with the use of luminescent or fluorescent filamentous fungi, for example genetically engineered strains of Aspergillus sp, Neurospora sp, etc. as a biosensor to detect a predetermined test substance measurable by the light output of the fungi .

In one aspect, the present invention provides a biosensor comprising luminescent or fluorescent filamentous fungi wherein the light output varies in response to the presence or absence of a predetermined test substance . ' The test substance detected and/or measured by the assay is suitably inorganic or organic chemical compounds, such as toxins, biocides or drugs. Mention may be made of metal ions, organophosphate, alcohol, cyanide, and an anaesthetic (eg. procaine) . Other similar test substances may however be selected to be the subject of the assay.

The use of luminescent or fluorescent filamentous fungi has several clear advantages over yeast or bacteria including: the increased complexity of filamentous fungi (filamentous fungi have two or three times the number of genes of a yeast and this- gives increased metabolic diversity as well as more genes and promoters to work with, enhancing biosensor flexibility) ; their capability to express exogenous proteins; and ability to grow in more diverse conditions with greater predictability. Additionally, filamentous fungi have also demonstrated improved longevity compared to yeast, and unlike yeast the filamentous fungi does not need to be maintained in a liquid suspension, making storage and transport easier. It is expected that the glycosylation of proteins by filamentous fungi will be closer to that experienced in plant and mammals as compared to yeast. A wide range of filamentous fungi are amenable to transformation, and heterologous expression.

The fungus is preferably selected from Aspergillus sp . or -Meurospora sp . However alternative species may be used including the pathogens Magnaporthe grisea and Sclerotinia sclerotiorum .

Optionally the luminescent or fluorescent protein is a foreign protein and the filamentous fungus is genetically engineered to express that protein and to be .luminescent or fluorescent, by introduction of the relevant gene. Alternatively, the fungus selected may be a naturally bioluminescent organism, and in this embodiment expression of the endogenous gene responsible for bioluminescence can conveniently be modified to be dependent . upon the presence or absence of a predetermined test substance.

The ge:ne for a luminescent or fluorescent protein may be obtained from firefly ( Photinus pyralyis) , crustaceans ( Cyridina hilgendorfi) , dinoflagellates (Nortilucus militaris, Gonyaulax polyhedra) or naturally luminescent fungi [Panellus stipticus) . Use of suitable proteins of bacterial origin is also possible.

Preferred luminescent proteins include luciferase proteins, especially marine luciferases, and photoproteins, such as the photoprotein obelin.' Examples of suitable luciferases include those from Gaussia and Pleuromamma : Suitable fluorescent proteins include those from Ptilosarcus, Renilla mullerei and Renilla reniformis . Any fluorescent or luminescent protein for which the encoding gene is optimized for filamentous fungi is also suitable. Alternatively the' gene can be optimized for mammalian expression. Exemplary genes expressing luminescent or fluorescent proteins are described in WO-A-99/49019 (Prolume Ltd) . The Gausssia princeps luciferase protein is a coelenterazine based luciferase and is unlike the firefly luciferase in that it does not require accessory high energy molecules such as ATP (adenosine triphosphate) (Prolume Ltd) .

In one embodiment the present invention utilises fungi expressing luciferases (e.g. Gaussia) which are not dependent upon ATP, and are resistant to pH extremes e.g. survive exposure to pH 3 or pH 11 overnight (0°C) (Prolume Ltd) . Suitably obelin or the Gaussia princeps luciferase is genetically engineered into Neurospora crassa , and optimised for mammalian codon usage. This mammalian gene can be successfully expressed in filamentous fungi. Gaussia luciferase may be expressed in other species of filamentous fungi including Aspergillus nidulans and Sclerotinia sclerotiorum (a plant pathogen) . Preferably the Gaussia luciferase gene is codon-optimised for codons preferred by filamentous fungi in order to increase expression and light output. Other novel luminescent and fluorescent proteins (e.g. the calcium-sensitive obelin photoprotein, and the Ptilosarcus green fluorescent protein) may also be expressed in filamentous fungi and optionally can be codon optimised. Obelin has similar properties to the calcium sensitive protein aequorin (from the jellyfish Aequorea. victorea) which has successfully been expressed in filamentous fungi. Genetic modifications other than codon optimisations , may be undertaken to enhance biosensor function, for example luciferase or fluorescent gene tagging e.g. with a signal secretory peptide or organelle .tags .

Biosensors that express the luminescent or fluorescent protein in response to specific stimuli (for example the presence of metal ions) may be produced by driving protein expression with constj;tutive or inducible promoters. The alcA promot-er is induced in response to ethanol utilisation (see Felenbok B (1991) "The ethanol utilisation regulation of Aspergillus nidulans the alcA-alcR system as a tool for expression of recombinant proteins". Journal of Biotechnology 17: 11-18; Flipphi M, Kocialkowska J, Felenbok B (2002) "Characteristics of physiological inducers of ethanol utilisation (al e) pathway in Aspergillus nidulans" . Biochemical Journal 36.4: 25-51).

The alcA promoter has been used to drive expression of Green Fluorescent Protein (GFP) in Aspergillus nidulans (see Fernandez -Abalos JM, Fox H, Pitt C, Wells B and Doonan JH (1998) "Plant adapted green • fluorescent protein is a versatile reporter for gene expression, protein localization and mitosis in the filamentous fungus, Aspergillus nidulans" . Molecular Microbiology 27: 121-130) . Aspergillus nidulans may be transformed with luciferase genes fused to the alcA promoter.

