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ABSTRACT
The following patent application refers to the methods and compositions relating to a novel use for enzymatic catalysis of C21-H30-O2 (delta-nine tetrahydrocannabinol (THC)) as an insect repellent, bactericide, and fungicide and dispensation methods as commercial reagent.
The bio-synthesis of cannabinoids represents a landmark achievment in the field of composting, vector removal and ecological reconstitution. Although the benefits have been known for millennia,the advent of modern bio-engineering techniques brings these small seeds of native wisdom to bear on a broader and more industrialized scale - removing dangerous molds and pestilences such as mosquitoes from swamped and flooded regions, raw sewage areas, and disaster sites where ensuing vermin and harmful vectors may cause greater damage than the initial catastrophes.
It is the ambition and intention of authors that tactical usage of this broad-sweeping technique may rapidly and at low cost satisfy a global demand in what may be termed a “grass-roots”bio-engineering project worthy of the 3rd Millennium;bringing to fruition micro-mass-productivity. Should a clear error be found either in spirit or in factual evidence presented please do not hesitate to contact me.
Aloha,
Anson Parker
Compositions And Methods Relating To Extensible Transgenic Vector Assembler, Pestilence Ridder, Plus Cannabinoid Producer
BACKGROUND::
[0001] The present invention relates the compositions and methods for controlling harmful pestilence in toilets and sewage, using modular extensible genetic techniques, and the facilitation of rapid, low-cost, cannabinoid production.
BACKGROUND OF THE INVENTION AND RELATED ARTS
[0002] Regions of standing wastewater harboring high concentrations of unprocessed, unfiltered rubbage and manure can be natural sites for disease. The need for low-impact, low-maintenance composting solutions is needed to address city sewers and streets around the world that routinely overflow with toxic bilge. Controlling pests in such regions may often require the use of manufactured chemicals - created along costly and inefficient energy gradients (USPTO 5,227,537). Many of these chemical treatments present long-term hazards to the environment in the forms of run-offcontamination, and build-up. Once introduced into a system these non-biodegradable inorganic compounds may not easily be eradicated. Although these methods may be suitable for certain bilge water,especially in treatment facilities where build-up and run-off are not concerns, they do not address the needs of farmers dealing with compost piles, nor sewage water running rampant through the streets of cities that have been obliterated by tsunamis, hurricanes, or tornadoes - wherein there may be an immediate need for rapid decomposition and pestilence ridding. Nor do these prior arts address the need for low impact conversion and or transmutation of toxins and pestilence, nor do these methods establish within the bilge an ecological breeding ground wherein one skilled in the artsmight hope to use the nutrient rich, albeit highly toxic, solution for the establishment of biological byproduct. On the other hand,allowing the degradation of the toxic bilge to take place naturally may not be a viable option - as the aforementioned pestilence may soon make a bid to use the nutrient source as a home. In the proposed invention one skilled in the arts would safely apply numerous vectors to convert the bilge into valuable natural resources while simultaneously defending the region from harmful pestilence both through the creation of anti-microbial substances and through direct competition for resources - in much the same way that acidophilus in yogurt out-competes other microbes.
With regards to the issue of treating filth dispersed deep in underground sewers and inaccessible areas - the invention makes a stark contrast to previous arts. Whereas most chemical reactions must obey the laws of Brownian motion or undergo energetically unfavorable processes such as pumping(USPTO 5,360,556) or heating (USPTO 6,753,536), enzymatic reactions enabled in motile vectors hold a decisive advantage as they can move through a liquid medium more easily. As one skilled in the artsappreciates the possibility of using a motile plant vector such as the sperm of the gingko would allow even greater motility for the vector. In the preferred embodiment of this invention catalystshosted in transgenic e. coli, transgenic tobacco root hair, and usedin modular extensible vectors controlling the synthesis of compoundssuch as tetrahydrocannabinolic acid (THCA), cannabigerolic acid(CBGA), cannabichromenic acid (CBMA), the associated long term costs of pestilence control may be reduced dramatically - while simultaneously enriching the soil with valuable nutrients for commercial crops. As one skilled in the arts will appreciate - the long term application of the proposed invention will manifest itself in stages - much as any great culture ranging from ancient cheese andyogurt cultures to present day bio-engineered vectors, each application of the invention may, in the spirit of evolution, lead to a unique bio-transformation specifically adapted to its environment. The proposed invention brings to the table a base level of safer transmutation of certain toxic fungi (Llewellyn 1977), (Turner 1981), infectious microbes, (Van Klingeren 1976), (Schmitz1973), and insect pests, (Quaghebeur,1981) as well as infectious disease transmitted throughinsects such as West Nile Virus (McPartland, 1993). The nature of this invention is energetically favorable, easily propagated, and low up-keep in cost making it also ideal for third-world implementation in the pursuit of cleaner, safer land. In cases of emergency the preferred embodiment might also serve as a possible source of the neuroprotectant delta-nine tetrahydrocannabinol (THC) through the application of heat such as sunlight or direct flame. In the event of a terrorist attack of neurotoxins, for instance, one might as a means of last resort set fire to the growth medium to convert THCA toTHC - which upon inhaling provides neuroprotection (Hampson 1998)(Van der Stelt 2001) (Mechoulam 2001) (USPTO 6,630,507). Whereas in prior arts Elsohy et al (USPTO 6,730,519) disclosed a method forreduced cost THC production they also rely on traditional abiotic,inorganic, energetically unfavorable means for THC extraction andpurification of THC. Moreover their claims depend on natural growthof Cannabis Sativa, a process that may take up to fifteen weeks. Clearly this is not an acceptable waiting period in the case of aterrorist attack. In an alternate embodiment of the invention aserum of raw nutrients, as opposed to raw sewage, were used as thebasic medium - in this case using modularized transgenic enzymatictechniques one skilled in the arts might produce several tons of THCin two to three days.
TERMINOLOGY::
[0003] The term “Transgenic Stilbene-carboxylatesynthase-like enzyme (TSCSL)” (see Fellermeier 1998) refers toany enzymatic reaction that yields Olivetolic Acid. The triggermechanism. In alternate embodiments of this invention it is linkedoperably to a bioluminescent and equipped with a unique “offswitch.”
