COMPOSITION

Abstract

The invention provides light-emitting compositions, including lasing and fluorescent compositions. The invention particularly relates to programmable biological substrates, which fluoresce and/or lase, and which have a wide variety of different applications. The invention extends to use of the fluorescent compositions and lasing compositions comprising programmable biological substrates in fabricating lasers, and in various biological imaging applications, such as in assays.

Claims

1-34. (canceled)

35. A method of using a laser, the method comprising disposing a gain medium in the laser and causing it to lase, wherein the gain medium comprises a biological substrate chemically modified at specific attachment sites with light-emitting labels.

36. A method according to claim 35, wherein the distance between adjacent labels on the biological substrate is such that they are unable to chemically react with each other, but can allow dipole-dipole interactions to occur between adjacent labels, and/or wherein the positions of the attachment sites create a repeating pattern along the substrate, and wherein the attachment sites are not randomly arranged along the substrate.

37. A method according to claim 35, wherein the labels are attached to specific, spaced-apart attachment sites, which are disposed along the structure of the biological substrate, optionally wherein the attachment sites are amino acids, or a side chain thereof, and are regularly spaced apart along the substrate, and wherein the distance between adjacent attachment sites is substantially the same along the substrate.

38. A method according to claim 35, wherein the average molecular diameter of the light-emitting labels is between about 0.5 nm and 2 nm and/or the average distance between adjacent light-emitting labels is between about 1 nm and 15 nm.

39. A method according to claim 35, wherein the light-emitting labels are capable of absorbing light of wavelength between 220 nm and 1000 m.

40. A method according to claim 35, wherein the light-emitting labels are attached to the substrate due to the presence of covalent bonds between the light-emitting labels and functional groups of amino acids present in the biological substrate.

41. A method according to claim 35, wherein the light-emitting labels (a) comprise a fluorophore, (b) are members of the xanthene family of dyes, (c) comprise GFP or quantum dots, (d) comprise rhodamine or a derivative thereof, and/or (e) comprise fluorescein or a derivative thereof.

42. A method according to claim 35, wherein more than one type or species of light-emitting labels are scaffolded to the substrate.

43. A method according to claim 35, wherein the biological substrate comprises a peptide, protein, nucleic acid, or any combination thereof.

44. A method according to claim 35, wherein the biological substrate is proteinaceous or comprises a protein-nucleic acid complex or conjugate.

45. A method according to claim 35, wherein the biological substrate comprises a wild-type or mutant biological substrate, including a bacteriophage, actin fiber, biomimetic compound, or other proteinaceous substrate.

46. A method according to claim 35, wherein the biological substrate comprises M13 filamentous bacteriophage (M13).

47. A method according to claim 35, wherein the biological substrate displays a fusion protein on its surface.

48. A method according to claim 35, wherein the biological substrate is configured to bind to a target.

49. A method according to claim 48, wherein the target is a biological target.

50. A method according to claim 48, wherein the gain medium further comprises the target, and the method further comprises detecting laser emission from the gain medium and quantifying the concentration of the target in the gain medium based upon the detected laser emission.

51. A method according to claim 35, wherein the laser is a dye laser.

Description

[0063] For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying diagrammatic drawings, in which:

[0064] FIG. 1a illustrates the M13 major coat protein assembly, and FIG. 1b illustrates the structure of the M13 major protein;

[0065] FIG. 2 illustrates the eigenstates of fluorescein. Radiative and non-radiative transitions are shown in solid and dash lines, respectively. Internal conversion is represented by the sinusoidal lines;

[0066] FIGS. 3a-c show the chemical modification of wild-type M13 to form M13-fluorophage. In particular, FIG. 3a shows NHS-fluorescein attached via an amide bond to the N-terminus or to the functional group of a lysine residue. FIG. 3b shows a model of a reaction between NHS-fluorescein and the amines exposed at the surface of M13. Residues with an amine are shown in bold. FIG. 3c is a schematic drawing detailing the position of addressable chemical linkage sites on the surface of M13;

[0067] FIG. 4 shows FLIM decay curves;