The copper metallothionein is expressed in response to copper ions and thus could form the basis of a copper biosensor through the expression of luciferase fused to the copper metallothionein promoter from Neurospora crassa (see Munger K, Germann UA, Lerch K (1985) "Isolation and structural organisation of the Neurospora crassa copper metallothionein gene". EMBO Journal 4: 2665-2668; and Schilling B, Linden RM, Kupper U and Lerch K (1992) "Expression of Neurospora crassa Laccase under control of the copper inducible metallothionein promoter", Current Genetics 22: 197- 203) . In- the genetic construct produced, the expression of the luminescent or fluorescent protein is desirably under the control of a gene promoter or enhancer sensitive to the presence of the predetermined substance to be assayed. Suitable promoters/enhancers are available from known in homologous or heterologous systems described in the literature. Alternatively suitable promoters/enhancers could be obtained through promoter trapping/tagging experiments which in turn will allow screening for promoters sensitive to libraries of compounds or environmental stimuli of interest.

A possible method to identify new inducible promoters is described below. Using an efficient transformation technique, randomly insert the luciferase gene with antibiotic resistance (luci£erase/hygromycin cassette) into . the fungi. Large inumbers (thousands) of transformants are then screened for luminescence increase in the presence of a pre-determined substance (e.g. heavy metal ions) .^" The strains which show increase in luminescence in the presence of the specific substance are then isolated, and the genetic sequence where the luciferase/hygromycin cassette is integrated is analysed to determine the inducible promoter.

In one embodiment, the presence of the predetermined test substance causes or promotes luminescence or fluorescence and an increase in optical output is observed when the test substance is brought into contact with the filamentous fungi. Conveniently, the increase in optical output is dependent on the concentration of test substance present . This would be an example of induced expression.

In an alternative embodiment the presence of the^' predetermined test substance prevents or decreases luminescence or fluorescence and a decrease in optical output is observed when the test substance is brought into contact with the filamentous fungi. Conveniently, the, decrease in optical output is dependent on the concentration of test substance present. This would typically have application to toxicity assays and in drug screening. ' Since the expression of luminescent or fluorescent protein is dependent on the biomass of the filamentous fungi, the light output can be correlated with the growth. Thus the assay provides a means of identifying compounds which affect spore germination and growth. This is particularly useful to study the mode of action of fungicides and toxic molecules .

In a further embodiment the assay can be used to identify, compounds which affect expression of those genes involved in disease, e.g. oncogenes in cancer, metabolic disorders etc. In this case, a gene encoding the luminescent or fluorescent protein is fused with promoters which are sensitive to substances which are linked to disease onset. Such an assay gives a quantitative method of identifying novel drugs that affect expression of gene targets involved in disease. Optionally the biosensor is in the form of an array which may be pre-populated with spores or mycelium of the biosensor fungus or may comprise actively growing biosensor fungi . Conveniently the array may be a 96, 384 or 1536 well plate, although any other plate format could be used.

Optionally the biosensor could include an indicator to confirm that the fungal biosensor cells are whole and viable. Suitable indicators include the fluorescent probe L-7010 (Molecular Probes Inc. Oregon)/ or FM4-64 (T-3166 Molecular Probes Inc. Oregon) or propidium iodide. Intact cells stained with FM4-64 exhibit low cytoplasmic staining. Cells with damaged plasma membrane rapidly take up the dye causing the cytoplasm to become highly fluorescent. This "change in fluorescence can be measured and gives: indication of cell viability, e.g. when testing compounds for their effect on spores.

A further novel aspect is the characteristic luminescent or fluorescent output which is termed a signature. If the luminescence or fluorescence can be repeatedly shown to give a similar response e.g. on application of a class of compounds, this is regarded as a signature. These different signatures can be assigned to different cellular responses: e.g. spore germination, cell division, transient expression, programmed cell death etc. The combination of luminescent or fluorescent signature with the use of multiple fluorescent probes gives the opportunity to measure multiple factors simultaneously- Examples of fluorescent probes that specifically target different organelles include DASPMI which targets mitochondria, DFFDA which targets the vacuolar network, and FM4-64 or FMl-43 which labels membranes and secretory vesicles in filamentous fungi (dyes all from Molecular Probes, Oregon, USA) .

The present invention also provides an assay to determine the presence of a predetermined test substance in a sample,' said assay comprising:

a) exposing the sample to a filamentous fungus having an ability to luminesce or fluoresce wherein the luminescent or fluorescent output will vary depending upon presence or absence of the test substance; and b) measuring the luminescent or fluorescent output from the fungi, and determining therefrom the presence of the test substance in the sample.

Optionally, the assay is performed using positive and negative standard controls i.e. with and without the predetermined test substance respectively and the luminescence or fluorescence compared to the sample under test. Optionally an array of filamentous fungi each sensitive for a different and distinct predetermined test substance is used in the assay.

Spores of the biosensor fungi may be immobilised in an array (for example the array may be within standard 96, 384 or 1536 well plates) for ease of storage, transport, fast preparation and measurement . Optionally freeze drying may be employed to preserve either spores or mycelium or both. Alternatively fungal spores or mycelium are immobilised on an array by use .of an adhesive and dried,! for example in a laminar flow hood.

The fungus may optionally be immobilised in a gel or matrix. Optionally the gel may be composed of a hydrogel, agar, agarose or other polymer.

Optionally, the filamentous fungus is in the form of a fibrous mat. Alternatively fungal spores are optionally coated onto beads.

The light output from the biosensors may be measured using a CCD camera (preferably cooled CCD) , photodiode or photomultiplier . Alternatively, plate reading luminometers may be used to measure light from the biosensors typically presented in the form of standard 96, 384 or 1536 well plates.

In one embodiment the fungi may also exhibit fluorescent properties and a focused laser beam may be used to selectively illuminate each individual biosensor in turn. The fluorescence may be measured as described above for the luminescence. A dichroic or filter may be used to select for specific fluorescent spectra. Alternatively a plate reading fluorometer may be used to quantify fluorescence from the biosensors, either exclusively or in combination with luminescence in the same assay. ^'

Figure 1 is a schematic representation of a suitable apparatus for use in the assay of the invention using (a) a conventional lens arrangement or (b) a fibre-optic taper , for focus onto a CCD.