[0004] The term “Transgenic GeranylpyrophosphatePrenylase (TOAP)” refers to any enzymatic reaction that yieldsCannabigerol (see Fellermeier 1998). In an alternate embodimentlinked operably to a bioluminescent and equipped with a unique “offswitch”.
[0005] The term “Transgenic Cannabigerolic AcidSynthase (TCAs)” (See Raharjo 2002) refers to any enzymaticreaction or nano-bot that synthesizes Cannabigerolic Acid. Inalternate embodiments of this invention it is linked operably to abioluminescent and equipped with a unique “off switch.”
[0006] The term “Transgenic Cannabidiolic AcidSynthase (TCBAs)” (see Taura F. 1996) refers to any enzymaticreaction that synthesizes Cannabidiolic Acid. In the preferredembodiment of this invention it is linked operably to abioluminescent and equipped with a unique “off switch.”
[0007] The term “Transgenic TetrahydrocannabinolicAcid Synthase (TTAs)” refers to any enzymatic reaction thatsynthesizes THCA, (see reference Taura 2004). In an alternateembodiment of this invention it is linked operably to abioluminescent and equipped with a unique “off switch.”
[0008] The term“Transgenic Cannabichromene Synthase (TCBMs). In an alternateembodiment of this invention it is linked operably to abioluminescent and equipped with a unique “off switch.”Such bioluminescent switch might include prior arts described inUSPTO 6,544,729, although one skilled in the arts might determine others more suitable.
[0009] Genetic“Off switch” - any of several dozen enzymes with knownlethality targeting specifically the aforementioned transgenic vectors - each with its own unique off switch. Including but in noway limited to switches described in USPTO 5,328,847.
[0010] The terms “wastewater, raw sewage, bilge water, manure, compost, toxic sludge, filth, festering rot, crud,crude, rubbage, and debris” refers to any medium that may needpestilence management.
[0011] The term “pestilence management”refers to the control - be it through repellence, extermination, or slowing of growth rate, of any or several of the following organismsAlabama argillacea (Riley 1885), Pieris brassicae (Beling 1932),Melolontha melolontha (Mateeva 1995), and Aphelenchoides composticola, (Grewal 1989), potato beetle (Leptinotarsadecemlineata) (Stratii 1976), mosquito larvae (Anopheles and Culexspecies)(Jalees et al. 1993), Chilo partellus, (a lepidopteran borer)(Bajpai and Sharma, 1992), Tetranychus urticae (Fenili andPegazzano, 1974). Japanese beetles (Metzger and Grant, 1932), Heterodera cajani (Mojumder et al. 1989), Ustilago species (Misra andDixit 1979, Singh and Pathak 1984), Neovossia indica (Gupta andSingh 1983), Curvularia (Upandhyaya and Gupta, 1989), Colletotrichumtruncatum (Kaushal and Paul, 1989), Aspergillus, Penicillium,Cladosporium, Drechslera, Fusarium, Cephalosporium, Rhizopus, Mucor and Curvularia (Pandey, 1982), gram (+) S. aureus, Bacillusmegaterium (Veliky and Genest 1972), gram (+) Corynebacterium speciesand gram (-) Pseudomonas and Agrobacterium species (Bel’tyukova1962), Trypanosoma brucei (Nok et al., 1994), Phomopsis ganjae(Charles and Jenkins 1914, McPartland 1983), Arctia caja (Rothschildet al., 1977) or any other known or unknown organism with undesirable traits.
[0012] The term “transgenically enhanced vector”(TEV) refers to any vector, its parental lineage or its offspringthat has been modified by the use of modern or Mendelian genetic techniques to produce a compound.
[0013] The term "operably linked" refers to a juxtaposition wherein the components so described are in arelationship permitting them to function in their intended manner. Acontrol sequence "operably linked" to a coding sequence isligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.
[0014] Floatation system - in the preferred embodimentfloating systems with roots embedded are used to suspend the transgenic roots as they convert cannabigerolic acid into cannabinoids.
[0015] The term "bioluminescent protein"refers to a protein capable of causing the emission of light through the catalysis of a chemical reaction. The term includes proteins that catalyze bioluminescent or chemiluminescent reactions, such as thosecausing the oxidation of luciferins. The term "bioluminescentprotein" includes not only bioluminescent proteins that occurnaturally, but also mutants that exhibit altered spectral or physicalproperties.
[0016] The term "transformed" refers to a cellinto which (or into an ancestor of which) has been introduced, by means of recombinant nucleic acid techniques, a heterologous nucleicacid molecule.
[0017] The term "transgenic" is used todescribe an organism that includes exogenous genetic material withinall of its cells. The term includes any organism whose genome hasbeen altered by in vitro manipulation of the early embryo orfertilized egg or by any transgenic technology to induce a specificgene knockout.
[0018] The term "transgene" refers any pieceof DNA which is inserted by artifice into a cell, and becomes part ofthe genome of the organism (i.e., either stably integrated or as astable extrachromosomal element) which develops from that cell. Sucha transgene may include a gene which is partly or entirelyheterologous (i.e., foreign) to the transgenic organism, or mayrepresent a gene homologous to an endogenous gene of the organism.Included within this definition is a transgene created by theproviding of an RNA sequence that is transcribed into DNA and thenincorporated into the genome. The transgenes of the invention includeDNA sequences that encode the fluorescent or bioluminescent proteinthat may be expressed in a transgenic non-human animal, the genesrequired for the synthesis of cannabinoids, and any additional genetic information necessary for the greater control of theinvention.