[0068] FIG. 5a shows fluorescence spectra, and FIG. 5b shows fluorescence over time under continuous pumping: only a fraction of the emitted fluorescence is allowed to reach the detector so that it does not become saturated; as such, only the relative change in intensity is important. Con. Dye=1 mg/ml; diluted dye=10 g/ml;

[0069] FIG. 6 is a schematic diagram of a tunable dye laser setup with a prism and fluorophage cuvette; and

[0070] FIG. 7 is a schematic diagram of a passive mode-locked continuous wave dye laser setup with fluorophage cuvette, acting as a gain medium;

[0071] FIG. 8 shows the use of BB-fluorophage as a secondary antibody;

[0072] FIG. 9 shows UV-Vis absorption spectrum used in fluorophage threshold experiments;

[0073] FIG. 10 is a schematic diagram of a laser setup for conducting threshold measurements;

[0074] FIG. 11 is a threshold curve for M13-fluorophage according to the invention;

[0075] FIG. 12 is a schematic representation of a genetic construct known as Tobacco Mosaic Virus (TMV) coat protein (CP) with C27A and C127C mutations;

[0076] FIG. 13 shows the structure of the TMV coat protein (TMVCP) showing the C27A and M127C mutations, and RNA packaged by the coat protein. Crystal structures were taken from protein data bank 2OM3;

[0077] FIG. 14 is a schematic representation of a genetic construct for a TMV RNA transcript;

[0078] FIG. 15 shows the structure of the helical rods made when the protein monomers from FIG. 12 self-assemble to package the RNA transcript. The view is from looking down the rod;

[0079] FIGS. 16-18 are transmission electron micrographs of recombinant TMV (rTMV). The rTMV molecules can be easily identified as the ring structures shown in each Figure; and

[0080] FIG. 19 shows reaction schemes used for the modification of rTMV with dyes.

EXAMPLES

[0081] As described in the Examples below, the inventors have developed a composition (referred to herein as a fluorophage) in which a light emitting label or dye molecule (e.g. fluorescein) is conjugated to a programmable biological substrate (e.g. M13 bacteriophage, or Tobacco Mosaic Virus). The light-emitting labels or dye molecules are regularly spaced-apart to form an array on the biological substrate, which thereby acts as a scaffold structure holding the dye molecules in position. The inventors have analysed the light-emitting and/or fluorescence characteristics of the fluorophage, and have used it as a dye in a dye laser, and instead of molecular dyes for biosensing and biological imaging applications. The lasing properties of the fluorophage make an excellent candidate as a light-emitting composition in a wide range of applications.

Example 1Preparation of an M13-Fluorophage

[0082] The dye used was 5-carboxyfluorescein, succinimidyl ester (5-FAM, SE) powder (Invitrogen). Herein, the term dye and light-emitting label are used interchangeably. It was dissolved in spectroscopic grade dimethyl sulfoxide (DMSO) (VWR) to a final concentration of 10 mg/ml. Referring to FIGS. 1a and 1b, there is shown the structure of the M13 major coat protein assembly. M13 was therefore propagated in a 400 ml culture of E. coli Top10f in Terrific Broth at 37 C. with shaking. The M13 was then purified using standard methods to a final titer of 2.20.910.sup.13 pfu/ml. All buffers were autoclaved and filtered sterilized to remove any contaminants larger than 0 22 m.

[0083] 19 l of the 5-FAM, SE stock dye was added to 100 l of the M13 phage stock and left for one hour at 37 C. with shaking in the dark to form the M13-fluorophage. The reaction was conducted at pH 7.5 to encourage single labelling of the coat proteins. The reaction was quenched with 1 M TRIS pH 8.5. The reaction was subjected to centrifugation to remove any insoluble reaction products or polymerised dye. The sample was then precipitated using 5% PEG-8000/200 mM NaCl and resuspended. The sample was subjected to further rounds of precipitation and resuspension until the supernatant was colourless by sight. The sample was then buffer-exchanged using a Zeba desalt spin column (Thermo Scientific) and subjected to a final precipitation with 5% PEG-8000/200 mM NaCl to concentrate the sample.