Figure 2 is a schematic diagram of (a) a biochip containing the biosensors of the invention and (b) biosensor. ; Figure 3 shows plasmid LBS6. Polylinker 1 contains T7, pnl, Apal, Xhol, Sail, Clal and HindII.1; Polylinker 2 contains: EcoRI, Pstl, Smal and BamHI ,- Polylinder 3 contains: Xbal, Notl, Sacl and T3.

Figure 4 shows restriction analysis of luciferase constructs. Insertion of Gaussia luciferase into LBS6 in the incorrect orientation gave fragments of sizes 4620, 1450 and 1160 bp (lanes 1 to 5) when digested with Xbal. Insertion of Pleuromamma luciferase into LBS6 produced constructs containing the luciferase insert in the correct (lanes 6,8,9 and 10 - fragments of 4690, 1732 and 906 bp using Xbal) and incorrect orientation (lane 7- fragments of 4690, 1482 and 1156 bp using Xbal) . Insertion of humanised Gaussia luciferase intθ'LBS6 in the desired orientation gave fragments of 5590 and 1700 bp in size after digestion with Notl (lane 12) .

Figure 5 shows the pPlLUC(2) (1) plasmid. Containing the pPlLUC Pleuromamma luciferase coding sequence in LBS6. Polylinker 1 contains: T7, Kpnl, Apal, Xhol, Sail, Clal and Hindlll; Polylinker 2 contains: EcoRI, Pstl and BamHI (the Smal site is no longer functional); Polylinker 3 contains: Xbal, Notl, Sact and T3.

Figure 6 shows the pcDNA3(2) (1) plasmid. Containing the pcDNA3 Gaussia luciferase coding sequence in LBS6. Polylinker 1 contains: T7, Kpnl, Apal, Xhol, Sail, .Clal arid Hindlll; Polylinker 2 contains: EcoRI ;. Pstl and BamHI (the Smal site is no longer functional); Polylinker 3 contains: Xbal, Notl, Sacl and T3.

Figure 7 shows quantitative in vivo analysis of luciferase luminescence over time in cot-2 strains of Neurospora crassa transformed with one of the two successful luciferase constructs, pPlLUC(2)(l) arid pcDNA3(2) (1) respectively containing Pleuromamma luciferase in LBS6 and humanised Gaussia luciferase in LBS6. The figure legend indicates the luciferase gene present (pPlLUC or pcDNA3) and the transformant number (e.g. pPlLUC#l is cot -2 transformant number 1, containing the Pleuromamma luciferase in LBS6) . . Figure 8 shows quantitative in vivo analysis of luciferase luminescence ^■ over time in wild type strains >of Neurospora crassa strains transformed with the luciferase construct pcDNA3(2) (1) containing humanised Gaussia luciferase in LBS6. The figure legend indicates the luciferase gene present (pcDNA3) , the strain (WT) and the transformant number (e.g. pcDNA3WT#l is wild type transformant number 1, containing humanised Gaussia luciferase in LBS6) . • . .

Figure 9 shows quantitative in vivo analysis of the total luciferase luminescence emitted in 60 minutes by co -2 strains of Neurospora crassa transformed with one of the two successful luciferase constructs, pPlLUC(2) (1) and pcDNA3(2) (1) respectively containing Pleuromamma luciferase in LBS6 and humanised Gaussia luciferase in LBS6. The figure legend indicates the luciferase gene, present (pPlLUC or pcDNA3) and the transformant number (e.g. pPlLUC#l is cot-2 transformant number 1, containing the Pleuromamma luciferase in LBS6) .

Figure 10 shows quantitative in vivo analysis of the total luciferase luminescence emitted in 60 minutes by wild type strains transformed with the successful luciferase construct, pcDNA3 (2) (1) containing humanised Gaussia luciferase in LBS6. The figure legend indicates the luciferase gene present (pcDNA3) , the strain (WT) and the transformant number (e.g. pcDNA3WT#l is wild type transformant number 1, containing humanised Gaussia luciferase in LBS6) .

Figure 11a is a bar graph showing average luminescence of the biosensor and supernatarit in the presence and absence of sodium chloride. Figure lib is a line graph showing luminescence of the biosensor and supernatant in the presence and absence of sodium chloride over time.

Figure 12 shows the luminescence of Neurospora crassa:, constitutively expressing Gaussia luciferase in the: presence of the luminescent substrate coelenterazine and different concentrations of CCCP (cyanide) .

Figure 13 shows the luminescence of Neurospora crassa constitutively expressing Gaussia luciferase in the presence of the luminescent substrate coelenterazine and different concentrations of procaine . This demonstrates that there is negligible response to procaine, and it does not appear to significantly inhibit germination cellular growth.

Describing the apparatus of the invention in more detail, Fig. la shows a laser source (1) directed through a line generating lens (2) and reflected from a mirrored surface (3) onto a specific portion of the biochip (4) . The laser light beam is thus focused onto an individual biosensor specific for a predetermined test substance. The focus of the laser beam may be redirected onto alternative biosensors for measurement of their fluorescent output, for example by movement of the mirror or relocation of the laser source. Such redirection of the laser beam is preferably automated or semi- automated. Where the laser beam causes fluorescence of the particular biosensor illuminated, the fluorescence (indicated .generally as arrow (F) ) is passed through a dichroic or filter (5) and focused by means of a conventional lens arrangement (6) onto a charged coupled device or. CCD (7). Likewise, luminescence output from the individual biosensors, (indicated generally as arrow (L) ) is focused by means of lens arrangement (6) onto CCD (7) . An output device, such_. as a computer, (not shown) displays the results.