[0019] The following terms are used to describe thesequence relationships between two or more polynucleotides:"reference sequence", "comparison window","sequence identity", "percentage identical to asequence", and "substantial identity". A "reference sequence" is a defined sequence used as a basis for a sequencecomparison; a reference sequence may be a subset of a largersequence, for example, as a segment of a full-length cDNA or genesequence, or may comprise a complete cDNA or gene sequence.Generally, a reference sequence is at least 20 nucleotides in length,frequently at least 25 nucleotides in length, and often at least 50nucleotides in length. Since two polynucleotides may each (1)comprise a sequence (i.e., a portion of the complete polynucleotidesequence) that is similar between the two polynucleotides, and (2)may further comprise a sequence that is divergent between the twopolynucleotides, sequence comparisons between two (or more)polynucleotides are typically performed by comparing sequences of thetwo polynucleotides over a "comparison window" to identifyand compare local regions of sequence similarity. A "comparisonwindow", as used herein, refers to a conceptual segment of atleast 20 contiguous nucleotide positions wherein a polynucleotidesequence may be compared to a reference sequence of at least 20contiguous nucleotides and wherein the portion of the polynucleotidesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less as compared to the referencesequence (which does not comprise additions or deletions) for optimalalignment of the two sequences. Optimal alignment of sequences foraligning a comparison window may be conducted by the local homologyalgorithm of Smith and Waterman (1981) Adv. Appl. Math. 2:482, by thehomology alignment algorithm of Needleman and Wunsch (1970) J. Mol.Biol. 48:443, by the search for similarity method of Pearson andLipman (1988) Proc. Natl. Acad. Sci. (U.S.A.) 85:2444, bycomputerized implementations of these algorithms (GAP, BESTFIT,FASTA, and TFASTA in the Wisconsin Genetics Software Package Release7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or byinspection, and the best alignment (i.e., resulting in the highestpercentage of homology over the comparison window) generated by thevarious methods is selected. The term "sequence identity"means that two polynucleotide sequences are identical (i.e., on anucleotide-by-nucleotide basis) over the window of comparison. Theterm "percentage identical to a sequence" is calculated bycomparing two optimally aligned sequences over the window ofcomparison, determining the number of positions at which theidentical nucleic acid base (e.g., A, T, C, G, U, or I) occurs inboth sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The terms"substantial identity" as used herein denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 30 percent sequence identity, preferably at least 50 to 60 percent sequence identity, more usually at least 60 percent sequence identity as compared to a reference sequence over a comparison window of at least20 nucleotide positions, frequently over a window of at least 25-50nucleotides, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the polynucleotide sequence which may include deletions or additions which total 20percent or less of the reference sequence over the window of comparison. As applied to polypeptides, the term "substantial identity" means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 30 percent sequence identity, preferably at least 40 percent sequence identity, more preferably at least 50 percent sequence identity, and most preferably at least 60 percent sequence identity. Preferably, residue positions which are not identical differ by conservative amino acid substitutions.Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine,leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and agroup of amino acids having sulfur-containing side chains is cysteineand methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine,lysine-arginine, alanine-valine, glutamic-aspartic, and asparagine-glutamine.
[0020] Since the list of technical and scientific terms cannot be all encompassing, any undefined terms shall be construed to have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. Furthermore, the singularforms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Forexample, reference to a "restriction enzyme" or a "high fidelity enzyme" may include mixtures of such enzymes and any other enzymes fitting the stated criteria, or reference to the method includes reference to one or more methods for obtaining cDNA sequences which will be known to those skilled in the art or will become known to them upon reading this specification.
OPERATIONS::
[0021] As one skilled in the arts may appreciate thevariety of vectors able to transform of the initial reagents (TSCSL,TOAP, TCAs, TTAs, TCBMs, TCBAs) into the desired reagents (THC, CBD,CBM) may result in hundreds or thousands of potential scenarios. Consider the heat that is generated in many compost conversion where temperatures may rise above 160 degrees Fahrenheit, in such cases itmay be expedient to use an thermophilic vector, particularly for theincubation of the TSCSL, and TOAP. In the preferred embodiment ofthe invention it should be noted that the TTAs, TCBMs, TCBAs, are used in either a plant or animal vector - since cannabinoids exhibits both anti-microbial and anti-fungal activity it may require anon-microbial and non-fungal host.
[0022] In its preferred embodiment begin with a gigantic pile of refuse, that may include fecal matter, untreated sewage water, and decaying animal parts. It may be to the advantage of the user to initiate the enzymatic activity in a more sterile environment with nutrients needed for the synthesis of precursors of cannabinoids to alleviate environmental pressures of the sludge. In such cases as necessary the resulting enzymes and precursor products may be added directly to the filthy sludge or set aside and used as the growth medium for the transgenic cannabinoid synthesis with the resulting cannabinoids added to the filth sludge after their synthesis is completed. See FIGURE 2
[0023] Expose the pile of refuse to TSCSLs, TOAPs, andTCs teas - brewed as per the guideline in the literature commonly asanyone skilled in the arts will appreciate - and genetically modified to include promoters operably linked to bioluminescent proteins tohelp indicate and monitor effectiveness of the treatment. This teais given from 24 hours to one month as indicated by bioluminescence(Figure 1) to finish blending in with the refuse - or as long as thebioluminescence appears active. (Figure 1. Step: Stilbene-like Synthase). These teas, when mixed with toxic bilge, enzymaticallysynthesize cannabinoids.
[0024] Next a root bed made of TTAs, TCBAs, and orTCBMs. Note the versatility of this invention. Any one of theaforementioned synthases, or indeed all three may be placed atop thepile bilge to create the desired reagents (ie THC, CBA, CBM). Alsonoteworthy is the elegant closed-loop nature of this system. Byinitiating the reaction with microbes that are not themselves immuneto the final product the system will eventually turn itself off - asthe reagent levels rise to higher levels the TSCSLs, TOAPs, and TCsdie.
In an alternate embodiment
[0025] The bucket containing the OAP is loosened atop apile of crud, that may consist of any decaying or decayed matter, andthat must consist of some decaying vegetable matter or livingvegetation.
[0026] The bucket containing the CAS is loosened atopthe pile of crud that previously received OAP treatment.
[0027] A blanket of roots from tobacco made of TTAs,TCBAs and or TCBMs are thrown over the crapulence and festeringtherein may it yield bountifully wee little cannabinoids.
[0028] This invention relates to the synthesis ofcannabinoids for the purpose of general pestilence riddance in filthyorganic and inorganic sludge. Through regulated enzymatic reactions,wherein cannabinoids with known anti-microbial, insecticidal,nematicidal, fungicidal properties and moreover nutritious, andneuroprotective, qualities are used to benefit regions where othercommercial chemical reagents would require mechanized dispersion and cleanup. In plain English for those skilled in the arts - the genesinvolved in the enzymatic formulation of cannabinoids are insertedinto foreign vectors thereby reproducing themselves and generatingsufficient quantities of cannabinoids to clear the region ofpestilence.