[0084] The M13-fluorophage samples and free 5-FAM, SE molecular dye samples were loaded into square capillaries with widths of 0.7 mm by capillary action. The fluorophage sample was shaken to move the sample into the middle of the capillary. The loaded capillaries were then left in the dark at 4 C.

[0085] Referring to FIGS. 3a-3c, there are shown the attachment of fluorescein (i.e. NHS-fluorescein) via an amide bond to the N-terminus or the functional group of a lysine residue. NHS-fluorescein is a N-Hydroxysuccinimide ester labelling reagent and can form stable amide bonds with primary amines, releasing N-hydroxysuccinimide as shown in 3a. There are two surface exposed primary amines on the surface of M13 as shown in 3b. These can be selectively modified because the amines on the N-terminus and lysine functional group have different pKa values. An array of dye molecules is formed because the chemically addressable sites form a well ordered array as shown in 3c.

Example 2Fluorescence Lifetime Imaging Microscopy (FLIM) using M13-Fluorophage

[0086] Fluorescence lifetime imaging microscopy (FLIM) was conducted on a Zeiss Axioskop META 510 NLO microscope with a Becker and Hickl FLIM system to test whether the M13-fluorophage prepared in Example 1 has decreased triplet state lifetimes and decreased steady state triplet state populations compared to free dye molecules in solution.

[0087] Referring to FIG. 4, there is shown FLIM decay curves of the free dye (5-FAM, SE) and the M13-fluorophage prepared in Example 1. As can be seen, there is one component to the fluorescence lifetime of free fluorescein dye molecules of approximately 3 ns but there were two approximately equal components to the fluorescence lifetime of M13-fluorophage of approximately 1 ns and approximately 2 ns. It will there be appreciated that the M13-fluorophage decays faster, and therefore has more deactivation pathways. This reduction in the lifetime for M13-fluorophage is due to the dipolar interactions between the scaffolded dyes, which open up more deactivation pathways.

[0088] In summary, this experiment measures the time taken for electrons in the excited states to return to the ground state. The shorter the time, the more deactivation pathways are available for electrons to return to the ground state. On the fluorophage of the invention there are more deactivation pathways because of the dipole-dipole interactions between neighbouring dyes. These dipole-dipole interactions are important for quenching the triplet state, and these experiments prove that they are there.

Example 3Confocal Microscopy using M13-Fluorophage

[0089] Fluorescence spectra and fluorescence time series were acquired using an Olympus Confocal Inverted Laser Scanning Microscope with a 10 objective lens to test whether the M13-fluorophages prepared in Example 1 have decreased photobleaching rates compared to free dye molecules in solution.

[0090] Referring to FIG. 5, there is shown fluorescence spectra (FIG. 5a), and fluorescence over time under continuous pumping (FIG. 5b). For each sample, the fluorescence spectra were measured. All of the samples were pumped with a 25 mW laser set at 40% power except the concentrated dye sample which was pumped with the same laser set at 1% power. As can be seen, the fluorescence peak of the concentrated free molecular dye solution (5-FAM, SE) and both of the M13-fluorophage samples were red-shifted compared to the peak of the diluted solution. The most red shifted fluorescence peak was the M13-fluorophage sample with 5% PEG-8000/200 mM NaCl. For each sample, the intensity of the fluorescence over time under continuous pumping with a 25 mW laser set at 40% power was measured. The mean separation of the dye molecules is significantly greater for the diluted dye sample than for the concentrated dye sample (see Table 1). The number of dyes per phage is an estimate, and is included to illustrate that the density of dyes on the surface of the phage is closer to that of the concentrated dye than the diluted dye. Consequently, collisions between dye molecules in the excited triplet state are much rarer for the diluted dye sample, which causes a reduction in the rate of photobleaching.

TABLE-US-00001 TABLE 1 Concentration and mean separation of dye molecules Concentration Mean separation Concentrated dye 1 mg/ml 9.2 nm Diluted dye 10 g/ml 42.5 nm M13-fluorophage 103 dyes per phage 4.5 nm

[0091] As shown in FIG. 5, the diluted molecular dye sample and the M13-fluorophage sample with 5% PEG-8000/200 mM NaCl underwent a comparable rate of photobleaching. However, the mean separation of the diluted dye molecules was approximately ten times greater than that of the dye molecules scaffolded to the surface of M13. For the M13-fluorophage, collisions between dye molecules in the excited triplet state are rare because the dyes are scaffolded. For the diluted dye sample, collisions are rare because the mean separation of the dye molecules is so great.