A modified arrangement is shown in Fig. lb in which a laser source (1) is again focused into a narrow beam. through use of a line generating lens .(2) and thereby reflected by mirror (3) onto an individual biosensor located on biochip (4) . Optionally a dichroic or filter (5) may be present to selectively filter any fluorescence produced through illumination by laser beam to select a particular optical wavelength. The light passes through a fibre optic taper (8) onto a CCD (7) for measurement. The fluorescence of the biosensor is indicated generally as arrow (F) . Where the biosensor provides luminescence, indicated generally as arrow (L) , this likewise passes through the fibre optic taper (8) onto CCD (7) . Preferably the CCD (7) is permanently attached, by means of glue, onto the fibre. optic taper (8) . The CCD device (7) is preferably thermoelectrically cooled and linked to a computer for direct visualisation of the biosensor outputs.

Fig. 2a shows a biochip (4) containing luminescent or fluorescent biosensors (9) embedded within a suitable matrix. A selectively permeable membrane ' (10) allows gas exchange (for example for the monitoring of toxic gases or pollution) or addition of substrate (for example by injection) . The. biosensor (9) /matrix combination is located on a transparent film (11) which allows transmission of light..from the biosensors to the imaging system as described above in Figs, la and lb.

Fig. 2b shows a magnified view of an individual biosensor (9) which comprises an inert bead (12) upon which are located the living biosensor cells (13) which may be, for example, a filamentous fungal spore.. In the embodiment illustrated addition of substrate causes luminescence and also, in the ' presence of laser exitation, fluorescence to be detected from the biosensor.

The present invention will now be further described by reference to the following, non-limiting, examples. ^' In the example reference is made to the following solutions and media: Liquid Vogel's Media: 4ml Vogel's*50 stock solution, 3 g sucrose, dH₂0 to 200 ml total.

Solid Vogel's Media: 4 ml Vogel's*50 stock solution, 3 g sucrose, 4 g agar, dH₂0 to 200 ml total. Vogel's*50 Stock Solution: ' Dissolve successively in 650 ml dH₂0: 125 g C₆H₅Na₃θ₇'"2H₂θ (tri- sodium citrate dehydrate) , 150 g KH₂P0₄ , (monopotassium phosphate, anhydrous) , 250 g NH₄N0₃: (ammonium nitrate anhydrous) , 10 g MgS0₄'7H₂0 (magnesium, sulphate) , 5 g CaCl₂"2H₂0 (dissolve first in 25 ml H₂0) then add: 5 ml Trace Element Solution, 2.5 ml Biotin solution (0.1 mg/ml in dH₂0) , dH₂0 to a volume of 1 litre plus 2 ml chloroform as a preservative.

Trace!Element Solution: 5 g citric acid"lH₂0, 5 g ZnS0₄ ^*7H0 (zinc sulphate) , 1 g Fe (NH₄) ₂ (S0₄) ₂ ^'6H₂0 (ferrous ammonium sulphate hexahydrate) 0.25 g CuS0₄'5H₂0 (cupric sulphate), 0.05 g MnS0₄"lH₂0 (manganese sulfate) , 0.05 g H₃B0₃ (anhydrous) (boric acid) , 0.05 g Na₂Mo0₄ ^*2H₂0 (molybdic acid sodium salt dihydrate) dH₂0 to final volume of 100 ml plus 2 ml chloroform.

Regeneration Medium (RM) : 4ml Vogel's*50 Stock Solution, 36.8 g sorbitol, 3 g agar, 170 ml dH₂0, autoclave then add 20 ml FGS*10. Plating Medium (PM) : 4 ml Vogel's*50 Stock Solution, 3 g agar, 176 ml dH₂0 autoclave then add 20 ml FGS*10.

FIGS*10: 100 g sorbose, 2.5 g fructose, 2.5 g glucose dH₂0 to a volume of 500 ml.

PTC: 40 g PEG 4000 (polyethylene glycol 4000), 5- ml 1 M Tris-HCl pH 8, 5 ml 1 M CaCl₂ dH₂0 to final vol. of 100 ml.

Protein Extraction Buffer: 5 ml 200 mM ethylene glycol-bis ( β-aminoethyl ether) -N,N,N' ,N' -tetraacetic acid (EGTA) pH 8 , 5 ml 1M Tris pH 7.4, 10 ml 5 M NaCl, 79.8 μl mercaptoethanol, dH₂0 to^' final volume of 100 ml.

STC: 18.2 g sorbitol, 5 ml 1 M Tris-HCl pH 8 , 5 ml 1 M CaCl₂ dH₂0 to final volume of 100 ml.

Protoplast Storage Solution: 8 ml STC, 2 ml PTC, 100 μl DMSO, mix components and filter sterilise.

Nutrient Agar: 0.8 g nutrient broth, 0.8 g agar dH₂0 to total volume of 100 ml.

Liquid LB Medium: 10 g Bacto® Tryptone, 5 g Bacto yeast extract 10 g NaCl dH₂0 to final volume of 1 litre. Solid LB Medium: As Liquid LB Medium plus 15 mg/ml agar.

TAE*50 Buffer for DNA gels: 242 g Tris Base, 57.1 g Glacial acetic acid 100 ml 0.5 M EDTA pH 8, dH₂0 to final volume of 1 litre.

Escherichia coli was grown on solid or in liquid Luria-Bertani (LB) medium at 37°C.' Solid nutrient agar (inoculated with a sterile wooden stick and stored at room temperature) or glycerol stocks (0.5ml. liquid LB culture + 0.5ml 80% glycerol mixed and stored at -80°C) were used for long-term storage of E. ..coli .

Two strains of Neurospora crassa were used in Example 2: (1) Oak Ridge wild-type 74-OR23 1A (FGSC No. 987) and (2) cot-2 (FGSC #1513 (mating type A) . Cultures were grown on Vogel's media (Vogel, 1956) containing 1.5% sucrose and 2% agar where appropriate. For the production of conidia (spores) , Neurospora crassa was grown for 1 to 2 weeks rat room temperature in 250ml flasks containing 50ml solid Vogel's media inoculated with conidia from the appropriate Neurospora crassa strain.