The advantages of this system are numerous. Whereascannabinoid synthesis may not easily take place in Cannabis sativadue to its illegality, this invention is highly preferable. Whereascannabinoid synthesis using inorganic techniques is not advantageous due to the inefficiency of inorganic and organic laboratory chemistry, this invention is highly preferable. Whereas most chemical synthesis routes for the creation of cannabinoids relate tothe creation of extremely pure cannabinoids, this invention merelycreates sufficient quantities as needed to rid a region of pestilence, and makes no claims whatsoever as to purity. Whereas thecost of creating cannabinoids synthetically would require large sums of money, as well as recurring costs for reagents, as well as a high degree of expertise and lab equipment, the invention described herein requires a single up-front cost to create the necessary vectors, and thereafter the invention may be distributed and applied to sludge and filth across the world with almost no requirements insofar a prioriknowledge.
[0029] Using closed-loop modular enzymatic reactions,wherein each phase of catalysis may be halted by another counter reaction, and wherein each phase of catalysis may be easily monitored for effectiveness allows one skilled in the arts to more safely andeffectively treat hazardous waste and the plethora of contagions therein. This invention refers to modular, in the sense that along the enzymatic pathway of choice each enzymatic building block is separated into a unique vector, uniquely identifiable by means bioluminescence and uniquely susceptible to a flavor of anti-microbial or anti-fungal such that the enzymatic process ofchoice may be halted at any given phase of production if desired. Modular may also or rather refer to the system as a whole, in that itshould, handled by one skilled in the arts, leave little or no traceof enzymatically active reagent and be a closed-loop system - withthe understanding that in nature there exists no such thing as anentirely closed-loop system, however, the preferred embodiment of this invention has in its design constructs a self-destruct or self-neutralizing mechanism for the living reagents. Thus in the preferred embodiment of the invention the catalysis of tetrahydrocannabinolic acid results in the recursive destruction of the initial vectors (Taura 2004).
[0030] Polyketide Synthesis converts 3 Malonyl CoA plus1 n-Hexanoyl-CoA to form OSCoA. This conversion may take placeinside the muck and sludge, or may take place in a contained area andafter the OSCoA
[0031] Stilbene Carboxylate Synthase-Like (STCSL), inthe case of Cannabis Sativa a Chalcone Synthase (CHS) that exhibitsStilbene Synthase (STS) activity in vivo and as per note in theliterature (Raharjo 2004) there is reason to believe that the sequisused in the preferred embodiment of this invention and refers to anyenzyme that generates 5-amylresorcinolic acid (olivetolic acid). While there are several enzymes capable of synthesizing olivetolic acid in the final analysis any enzyme capable of Olivetolic Acidsynthesis will is sufficient. In the preferred embodiment the STCSLis inserted into the mitochondrial genome using the protofectiontechnique (Khan 2004). The STCSL should be operably linked to bioluminescent protein to facilitate the monitoring of activity. Thevector of the STCSL should also, in the preferred embodiment, have anoperably linked In an alternate embodiment of this inventionolivetolic acid is synthesized through inorganic techniques and thus added to the filthy sludge as a trigger molecule. In this manner the invention would have a limiting reagent from the offset, restricting the final output of pestilence ridders in such cases whereinlimitations might be preferable. In another alternate embodiment ofthe invention the STCSL is chimeric with GOAP, or a pestilence ridding molecule.
[0032] Geranylpyrophosphate Olivetolic Acid Prenylase(GOAP) (Fellermeier 1998) is an integral part of this invention, andconverts olivetolic acid into cannabigerolic acid. As one skilled inthe art may appreciate any enzyme capable of yielding cannabigerolicacid is sufficient. In the preferred embodiment the GOAP is loadedinto the vector in the manner described in Fellermeier’s work. In the preferred embodiment of this invention the GOAP is operablylinked to a bioluminescent protein such as GFP or aequorin, and thusits activation is more easily monitored with minimal technicalexpertise. The GOAP is also operably linked to a promoter capable ofup-regulating GOAP and thereby amplifying GOAP production.
[0033] Products made from these transgenic vectors should produce THCA, and, in addition, other precursor molecules aswell as the necessary enzymes and proteins requisite for theaforementioned production, such as, tetrahydrocannibigerolic acidsynthase, cannabigerolic acid synthase (CBGAS), cannabidiolic acidsynthase (CBDAS), cannabichromenic acid synthase (CBRMAS),tetrahydrocannibinolic acid (THCA), olivetolic acid, polyketidesynthase, and cannabigerolic acid synthase. Also disclosed is the unique and novel application of the TTAs in the function of a compost toilet additive and for the low-impact, sustainable, macrobioticcontrol of pests including Alabama argillacea (Riley 1885), Pierisbrassicae (Beling 1932), Melolontha melolontha (Mateeva 1995), and Aphelenchoides composticola, (Grewal 1989).
[0034] First the transgenically enhanced vectors (TEVs)as necessary and leading up to the cannabigerolic acid phase of biosynthesis (Figure 1) are added into the growth medium and let torest for anywhere from 12 hours to several days with a temperature range of 25-35 degrees centigrade, and also depending on the volumeof waste, the thickness of the muck, and the general nature of the festering filth. If time is of the essence one may speed up growthtimes by dispersing units of TEVs around the afflicted region throughartificial or assisted means. If precision in timing is desired itmay be convenient to include a bioluminescent protein operably linkeda functional promoter to the TEVs similar in methods to (USPTO6,544,729) and created such as to reflect the activity of the TEVs.
[0035] Next the transgenic plant vector is placed atopthe festering sludge. The transgenic plant vector releasescannabinoids into the sludge, and as it appropriates greater theproduct of transgenic E. Coli(s) so shall it release cannabinoids -all the while eradicating both the transgenic E. Coli vector as wellas the numerous pathogens, microbes, insects, fungi etc... that are defenseless against the cannabinoids.