[0092] The M13-fluorophage sample without 5% PEG-8000/200 mM NaCl had significantly lower rates of photobleaching than the concentrated molecular dye sample despite having dye molecules scaffolded to a comparable mean separation. This indicates that the dye molecules on the surface of the M13-fluorophage were not able to exchange electrons with neighbouring dye molecules.

[0093] The rate of photobleaching is reduced for M13-fluorophage samples containing 5% PEG-8000/200 mM NaCl. PEG-8000 is a molecular crowding agent that causes the local concentration of M13-fluorophage to increase. M13 forms liquid crystals at high concentrations so it is reasonable to anticipate that M13-fluorophage is also capable of forming crystal structures. The hydrogen bonding network between M13-fluorophage in a liquid crystal is likely to prevent fluorophage-fluorophage collisions, which explains why M13-fluorophage in solutions with 5% PEG-8000/200 mM NaCl undergo an even greater reduction in their rate of photobleaching.

[0094] Over time, the intensity of the fluorescence would tend towards zero if the dyes were unable to diffuse away from the laser path. However, as the dyes can diffuse away from the laser path, the intensity reaches an asymptote where the rate of fresh dye diffusing into the path of the laser matches the rate of photobleached dye out of the path. This effect is more pronounced for the molecular dyes because they are much smaller than M13-fluorophage and so can more readily diffuse out of the path of the laser beam.

Example 4Use of Fluorophage in Dye Lasers

[0095] Based on the data produced from Examples 2 and 3, the inventors have shown that the fluorophages of the invention, as prepared by Example 1, can be readily used instead of molecular dyes in dye laser systems. Two types of dye lasers with commercial value are tunable lasers and ultrashort pulse lasers, which are illustrated in FIGS. 6 and 7, respectively. The inventors have shown that a dye cell containing the fluorophage of the invention can be placed in the beam path of either laser system, which benefit from a number of advantages, as discussed below.

[0096] (i) Tunable Lasers

[0097] Tunable dye lasers can emit at a broad range of wavelengths, and an example of such a laser 2 is shown in FIG. 6. The fluorophage 12 is pumped by an excitation beam 4 of the appropriate wavelength. The excitation beam 4 is passed through a dispersive element, in this case a prism 6, so that the beam refracts towards a first mirror 8. The excitation beam is reflected towards a cell 10 containing the fluorophage 12. Emission from the fluorophage 12 is reflected at mirrors 14 and 8 towards the prism 6. The emission from the fluorophage 12 is at a different wavelength to the excitation beam so the angle of refraction from the prism 6 is different. Mirror 18 is rotated to tune the optical cavity so that losses for one particular wavelength are much smaller than for other wavelengths. These mirrors 8, 14, 18 and the prism 6 make up the optical cavity. Mirror 14 is not 100% reflective so output light 16 is transmitted through mirror 14.

[0098] Tunable lasers 2 are especially useful in applications where scattering is observed as a function of wavelength. Dye lasers can be used to make tunable ultraviolet and infrared lasers sources by frequency mixing in non-linear materials. Applications of tunable dye lasers, where the fluorophage dye cell shown in FIG. 6, could be employed include: [0099] Raman scattering experiments; [0100] Observing the resonant scattering from atoms and molecules; [0101] Atomic absorption and fluorescence spectroscopy; [0102] Optical pumping at wavelengths inaccessible to diode lasers; [0103] Photomagnetism experiments; [0104] Generation of tunable UV and IR laser light; [0105] Photochemistry; [0106] LIDAR; and [0107] Laser spectroscopy.