Example 1 : Construction of Plasmids for the expression of luciferase in Neurospora .

The suitability of three luciferase genes for generation of bioluminescent filamentous fungi was assessed. The 3 luciferase genes were: - Pleuromamma luciferase (pPlLUC) - Gaussia princeps luciferase (pGLUC) - humanised Gaussia princeps luciferase (pcDNA3)

Genes, plasmids and DNA

Plasmids containing Gaussia princeps luciferase, human codon optimised Gaussia princeps luciferase and Pleuromamma luciferase in pUC19 were obtained from Prolume LTD (Pinetop, AZ, USA) . http : //www.nanolight . com/nanoligh.htm http: //www.nanolight . com/nanόlight/gaussia_luciferas e.htm

All manipulation of nucleic acids was performed according to standard molecular biology procedures as described by Sambrook et al : (1989) .

The three luciferase genes were excised from the plasmids provided from NanoLight and inserted into plasmid LBS6 (shown in Fig. 3) before being cloned in E. coli using the following protocol. The entire luciferase coding sequence in each of pGLUC, pcDNA3 and pPlLUC was excised from its respective plasmid. In the case of pGLUC and pPlLUC, plasmid DNA was digested with EcoRI and Hindlll. In the case of pcDNA3 , BamHI and Xbal were used. These digestions separated the vector DNA, pUC19 (2686 bp) , from the luciferase coding sequence insert contained in the original plasmids. In the case of pGLUC, the digest liberated a 541 bp fragment containing the entire Gaussia luciferase coding sequence. In the case of pcDNA3 a 590 bp fragment was freed. This fragment contained the humanised Gaussia luciferase coding sequence. In the case of pPlLUC a 628 bp fragment was excised. This fragment contained the Pleuromamma luciferase coding sequence. The digests were cleaned by agarose-gel electrophoresis and the bands corresponding to the luciferase coding sequences were purified from the gel using a QIAquick Gel Extraction Kit (QIAGEN Ltd., UK) according to the manufacturer's instructions. Overhangs on the double-stranded DNA inserts*were filled in using T4 DNA Polymerase (New Englandi'Ωiolabs Inc., USA) according to the manufacturer's instructions.

Plasmid LBS6 (kindly provided by D. Ebbole and constructed by L. B. Shrode) was linearised at polylinker 2 using Smal . LBS6 contains the cpc-1 promoter modified for constitutive expression by deletion of the two upstream open reading frames. After gel purification, linearised LBS6 was dephosphorylated using calf intestinal alkaline phosphatase (CIP) (New England Biolabs Inc., USA) according to the manufacturer's instructions. The blunt ended inserts were then ligated into LBS6 at polylinker 2 between the native Neurospora cpc-1 promoter (modified for constitutive expression) and the TrpC terminator ^' from Aspergillus . Ligation was done with T4 DNA ligase (New England Biolabs Inc., USA) according to the manufacturer's instructions. Each ligation reaction mixture was then used to transform 100 μl of competent E. coli cells, which were then screened for survival on chloramphenicol amended (170μg/ml) LB media. Surviving bacterial colonies' were picked and grown overnight at 37°C in chloramphenicol amended (170 μg/ml) liquid LB media. Plasmid DNA was extracted using a QIAGEN Plasmid Mini Kit (QIAGEN Ltd., UK) and subjected to restriction analysis to determine the number and orientation of the inserts.

Competent cells were produced according to Sambrook et al . (1989) and stored at -80°C. For transformation, competent cells were thawed on ice. 20 μl DNA solution was gently mixed with 100 μl competent cells and incubated on ice for 30. minutes. Cells were subjected to heat shock for 2 minutes at 42°C and incubated on ice for 5 minutes. 1 ml liquid LB medium was added to the transformation mix, which was incubated for 1 hour at 37°C, 160 rpm to allow expression of the chimeric DNA. Solid chloramphenicol amended (170μg/ml) LB plates were inoculated with 50μl and lOOμl of the transformation mix. The remaining mixture was centrifuged in a desktop microfuge at maximum speed for 1 minutes. The pellet was resuspended in lOOμl of supernatant and applied to another chloramphenicol/LB plate. The resulting LB plates were incubated upside-down at 37°C overnight.

LBS6 confers chloramphenicol resistance on cells incorporating it and therefore only transformed cells survive. Plasmid DNA from bacteria transformed with the luciferase insert from pGLUC and pPlLUC plus LBS6 was digested with Xbal . Based on the map of LBS6 (Figure 3) and the sequence of the inserts, restriction fragment patterns were calculated for various ligation scenarios . The expected restriction products were as follows:

Substrate Fragment sizes in bp LBS6 only (Xbal digest) - 2010 and 4690

(Notl digest) - 6700 (linear)

LBS6 + 'pPILUC (correct) 4690, 1732, 906. (incorrect) 4690, 1482, 1156

LBS6 + pGLUC (correct) 4620, 1730, 880. (incorrect) 4620, 1450, 1160

LBS6 + pcDNA3 (correct) 5590, 1700,

(incorrect) 6140, 1150.

In addition there is the possibility of multiple inserts occurring. These variants would also be differentiated using the above restriction analysis

In the case of the luciferase insert from pPILUC, one luciferase insert in LBS6 in the desired orientation (i.e. cpc-1 promotor, luciferase ATG start codon followed by the rest of the luciferase coding sequence and finally the TrpC terminator) should yield 3 restriction fragments: one of 4690, one of 1732 and one of 906 bp . A single insert in the incorrect orientation would give fragments of 4690, 1482 and 1156 bp . Restriction analysis (see Figure 4) revealed that, lanes 7, 9, 10 and 11 contained plasmids with single inserts in the correct orientation. Lane 8 contained a plasmid with a single insert in the incorrect orientation. The colony from which the DNA in lane 10 was extracted, grown and glycerol stocks and nutrient agar stabs were made for long term storage. This construct was named pPlLUC(2) (1) and is shown in Figure 5. Plasmid DNA extracted from E. coli colonies transformed with the luciferase fragment from pGLUC . ligated into LBS6 , was digested with Xbal. All the colonies tested contained single inserts orientated in the incorrect direction. Correct orientation would have given restriction fragments of 4620, 1730 and 880 bp when digested with Xbal. Incorrect orientation, as shown in Figure' 4, lanes 2 to 6, gave three bands of sizes 4620, 1450 and 1160 bp. None of the bacterial colonies from which this DNA ' was extracted were saved.