[0036]We do hereby claim that enzymatically biosynthesizeddelta-9-tetrahydrocannabinol and delta-9-tetrahydrocannabinolic-acidprovides a novel and unique technique for ridding sludge and sewage containing bacterium, insects and their larvae, fungi, and other non-human organisms. This claim extends to all enzymatically generated cannabinols grown in any non-human organism specificallyfor the purpose of pestilence killing or minimization, with especial claim to the techniques illustrated in the summary of the inventionand Table 1, wherein one skilled in the arts may clearly review the methods for said endeavor.
[0037]We do hereby claim that the methods of enzymatically generating delta-9-tetrahydrocannabinol and delta-9-tetrahydrocannabinolic-acid are both unique and novel and of substantial benefit to humanity as alow cost strategy for generating industrial quantities of said compounds.
[0040] Table 1 Enzyme Reagents Product Time Temp Vector Key References Accession # Polyketide STCSL / CHS 3 MalonylCo-A & 1 n-Hexanoyl-CoA OSCoA OSCoA Olivetolic Acid 1-24 hrs 25-35 C E. Coli M15 Raharjo, 2004 AY082343 CBDAs Cannabigerolic Acid Cannabichromenic Acid 24-48 hrs 25-35 C Tobacco Root Hairs Morimoto, 1999 Prenylase Olivetolic Acid + GPP Cannabigerolic Acid 1-24 hrs 25-35 C E. Coli M15 Fellermeier, 1998 CBCAs Cannabigerolic Acid Cannabidiolic Acid 24-48 hrs 25-35 C Tobacco Root Hairs Morimoto, 1999 THCAs Cannabigerolic Acid Tetrahydrocannabinolic Acid 24-48 hrs 25-35 C Tobacco Root Hairs Taura, 1995 & Sirikantaramas 2004 AB057805
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ABSTRACT
The following patent application refers to the methods and compositions relating to a novel use for enzymatic catalysis of C21-H30-O2 (delta-nine tetrahydrocannabinol (THC)) as an insect repellent, bactericide, and fungicide and dispensation methods as commercial reagent.
The bio-synthesis of cannabinoids represents a landmark achievement in the field of composting, vector removal and ecological reconstitution. Although the benefits have been known for millennia,the advent of modern bio-engineering techniques brings these small seeds of native wisdom to bear on a broader and more industrialized scale - removing dangerous molds and pestilences such as mosquitoes from swamped and flooded regions, raw sewage areas, and disaster sites where ensuing vermin and harmful vectors may cause greater damage than the initial catastrophes.
It is the ambition and intention of authors that tactical usage of this broad-sweeping technique may rapidly and at low cost satisfy a global demand in what may be termed a “grass-roots”bio-engineering project worthy of the 3rd Millennium;bringing to fruition micro-mass-productivity. Should a clear error be found either in spirit or in factual evidence presented please do not hesitate to contact me.
Aloha,
Anson Parker
Compositions And Methods Relating To Extensible Transgenic Vector Assembler, Pestilence Ridder, Plus Cannabinoid Producer
BACKGROUND::
[0001] The present invention relates the compositions and methods for controlling harmful pestilence in toilets and sewage, using modular extensible genetic techniques, and the facilitation of rapid, low-cost, cannabinoid production.
BACKGROUND OF THE INVENTION AND RELATED ARTS
[0002] Regions of standing wastewater harboring high concentrations of unprocessed, unfiltered rubbage and manure can be natural sites for disease. The need for low-impact, low-maintenance composting solutions is needed to address city sewers and streets around the world that routinely overflow with toxic bilge. Controlling pests in such regions may often require the use of manufactured chemicals - created along costly and inefficient energy gradients (USPTO 5,227,537). Many of these chemical treatments present long-term hazards to the environment in the forms of run-offcontamination, and build-up. Once introduced into a system these non-biodegradable inorganic compounds may not easily be eradicated. Although these methods may be suitable for certain bilge water,especially in treatment facilities where build-up and run-off are not concerns, they do not address the needs of farmers dealing with compost piles, nor sewage water running rampant through the streets of cities that have been obliterated by tsunamis, hurricanes, or tornadoes - wherein there may be an immediate need for rapid decomposition and pestilence ridding. Nor do these prior arts address the need for low impact conversion and or transmutation of toxins and pestilence, nor do these methods establish within the bilge an ecological breeding ground wherein one skilled in the artsmight hope to use the nutrient rich, albeit highly toxic, solution for the establishment of biological byproduct. On the other hand,allowing the degradation of the toxic bilge to take place naturally may not be a viable option - as the aforementioned pestilence may soon make a bid to use the nutrient source as a home. In the proposed invention one skilled in the arts would safely apply numerous vectors to convert the bilge into valuable natural resources while simultaneously defending the region from harmful pestilence both through the creation of anti-microbial substances and through direct competition for resources - in much the same way that acidophilus in yogurt out-competes other microbes.
With regards to the issue of treating filth dispersed deep in underground sewers and inaccessible areas - the invention makes a stark contrast to previous arts. Whereas most chemical reactions must obey the laws of Brownian motion or undergo energetically unfavorable processes such as pumping(USPTO 5,360,556) or heating (USPTO 6,753,536), enzymatic reactions enabled in motile vectors hold a decisive advantage as they can move through a liquid medium more easily. As one skilled in the artsappreciates the possibility of using a motile plant vector such as the sperm of the gingko would allow even greater motility for the vector. In the preferred embodiment of this invention catalystshosted in transgenic e. coli, transgenic tobacco root hair, and usedin modular extensible vectors controlling the synthesis of compoundssuch as tetrahydrocannabinolic acid (THCA), cannabigerolic acid(CBGA), cannabichromenic acid (CBMA), the associated long term costs of pestilence control may be reduced dramatically - while simultaneously enriching the soil with valuable nutrients for commercial crops. As one skilled in the arts will appreciate - the long term application of the proposed invention will manifest itself in stages - much as any great culture ranging from ancient cheese andyogurt cultures to present day bio-engineered vectors, each application of the invention may, in the spirit of evolution, lead to a unique bio-transformation specifically adapted to its environment. The proposed invention brings to the table a base level of safer transmutation of certain toxic fungi (Llewellyn 1977), (Turner 1981), infectious microbes, (Van Klingeren 1976), (Schmitz1973), and insect pests, (Quaghebeur,1981) as well as infectious disease transmitted throughinsects such as West Nile Virus (McPartland, 1993). The nature of this invention is energetically favorable, easily propagated, and low up-keep in cost making it also ideal for third-world implementation in the pursuit of cleaner, safer land. In cases of emergency the preferred embodiment might also serve as a possible source of the neuroprotectant delta-nine tetrahydrocannabinol (THC) through the application of heat such as sunlight or direct flame. In the event of a terrorist attack of neurotoxins, for instance, one might as a means of last resort set fire to the growth medium to convert THCA toTHC - which upon inhaling provides neuroprotection (Hampson 1998)(Van der Stelt 2001) (Mechoulam 2001) (USPTO 6,630,507). Whereas in prior arts Elsohy et al (USPTO 6,730,519) disclosed a method forreduced cost THC production they also rely on traditional abiotic,inorganic, energetically unfavorable means for THC extraction andpurification of THC. Moreover their claims depend on natural growthof Cannabis Sativa, a process that may take up to fifteen weeks. Clearly this is not an acceptable waiting period in the case of aterrorist attack. In an alternate embodiment of the invention aserum of raw nutrients, as opposed to raw sewage, were used as thebasic medium - in this case using modularized transgenic enzymatictechniques one skilled in the arts might produce several tons of THCin two to three days.