[0108] (ii) Ultrashort Pulse Lasers

[0109] With reference to FIG. 7, picosecond and femtosecond lasers 20 are useful for making time resolved measurements. The fluorophage 12 is pumped by an excitation beam 22 of the appropriate wavelength. The excitation beam 22 is passed through a dispersive element, in this case a prism 24, so that the beam refracts towards a first mirror 26. The excitation beam is reflected towards a cell 28 containing the fluorophage 12 Emission from the fluorophage 12 is reflected at mirrors 32 and 26 towards the prism 24. The emission from the fluorophage 12 is at a different wavelength to the excitation beam so the angle of refraction from the prism 24 is different. The emission from the fluorophage is reflected at mirrors 34, 36 towards a saturable absorber 38. The saturable absorber is responsible for passively mode-locking the system. Mirror 40 makes up the optical cavity along with mirrors 32, 26, 34, 36 and the prism 24. Mirror 40 is not 100% reflective so output light 42 is transmitted through mirror 40.

[0110] Ultrashort pulse lasers 20 can be used to measure the relaxation times of atoms and molecules and to monitor chemical reactions. Applications of ultrashort pulse lasers where the fluorophage dye cell can be employed include: [0111] Time resolved fluorescence; [0112] Non-linear spectroscopy; and [0113] Pump-probe experiments.

[0114] The inventors have shown that fluorophage dye lasers 2, 20 have a number of major advantages over conventional dye lasers, including:

[0115] (i) The fluorophage dye does not photobleach as quickly as conventional dye molecules, so they do not need to be pumped through the optical cavity;

[0116] (ii) The fluorophage dye could be cast into viral films to make a solid state gain medium;

[0117] (iii) Tunable fluorophage dye lasers are able to sustain longer pulse lengths than conventional dye lasers.

[0118] (iv) The previous generation of dye lasers required the operator to pump toxic solvents around the lasers system, and so they developed a reputation for not being user friendly.

[0119] The fluorophage lasers 2, 20 shown in FIGS. 6 and 7 overcome the limitations of the previous generation of dye lasers because they do not require dye to be pumped through the optical cavity.

[0120] The market for lasers is very broad because lasers have so many applications. Laser manufacturers, such as Coherent and Spectra-Physics (Newport Corp), are especially keen to acquire and develop technologies that give them an advantage over the competition. Ti:Sapphire lasers are the industry standard for generating ultrashort pulses. However, even after frequency doubling, these lasers have wavelengths greater than 350 nm, which is too long to excite intrinsic fluorescence in proteins.

Example 5Use of Fluorophage in Biosensors and in Biological Imaging

[0121] The fluorophage of the invention can also be used instead of molecular dyes in a range of biosensing and biological imaging applications. For example, protein fusions can be displayed at the surface of the fluorophage.

[0122] Referring to FIG. 8, there is shown the use of the fluorophage in a biological assay. The g3p coat protein of the fluorophage has been genetically engineered to display the BB-domain from protein A of Staphylococcus aureus in a construct denoted as BB-M13 fluorophage 50. This BB-M13 fluorophage 50 then binds IgG antibodies 52 bound to a support surface 54 and is used in the same way as secondary antibodies to detect proteins in biological assays, including Western blots and ELISA. As shown in FIG. 8a, a solution containing the fluorophage 50 is added to the antibodies 52 bound on the support surface 54 to allow binding, as shown in FIG. 8b. The support 54 is then washed to remove unbound fluorophage 50, as shown in FIG. 8c. Finally, the amount of bound fluorophage 50 can then be quantified based on the fluorescence of the fluorophage 50, as shown in FIG. 8d. Importantly, the amount of bound fluorophage can be quantified by the laser emission of the fluorophage. For example, the fluorophage 50 can be excited by a laser 60, and laser emission 62 can be detected via a photodiode 64. This would not be possible usually because conventional dyes would be too sensitive to photobleaching. An assay based on laser emission would have a much superior signal to noise ratio. The main advantage of this method is the far greater sensitivity to lower protein concentrations and a more quantifiable, repeatable emission compared to using the chemiluminescence from an enzyme-based secondary antibody system.