Plasmid DNA from E. coli colonies transformed with the humanised Gaussia luciferase insert from pcDNA3 ligated into LBS6 was digested with Notl to determine the number and orientation of luciferase inserts. The resulting two bands of 5590 and 1700 bp in size, . indicated one luciferase insert in the correct orientation. If the insert were in the incorrect orientation the bands would be 6140 and 1150 bp in size. Thus, Figure 4 shows that lane 13 contains DNA from a colony that contains the desired pcDNA3/LBS6 construct. This colony was grown and glycerol stocks and nutrient agar stabs were made for long term storage. This construct was named pcDNA(i) (1) and is shown in Figure 6.

Example 2: Transformation of Neurospora Strains

Wild type and cot -2 protoplasts were made from conidia (spores) . Neurospora protoplasts were produced and transformed according to a method adapted from Vollmer and Yanofsky (1986) . Transformed protoplasts were selected and homokaryons purified by growth. for three generations on hygromycin-amended media (150 μg hygromycin/ml media) .

Cot-2 is a colonial temperature sensitive mutant of Neurospora crassa . Cot-2 grows normally and is almost- indistinguishable from wild type at 25°C, but is temperature sensitive at 34°C and above, resulting in cessation of radial growth of the colony, hyperbranched phenotype and produces smaller dense : colonies . This phenotype is useful due to the ability to halt growth. Growth resumes as normal when the temperature is shifted back to 25°C.

Conidia produced as described in '' Fungal Strains and Culture Conditions' were harvested in 50 ml liquid Vogel's media and the resulting solution was passed . through a funnel containing a cheesecloth filter into a l l flask and incubated at 4°C overnight for rehydration. Germination of the conidia was initiated by incubating at 24°C on a shaker (120 rpm) . Once a large proportion of the conidial population had produced ^• germ tubes 1 to 4 conidial diameters in length, the solution was decanted into sterile 50 ml tubes and centrifuged at room temperature at 1400 rpm .for 8 minutes. For each tube, the supernatant was removed, the pellet resuspended in 30 ml sterile distilled water (dH₂0) , and the centrifugation repeated. This wash was performed twice more. After the final wash conidia from all the tubes were combined, resuspended in 1 mg Novozyme™ 234 Cell Wall Lysing Enzyme (Calbiochem-Novabiochem Corporation La Jolla, CA 92039-2087) in 2 ml 1 M sorbitol per 2xl0⁹ conidia (filter sterilised) arid incubated horizontally on a shaker at 55 rpm, 31°C.

Once protoplasts had formed (i.e. spherical cells that burst upon addition of dH₂0 are visible when the solution is examined under the microscope) the ^■ solution was centrifuged for 10 minutes at 800 rpm at 4°C. The supernatant was removed and the protoplasts washed twice by re-suspending in 10 ml chilled 1 M sorbitol and repeating the centrifugation. The pellet was then resuspended in 10 ml chilled STC (i.e. STC "on. ice") and the number of protoplasts per ml estimated using a haemocytometer . The solution was centrifuged once more and the pellet resuspended in a volume of storage solution that gave a final concentration of approximately 10⁷ protoplasts per ml. Protoplasts were divided into 400 μl aliquots and stored at -80°C. 1 For each transformation, 20 μl of heparin (5 mg ml^"1)

2 plus 3 μg of DNA were added to 100 μl of protoplasts

3 and incubated on ice for 30 minutes. 1 ml of PTC was

4 added to the reaction mixture, which was then

5 incubated at room temperature for 20 minutes. The

6 reaction mixture was mixed with 8 to 10 ml RM,

7 poured into a Petri dish containing 15 to 20 ml

8 hygromycin (150 μg hygromycin/ml) amended PM and

9 incubated at 34°C or 24°C for 5 to 10 days. 10^'

11 Plasmid DNA extracted from the two successful

12 luciferase constructs, pPILUC (2) (1) and pcDNA3 (2) :(1)

13 (depicted in Figures 5 and 6 respectively)

14 containing the Pleuromamma luciferase in LBS6.and

15 the humanised Gaussia luciferase in LBS6, was used

16 to transform both wild-type and cot-2 JVeurospora

17 protoplasts. Successful transformation was achieved.

18 using ibcth constructs, based on the ability of

19 transformed colonies to grow on hygromycin (150

20 μg/ml);; amended media. 21

22 Example 3 : Quantitative in vivo analysis of

2.3 luciferase luminescence

24

25 The literature revealed that most quantitative

26 . luciferase measurement is currently performed on .

27 crude protein extracts in vitro using Q/A. quick spin

28 columns (Qiagen Ltd; UK) . Bhaumik and Gambhir (2002) 29 have recently done some in vivo luciferase imaging

30 in mice. Other useful information was found in an

31 article published by Kennedy et al . (1997) Based on •

32 this work along with past experience of the quantitative measurement of aequorin luminescence in living fungi . A method was developed to quantitatively measure luciferase luminescence in living Neurospora hyphae .