TERMINOLOGY::
[0003] The term “Transgenic Stilbene-carboxylatesynthase-like enzyme (TSCSL)” (see Fellermeier 1998) refers toany enzymatic reaction that yields Olivetolic Acid. The triggermechanism. In alternate embodiments of this invention it is linkedoperably to a bioluminescent and equipped with a unique “offswitch.”
[0004] The term “Transgenic GeranylpyrophosphatePrenylase (TOAP)” refers to any enzymatic reaction that yieldsCannabigerol (see Fellermeier 1998). In an alternate embodimentlinked operably to a bioluminescent and equipped with a unique “offswitch”.
[0005] The term “Transgenic Cannabigerolic AcidSynthase (TCAs)” (See Raharjo 2002) refers to any enzymaticreaction or nano-bot that synthesizes Cannabigerolic Acid. Inalternate embodiments of this invention it is linked operably to abioluminescent and equipped with a unique “off switch.”
[0006] The term “Transgenic Cannabidiolic AcidSynthase (TCBAs)” (see Taura F. 1996) refers to any enzymaticreaction that synthesizes Cannabidiolic Acid. In the preferredembodiment of this invention it is linked operably to abioluminescent and equipped with a unique “off switch.”
[0007] The term “Transgenic TetrahydrocannabinolicAcid Synthase (TTAs)” refers to any enzymatic reaction thatsynthesizes THCA, (see reference Taura 2004). In an alternateembodiment of this invention it is linked operably to abioluminescent and equipped with a unique “off switch.”
[0008] The term“Transgenic Cannabichromene Synthase (TCBMs). In an alternateembodiment of this invention it is linked operably to abioluminescent and equipped with a unique “off switch.”Such bioluminescent switch might include prior arts described inUSPTO 6,544,729, although one skilled in the arts might determine others more suitable.
[0009] Genetic“Off switch” - any of several dozen enzymes with knownlethality targeting specifically the aforementioned transgenic vectors - each with its own unique off switch. Including but in noway limited to switches described in USPTO 5,328,847.
[0010] The terms “wastewater, raw sewage, bilge water, manure, compost, toxic sludge, filth, festering rot, crud,crude, rubbage, and debris” refers to any medium that may needpestilence management.
[0011] The term “pestilence management”refers to the control - be it through repellence, extermination, or slowing of growth rate, of any or several of the following organismsAlabama argillacea (Riley 1885), Pieris brassicae (Beling 1932),Melolontha melolontha (Mateeva 1995), and Aphelenchoides composticola, (Grewal 1989), potato beetle (Leptinotarsadecemlineata) (Stratii 1976), mosquito larvae (Anopheles and Culexspecies)(Jalees et al. 1993), Chilo partellus, (a lepidopteran borer)(Bajpai and Sharma, 1992), Tetranychus urticae (Fenili andPegazzano, 1974). Japanese beetles (Metzger and Grant, 1932), Heterodera cajani (Mojumder et al. 1989), Ustilago species (Misra andDixit 1979, Singh and Pathak 1984), Neovossia indica (Gupta andSingh 1983), Curvularia (Upandhyaya and Gupta, 1989), Colletotrichumtruncatum (Kaushal and Paul, 1989), Aspergillus, Penicillium,Cladosporium, Drechslera, Fusarium, Cephalosporium, Rhizopus, Mucor and Curvularia (Pandey, 1982), gram (+) S. aureus, Bacillusmegaterium (Veliky and Genest 1972), gram (+) Corynebacterium speciesand gram (-) Pseudomonas and Agrobacterium species (Bel’tyukova1962), Trypanosoma brucei (Nok et al., 1994), Phomopsis ganjae(Charles and Jenkins 1914, McPartland 1983), Arctia caja (Rothschildet al., 1977) or any other known or unknown organism with undesirable traits.
[0012] The term “transgenically enhanced vector”(TEV) refers to any vector, its parental lineage or its offspringthat has been modified by the use of modern or Mendelian genetic techniques to produce a compound.
[0013] The term "operably linked" refers to a juxtaposition wherein the components so described are in arelationship permitting them to function in their intended manner. Acontrol sequence "operably linked" to a coding sequence isligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.
[0014] Floatation system - in the preferred embodimentfloating systems with roots embedded are used to suspend the transgenic roots as they convert cannabigerolic acid into cannabinoids.
[0015] The term "bioluminescent protein"refers to a protein capable of causing the emission of light through the catalysis of a chemical reaction. The term includes proteins that catalyze bioluminescent or chemiluminescent reactions, such as thosecausing the oxidation of luciferins. The term "bioluminescentprotein" includes not only bioluminescent proteins that occurnaturally, but also mutants that exhibit altered spectral or physicalproperties.
[0016] The term "transformed" refers to a cellinto which (or into an ancestor of which) has been introduced, by means of recombinant nucleic acid techniques, a heterologous nucleicacid molecule.