[0123] In the case of a lasing fluorophage, this can offer an entirely new means of obtaining contrast in an image. Organic dyes remain the molecules of choice for biological imaging, where fluorescent labels are necessary for the confocal microscopy of biomolecules and some biosensors and immunobiological assays. Inorganic quantum dot labels are making inroads, but they must be packaged to mitigate against toxicity, and subsequently functionalized to attach to the sites of interest, yielding moieties much more unwieldy than organic dye molecules. In particular, derivatives of dyes are available which allow the dye to be conjugated to the functional groups on proteins. Nonetheless, the photobleaching of dyes in confocal microscopy can prove problematic in some cases.

[0124] The inventors have shown that the fluorophage of the invention can make a big impact in the biosensing and biological imaging fields. Advantages of fluorophage dyes over conventional dyes include:

[0125] (i) The fluorophage is less susceptible to photobleaching than conventional dyes;

[0126] (ii) The fluorophage is brighter than single dye molecules because there are over a hundred (or thousand) molecular dyes attached to the biological substrate per fluorophage;

[0127] (iii) The fluorophage can be programmed to bind to biological and non-biological targets;

[0128] (iv) Unlike assays that use enzyme-conjugated secondary antibodies, the amount of fluorescent emission from a fluorophage does not depend on temperature, exposure time or other experimental variables.

[0129] Dyes are used ubiquitously as fluorescent labels for assays and confocal microscopy in bio labs worldwide. The fluorophage of the invention represents a better alternative to molecular dyes for many of these applications. Optical based assays are sold by many biotech companies, including Thermo Scientific and Merck. In addition, since a single fluorophage can act as a laser, because fluorophages are less susceptible to photobleaching, this would be revolutionary for biosensing and microscopy, since the signal to noise ratio would be greatly enhanced in comparison to the fluorescence of a single dye molecule.

Example 6Demonstration of a Fluorophage Laser

[0130] Preliminary evidence that the compositions of the invention could sustain laser action was acquired. Fluorescein isothiocyanate was titrated into a M13 phage solution prepared as in the previous examples. Reactions were quenched with 1M pH 7 TRIS. Insoluble products were removed through two centrifugation steps. After the second centrifugation step, only a small pellet precipitated to the side of the tube. Fluorophage was separated from smaller molecular weight products by 5% PEG-8000/200 mM NaCl precipitation.

[0131] As shown in FIG. 9, the UV-Vis absorption spectrum of the sample contains a characteristic fluorescein peak close to 490 nm (Digilab Hitachi U-1800 spectrophotometer). The ratio of the characteristic M13 phage peak at 269 nm and the fluorescein peak at 490 nm indicates that there were approximately 265 dyes per phage. The fluorophage solution had an optical density equivalent to a 23.7 g/ml fluorescein solution.

[0132] A dye laser 100 was built to test whether the composition was capable of sustaining laser action, and this is shown in FIG. 10. The fluorophage 12 is stored in a dye reservoir 102 and continuously pumped via peristaltic pump 104 through silicone tubing 106 to/from a flow cuvette 108. The continuous circulation of the fluorphage 12 is represented in the Figure by the arrow shown as dye flow. An excitation beam 4 of the appropriate wavelength is then directed towards the fluorophage 12 within the cuvette 108. The excitation beam 4 exits the cuvette 108 towards an aluminium confocal mirror (f=150 mm) 112 and then to a flat mirror 110. The beam 4 then passes through a phototransistor 114 and ultimately to a detector 116.

[0133] Threshold behaviour was observed, which is a characteristic property of a laser, as shown in FIG. 11. Hence, the inventors have demonstrated that the fluorophage is clearly capable of lasing.

Example 7Engineering an Improved Biological Substrate for Lasing

[0134] The fluorophage concept was demonstrated using M13 because it is a readily available, well-understood biological substrate, and it clearly showed that the M13 could act as a good biological substrate for producing a light emitting (i.e. lasing) composition. However, the inventors then set out to show that other biological substrates could also be used. Using principles taken from synthetic biology, multiple biological components can be augmented to fabricate an improved biological substrate.

[0135] Dyes or light-emitting molecules can be attached to specific sites on the coat protein of a plant virus called Tobacco Mosaic Virus (TMV) such that there is no contact quenching between neighbouring dyes. This has inspired the design and fabrication of a recombinant Tobacco Mosaic Virus-like particle (rTMV) that can be expressed in E. coli.