Neurospora cultures were grown in the dark for 18 hours at 24°C in 96 well opaque white microtiter plates (DYNEX Technologies, Inc., Chantily, UK) covered by a microplate lid. Each well had a capacity of 350 μl .' Cultures consisted of 100 μl liquid Vogel's media containing 1x10^s conidia ml^"1. This concentration was previously determined to give near-optimal luminescence in aequorin expressing Neurospora strains (Zelter and Read, unpublished data) . In order to synchronise conidial germination, conidia were incubated in Vogel's media overnight at 4°C before use. 30 nmol aliquots of native coelenterazine (NanoFuel™ Coelenterazine, NanoLight™ Technology, Prolume LTD., Pinetop, AZ, USA) were first dissolved in 25 μl pre-cooled methanol in the dark before the addition of 575 μl liquid Vogel's media, giving a final concentration 50 μM. 25 μl of this solution was added to the fungal cultures in the dark, immediately before luminometry. The final coelentrazine concentration was 10 μM. Luminescence of the samples was measured from the moment the microwell plate was placed in the luminometer (about 1 to 2 minutes after addition of coelentrazine) . A repetitive protocol was used to measure the kinetics of light emission over time in all the sample wells simultaneously. Measurement temperature was 24°C, cycle time was 56.6 seconds, integration time was 1 second and total measuring time was 60 minutes. No sample injections were performed using the luminometer' s inbuilt injectors.

All luminometry was carried out in an EG&G Berthold LB96P Microlumat luminometer (Bad Wildbad, Germany) equipped with two 100 μl injectors and calibrated to the optimal working voltage of 1496 V. The luminometer was controlled by a PC running Berthold ' s WinGlow software. ^{• ' "} . ^• Initial transformations and luminescence analysis was performed using cot-2. Figures 7 and 8 show that none of the pPlLUC(2) (1) transformants produced significant luminescence compared to the untransformed control (UT control) . However, several of the' pcDNA3 (2) (1) transformants did produce significant luminescence. The best three cot-2 transformants, pcDNA3#18, pcDNA3#26 and pcDNA3#29 (in descending order) , were kept for further use. It should also be noted that all the luminescent strains snowed maximum luminescence immediately after 'addition of the coelentrazine, and that luminescence decreased from that point . onwards .

Subsequent analysis of luminescence in wild type transformants was performed only on pcDNA3(2) (1) containing cultures. Figures 9 and 10 show that of the 10 transformants tested, 4 strains showed luminescence clearly greater than the untransformed control. The best three wild type transformants, pcDNA3WT#9, pcDNA3WT#10 and pcDNA3WT#5 (in descending order) , were kept for further use.

Example 4 : Sodium ion Dependent Biosensor

Methods Conidia of strain pcDNA3#29 were suspended in Vogel's sucrose medium and 200 μl portions were grown for 18 hours at 25 degrees Celsius in a 96 well plate. In addition to fungal cultures, the supernatant liquid was taken from cultures of pcDNA3#29 that had been grown in liquid Vogel's medium (2% sucrose) for 18 hours at 25 degrees Celsius. Sodium chloride of 1.0% w/v was added to 50% of the wells tested, the other wells contained only nutrient solution + fungus, or supernatant. The 96 well plates were then placed in a Berthold Mithras (TM) Luminometer and 10 μM Coelenterazine was_. added to each well using a robotic injector. Immediately afterwards luminescence was measured for 1 second per well. The process was repeated at 6 minute intervals for a total of 20 hours.

Results • Figure 11a shows a bar graph showing average luminescence of the biosensor in the presence and absence of NaCl. The luminescence of both the biosensor and the supernatant is approximately three times brighter in the presence of sodium chloride. Figure lib shows a comparison of the biosensor and supernatant in the presence or absence of NaCl over time. Immediately following addition of the luminescent substrate coelenterazine, luminescence emission is extremely high. After a period of time (1-2 hours) this is reduced. ^' In the case of the fungal biosensor, the luminescence subsequently increases, presumably as the biomass increases and more Gaussia luciferase is expressed. Conversely, the supernatant luminescence is initially bright, but after 1-2 hours is reduced to effectively zero, and does not recover, presumably because the luciferase is depleted / de-activated. This demonstrates that the fungal biosensor produces a . characteristic luminescence output when actively growing.

The gene construct used to engineer the biosensor contained a secretory peptide and this was believed to result in secretion of the luciferase into external medium, exemplified in the supernatant_. assay. It has previously been demonstrated that the Gaussia, luciferase is sodium dependent and exhibits 3 fold brightness in the presence of (Prolume Ltd) .

The present invention shows that when the Gaussia luciferase is expressed in a filamentous fungus, the presence of sodium chloride increases luminescence. The filamentous fungus is capable of growing in the presence/absence of sodium chloride and this may provide a novel method of control in biosensor assays. The supernatant was also affected by the presence of 1.0% w/v sodium chloride resulting in approximately 3 times brighter luminescence. Conclusions Plasmids containing Pleuromamma luciferase and humanised Gaussia princeps luciferase in'LBS6, a plasmid driving constitutive expression in Neurospora crassa, were .successfully produced. These plasmids were transformed into two strains of Neurospora , wild type and the morphological mutarit cot-2.

A method was developed that, enabled the quantitative measurement luciferase luminescence in living Neurospora cultures . This method was used to determine the three most luminescent transformants in each strain. The three most luminescent cot-2 strains were: (1) pcDNA3#18, (2) pcDNA3#26 and (3) pcDNA3#29. The three best wild type strains were: (1) pcDNA3WT#9, (2) pcDNA3WT#10 and (3) pcDNA3WT#5. Although the correct vector for expression of Pleuromamma luciferase in Neurospora was produced, Pleuromamma luciferase appears not to be functional in Neurospora . This could be a result of factors such as codon usage that is incompatible with that of Neurospora .