[0017] The term "transgenic" is used todescribe an organism that includes exogenous genetic material withinall of its cells. The term includes any organism whose genome hasbeen altered by in vitro manipulation of the early embryo orfertilized egg or by any transgenic technology to induce a specificgene knockout.
[0018] The term "transgene" refers any pieceof DNA which is inserted by artifice into a cell, and becomes part ofthe genome of the organism (i.e., either stably integrated or as astable extrachromosomal element) which develops from that cell. Sucha transgene may include a gene which is partly or entirelyheterologous (i.e., foreign) to the transgenic organism, or mayrepresent a gene homologous to an endogenous gene of the organism.Included within this definition is a transgene created by theproviding of an RNA sequence that is transcribed into DNA and thenincorporated into the genome. The transgenes of the invention includeDNA sequences that encode the fluorescent or bioluminescent proteinthat may be expressed in a transgenic non-human animal, the genesrequired for the synthesis of cannabinoids, and any additional genetic information necessary for the greater control of theinvention.
[0019] The following terms are used to describe thesequence relationships between two or more polynucleotides:"reference sequence", "comparison window","sequence identity", "percentage identical to asequence", and "substantial identity". A "reference sequence" is a defined sequence used as a basis for a sequencecomparison; a reference sequence may be a subset of a largersequence, for example, as a segment of a full-length cDNA or genesequence, or may comprise a complete cDNA or gene sequence.Generally, a reference sequence is at least 20 nucleotides in length,frequently at least 25 nucleotides in length, and often at least 50nucleotides in length. Since two polynucleotides may each (1)comprise a sequence (i.e., a portion of the complete polynucleotidesequence) that is similar between the two polynucleotides, and (2)may further comprise a sequence that is divergent between the twopolynucleotides, sequence comparisons between two (or more)polynucleotides are typically performed by comparing sequences of thetwo polynucleotides over a "comparison window" to identifyand compare local regions of sequence similarity. A "comparisonwindow", as used herein, refers to a conceptual segment of atleast 20 contiguous nucleotide positions wherein a polynucleotidesequence may be compared to a reference sequence of at least 20contiguous nucleotides and wherein the portion of the polynucleotidesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less as compared to the referencesequence (which does not comprise additions or deletions) for optimalalignment of the two sequences. Optimal alignment of sequences foraligning a comparison window may be conducted by the local homologyalgorithm of Smith and Waterman (1981) Adv. Appl. Math. 2:482, by thehomology alignment algorithm of Needleman and Wunsch (1970) J. Mol.Biol. 48:443, by the search for similarity method of Pearson andLipman (1988) Proc. Natl. Acad. Sci. (U.S.A.) 85:2444, bycomputerized implementations of these algorithms (GAP, BESTFIT,FASTA, and TFASTA in the Wisconsin Genetics Software Package Release7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or byinspection, and the best alignment (i.e., resulting in the highestpercentage of homology over the comparison window) generated by thevarious methods is selected. The term "sequence identity"means that two polynucleotide sequences are identical (i.e., on anucleotide-by-nucleotide basis) over the window of comparison. Theterm "percentage identical to a sequence" is calculated bycomparing two optimally aligned sequences over the window ofcomparison, determining the number of positions at which theidentical nucleic acid base (e.g., A, T, C, G, U, or I) occurs inboth sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The terms"substantial identity" as used herein denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 30 percent sequence identity, preferably at least 50 to 60 percent sequence identity, more usually at least 60 percent sequence identity as compared to a reference sequence over a comparison window of at least20 nucleotide positions, frequently over a window of at least 25-50nucleotides, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the polynucleotide sequence which may include deletions or additions which total 20percent or less of the reference sequence over the window of comparison. As applied to polypeptides, the term "substantial identity" means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 30 percent sequence identity, preferably at least 40 percent sequence identity, more preferably at least 50 percent sequence identity, and most preferably at least 60 percent sequence identity. Preferably, residue positions which are not identical differ by conservative amino acid substitutions.Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine,leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and agroup of amino acids having sulfur-containing side chains is cysteineand methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine,lysine-arginine, alanine-valine, glutamic-aspartic, and asparagine-glutamine.
[0020] Since the list of technical and scientific terms cannot be all encompassing, any undefined terms shall be construed to have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. Furthermore, the singularforms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Forexample, reference to a "restriction enzyme" or a "high fidelity enzyme" may include mixtures of such enzymes and any other enzymes fitting the stated criteria, or reference to the method includes reference to one or more methods for obtaining cDNA sequences which will be known to those skilled in the art or will become known to them upon reading this specification.
OPERATIONS::
[0021] As one skilled in the arts may appreciate thevariety of vectors able to transform of the initial reagents (TSCSL,TOAP, TCAs, TTAs, TCBMs, TCBAs) into the desired reagents (THC, CBD,CBM) may result in hundreds or thousands of potential scenarios. Consider the heat that is generated in many compost conversion where temperatures may rise above 160 degrees Fahrenheit, in such cases itmay be expedient to use an thermophilic vector, particularly for theincubation of the TSCSL, and TOAP. In the preferred embodiment ofthe invention it should be noted that the TTAs, TCBMs, TCBAs, are used in either a plant or animal vector - since cannabinoids exhibits both anti-microbial and anti-fungal activity it may require anon-microbial and non-fungal host.
[0022] In its preferred embodiment begin with a gigantic pile of refuse, that may include fecal matter, untreated sewage water, and decaying animal parts. It may be to the advantage of the user to initiate the enzymatic activity in a more sterile environment with nutrients needed for the synthesis of precursors of cannabinoids to alleviate environmental pressures of the sludge. In such cases as necessary the resulting enzymes and precursor products may be added directly to the filthy sludge or set aside and used as the growth medium for the transgenic cannabinoid synthesis with the resulting cannabinoids added to the filth sludge after their synthesis is completed. See FIGURE 2
[0023] Expose the pile of refuse to TSCSLs, TOAPs, andTCs teas - brewed as per the guideline in the literature commonly asanyone skilled in the arts will appreciate - and genetically modified to include promoters operably linked to bioluminescent proteins tohelp indicate and monitor effectiveness of the treatment. This teais given from 24 hours to one month as indicated by bioluminescence(Figure 1) to finish blending in with the refuse - or as long as thebioluminescence appears active. (Figure 1. Step: Stilbene-like Synthase). These teas, when mixed with toxic bilge, enzymaticallysynthesize cannabinoids.