[0136] Two genetic constructs were designed and constructed using synthetic genes and standard molecular cloning techniques. The first construct (shown in FIG. 12) is a codon-optimised TMV coat protein gene with mutations at C27A and N127C with an IPTG inducable T5 promoter. The mutations introduce a unique attachment site for thiol-reactive molecular probes, including dyes and other light-emitting molecules. The crystal structure of the TMV coat protein is shown in FIG. 13, and shows that the attachment sites are introduced near to the RNA binding site. For the coat protein to self-assemble into rods of controllable size, a packaging RNA transcript is required.

[0137] The second construct shown in FIG. 14 is a gene containing no ribosome binding sites (rbs) with the TMV coat protein specific origin of assembly sequence flanked by 5 and 3 end protection sequences under the control of an L-arabinose induceable pBAD promoter. A 5 hairpin looppHP17 with the rbs removedwas incorporated for 5 end protection because it has been demonstrated to improve the half-life of RNA transcripts to nearly 20 min. The 3 end tRNA-like structure from the native TMV genome was included in this synthetic construct to offer 3 end protection. A strong terminator from the Register of Standard Biological Parts, BBa_B0015, ensures homogeneity in the length of RNA transcripts. Restriction sites are available for the cloning of non-coding spacer DNA which would increase the length of the rTMV. However, no spacer DNA was included in the example provided here.

[0138] When co-expressed, the RNA transcript and the coat protein form rTMV. The crystal structure of the native virus (shown in FIG. 15) shows that the attachment sites are on the underside of the protein after assembly. As such, dyes attached at these sites cannot interact with dyes attached in the layer above them because the protein sterically hinders this interaction. The helix between the proline at position 20 and the alanine at position 30 can also be seen to sterically hinder potential interactions between dyes attached to neighbouring coat proteins. Both constructs were transformed into E. coli Top 10 cells using standard techniques and single colonies were isolated. An overnight culture of these cells in Terrific Broth was grown overnight at 37 C. in a shaking incubator and was added to 50 ml expression medium in a baffled flask to a final concentration of 1%. The expression medium contained 36 g/L yeast extract, 18 g/L peptone, 2m1/L glycerol, 100 mM TRIS buffer pH7, 25 g/ml ampicillin and 10 g/ml kanamycin. After a three hour pre-induction phase at 37 C. in a shaking incubator at 250 rpm, L-arabinose and IPTG were added to final concentrations of 0.01% and 1 mM respectively. After six hours, cells were harvested and lysed overnight at 4 C. in a lysozyme, DNase I lysis solution. After centrifugation for 30 min at 10,400g (Eppendorf 5810 R) to remove the insoluble fraction of the lysate, the supernatant was subjected to a 4 g/10 ml CsCl density gradient in an ultracentrifuge (Beckman Coulter Optima L-100 XP, Type 70.1 Ti rotor) at 35,000 rpm. A clear band was formed between the anticipated locations of the RNA and protein fractions, which was extracted. The density of this band was consistent with a RNA-protein complex, such as rTMV. After desalting in a PD-10 column (GE Healthcare), samples were inspected by transmission electron microscopy. rTMV rods can be readily identified by their characteristic o shape (see FIGS. 16-18), confirming the successful fabrication of rTMV.

Example 8rTMV Fluorophage Lasers

[0139] The inventors then used the following steps for the production of an rTMV fluorophage laser. Light emitting molecules, including dyes, having a thiol reactive moiety were attached to the cysteine residue. The reaction schemes are outlined in FIG. 19. The Figure shows the reaaction between a cysteine residue on the rTMV and a thiol group on the probe (e.g. maleimide) in a bioconjugation reaction. FIG. 19 shows three alternative thiol containing light-emitters, including fluorescein, tetramethylrhodamine and 7-diethylamino-3-(((ethyl)amino)carbonyl)coumarin. After purification in a size exclusion column or in a density gradient, the chemically modified rTMV was then tested for its lasing characteristics in the same way M13-fluorophage was tested in Example 6.