Transformants that showed luminescence tended to produce most light immediately after the addition of coelenterazine. This indicates that the fungus takes up- the coelenterazine rapidly. Consequent reduction of luminescence may be due to decreasing levels of coelenterazine, or to the scarcity of other, as yet unidentified, components that are necessary for luciferase activity. Example 5: fungal luminescence in the presence of a toxicant

Materials and Methods Neurospora crassa constitutively expressing the Gaussia luciferase (Strain PCDNA-329) was used throughout these experiments . Spores were harvested from cultures grown on nutrient agar (2% sucrose and Vogel's salt solution) containing hygromycin (0.3%) . Approx 0.2 mg of spores were . suspended in 10 ml of liquid nutrient broth (2% sucrose, 2% Vogel's. salt solution and 1% sodium chloride) . 150 μl portions were pipetted into wells of a 96 well plate and 50 μl of the luminescent substrate coelenterazine was added to give a final concentration of (20 μg/ml) . Serial dilutions (in 50 μl portions) of the toxicant were then added to wells, and 50 μl of distilled H₂0 was added to other, wells as a control. The plate was then placed in a "Berthold Luminograph" luminometer and the light output of each well was measured for 1 second. The cycle was repeated over 18 hours, with measurements every 30 minutes. Data was then. exported from Berthold Winglow software and plotted using. Microsoft Excel.

Spores were hydrated in the presence of a different compounds and the luminescence was measured over a period of 20 hours.^'

Results The results are shown in Figs. 12 and 13. The . average control line is the standard luminescence curve observed following germination of untreated, healthy spores . The increase in light reflects the increase in cell density and expression of luminescent protein. The fall in luminescence around 18 hours is due to depletion of the luminescent substrate .

Figure 12 shows luminescence of the fungal biosensor over time in the presence of 0.0015, 0.015, 0.15, 1.5 or 15 ppm of the toxic compound CCCP (cyanide)

High concentrations of CCCP resulted in complete inhibition of light due to cellular death. The lowest concentration (7.3 nM or 1.5 parts per billion) showed 50 % inhibition after approximately 4 hours . ^' Figure 13 shows the luminescence of the fungal biosensor over time in the presence of 0.02., 0.2, 2.0, 20 or 200 ppm of the anaesthetic procaine. In the presence of procaine, an anaesthetic, only minimal reduction in luminescence was noted at all concentrations tested, indicating low cytotoxicity.

References

Bhaumik, S and Gambhir, S. S. 2002. Optical imaging of Renilla luciferase reporter gene expression in living mice. PNAS 99: 377-382 Kennedy, H. J. , Viollet, B., Rafiq, I., Kahn, A. and Rutter, G., A. 1997. Upstream stimulatory factor-2 (USF2) activity is required for glucose stimulation if L-pyruvate kinase promoter activity in single living islet β-cells. JBC 272: 20636-20640.

Sambrook, J., Fritsch, E. F., and Maniatis, T. 1989. Molecular Cloning : A Laboratory manual , 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

Vogel, H. J. 1956. A convenient growth medium for Neurospora (medium N) . Microbiol . Genet . Bull . 13: 42-43

Vollmer, S. J., and Yanofsky, C. 1986. Efficient ^■ cloning of genes of Neurospora crassa . Proc . Natl . Acad . 'iSci . USA 83 ;^■ 4869-4873.

Claims

1. A biosensor comprising luminescent or fluorescent filamentous fungi wherein the light output varies in response to the presence or absence of. a predetermined test substance.

2. The biosensor claimed in Claim 1^' comprising fluorescent filamentous fungi .

3. The biosensor claimed in Claim 1 comprising luminescent filamentous fungi.

4. The biosensor claimed in Claim 3 wherein said filamentous 'fungi produces a luciferase.

5. The biosensor claimed in Claim 4 wherein said luciferase is a Gaussia luciferase or . a Pleuromamma luciferase.

6. The biosensor as claimed in any one of Claims 1 to 5 wherein the fungus is Aspergillus sp . , Neurospora sp . , Magnaporthe grisea or Sclerotinia sclerotiorum .

7 . The biosensor as claimed in any one of Claims 1 to 6 wherein the luminescence or fluorescence of the fungi i s ATP independent .

8. The biosensor as claimed in any one of Claims 1 to 7 wherein expression of a gene encoding a luminescent or fluorescent protein in the fungus is driven by an inducible promoter or enhancer.

9. The biosensor as claimed in Claim 8 wherein the fungus is genetically engineered to comprise a coristitutive or inducible promoter or enhancer genetically linked to the gene encoding the luminescent or fluorescent protein.

10. The biosensor as claimed in any one of Claims 1 to 9 wherein the luminescence or fluorescence of said filamentous fungi is increased in the presence of said test .-substance.

11- tThe biosensor as claimed in any one of Claims -1 to 9 wherein the luminescence or rfluorescence of said filamentous fungi is -decreased in the presence of said test -.substance.

12. The biosensor as claimed in any one of Claims 1 to 11 wherein the filamentous fungi is in the form of an array.

13. The biosensor^' as claimed in any one of Claims 1 to 12 which includes an indicator to show the viability of the fungus.

1 14. An assay to determine the presence of a

2 predetermined test substance in a sample, said

3 assay comprising: 4

5 a) exposing the sample to a filamentous

6 fungus having an ability to luminesce or

7 fluoresce wherein the luminescent or

8 fluorescent output will vary depending

9 upon presence or absence of the test

10 substance; and

11 b) measuring the luminescent or fluorescent

12 output from the fungi, and determining 13_. therefrom the presence of the test

14 substance in the sample .

15

16 15. The assay as claimed Claim^' 14 further

17 including positive' and negative controls . 18

19 16. The assay as claimed in either one of. Claims 20 14 and 15 wherein spores of the fungus are

21 immobilised in an array.

22

23 17. The assay as claimed in either one of Claims

24 14 and 15 wherein said fungus is in the form

25 of a fibrous mat. 26

27 18. The assay as claimed in any one of Claims 14

28 to 17 wherein the light output is measured by

29 a CCD camera, photodiode, photomultiplier or

30 luminometer.