[0024] Next a root bed made of TTAs, TCBAs, and orTCBMs. Note the versatility of this invention. Any one of theaforementioned synthases, or indeed all three may be placed atop thepile bilge to create the desired reagents (ie THC, CBA, CBM). Alsonoteworthy is the elegant closed-loop nature of this system. Byinitiating the reaction with microbes that are not themselves immuneto the final product the system will eventually turn itself off - asthe reagent levels rise to higher levels the TSCSLs, TOAPs, and TCsdie.
In an alternate embodiment
[0025] The bucket containing the OAP is loosened atop apile of crud, that may consist of any decaying or decayed matter, andthat must consist of some decaying vegetable matter or livingvegetation.
[0026] The bucket containing the CAS is loosened atopthe pile of crud that previously received OAP treatment.
[0027] A blanket of roots from tobacco made of TTAs,TCBAs and or TCBMs are thrown over the crapulence and festeringtherein may it yield bountifully wee little cannabinoids.
[0028] This invention relates to the synthesis ofcannabinoids for the purpose of general pestilence riddance in filthyorganic and inorganic sludge. Through regulated enzymatic reactions,wherein cannabinoids with known anti-microbial, insecticidal,nematicidal, fungicidal properties and moreover nutritious, andneuroprotective, qualities are used to benefit regions where othercommercial chemical reagents would require mechanized dispersion and cleanup. In plain English for those skilled in the arts - the genesinvolved in the enzymatic formulation of cannabinoids are insertedinto foreign vectors thereby reproducing themselves and generatingsufficient quantities of cannabinoids to clear the region ofpestilence.
The advantages of this system are numerous. Whereascannabinoid synthesis may not easily take place in Cannabis sativadue to its illegality, this invention is highly preferable. Whereascannabinoid synthesis using inorganic techniques is not advantageous due to the inefficiency of inorganic and organic laboratory chemistry, this invention is highly preferable. Whereas most chemical synthesis routes for the creation of cannabinoids relate tothe creation of extremely pure cannabinoids, this invention merelycreates sufficient quantities as needed to rid a region of pestilence, and makes no claims whatsoever as to purity. Whereas thecost of creating cannabinoids synthetically would require large sums of money, as well as recurring costs for reagents, as well as a high degree of expertise and lab equipment, the invention described herein requires a single up-front cost to create the necessary vectors, and thereafter the invention may be distributed and applied to sludge and filth across the world with almost no requirements insofar a prioriknowledge.
[0029] Using closed-loop modular enzymatic reactions,wherein each phase of catalysis may be halted by another counter reaction, and wherein each phase of catalysis may be easily monitored for effectiveness allows one skilled in the arts to more safely andeffectively treat hazardous waste and the plethora of contagions therein. This invention refers to modular, in the sense that along the enzymatic pathway of choice each enzymatic building block is separated into a unique vector, uniquely identifiable by means bioluminescence and uniquely susceptible to a flavor of anti-microbial or anti-fungal such that the enzymatic process ofchoice may be halted at any given phase of production if desired. Modular may also or rather refer to the system as a whole, in that itshould, handled by one skilled in the arts, leave little or no traceof enzymatically active reagent and be a closed-loop system - withthe understanding that in nature there exists no such thing as anentirely closed-loop system, however, the preferred embodiment of this invention has in its design constructs a self-destruct or self-neutralizing mechanism for the living reagents. Thus in the preferred embodiment of the invention the catalysis of tetrahydrocannabinolic acid results in the recursive destruction of the initial vectors (Taura 2004).
[0030] Polyketide Synthesis converts 3 Malonyl CoA plus1 n-Hexanoyl-CoA to form OSCoA. This conversion may take placeinside the muck and sludge, or may take place in a contained area andafter the OSCoA
[0031] Stilbene Carboxylate Synthase-Like (STCSL), inthe case of Cannabis Sativa a Chalcone Synthase (CHS) that exhibitsStilbene Synthase (STS) activity in vivo and as per note in theliterature (Raharjo 2004) there is reason to believe that the sequisused in the preferred embodiment of this invention and refers to anyenzyme that generates 5-amylresorcinolic acid (olivetolic acid). While there are several enzymes capable of synthesizing olivetolic acid in the final analysis any enzyme capable of Olivetolic Acidsynthesis will is sufficient. In the preferred embodiment the STCSLis inserted into the mitochondrial genome using the protofectiontechnique (Khan 2004). The STCSL should be operably linked to bioluminescent protein to facilitate the monitoring of activity. Thevector of the STCSL should also, in the preferred embodiment, have anoperably linked In an alternate embodiment of this inventionolivetolic acid is synthesized through inorganic techniques and thus added to the filthy sludge as a trigger molecule. In this manner the invention would have a limiting reagent from the offset, restricting the final output of pestilence ridders in such cases whereinlimitations might be preferable. In another alternate embodiment ofthe invention the STCSL is chimeric with GOAP, or a pestilence ridding molecule.
[0032] Geranylpyrophosphate Olivetolic Acid Prenylase(GOAP) (Fellermeier 1998) is an integral part of this invention, andconverts olivetolic acid into cannabigerolic acid. As one skilled inthe art may appreciate any enzyme capable of yielding cannabigerolicacid is sufficient. In the preferred embodiment the GOAP is loadedinto the vector in the manner described in Fellermeier’s work. In the preferred embodiment of this invention the GOAP is operablylinked to a bioluminescent protein such as GFP or aequorin, and thusits activation is more easily monitored with minimal technicalexpertise. The GOAP is also operably linked to a promoter capable ofup-regulating GOAP and thereby amplifying GOAP production.
[0033] Products made from these transgenic vectors should produce THCA, and, in addition, other precursor molecules aswell as the necessary enzymes and proteins requisite for theaforementioned production, such as, tetrahydrocannibigerolic acidsynthase, cannabigerolic acid synthase (CBGAS), cannabidiolic acidsynthase (CBDAS), cannabichromenic acid synthase (CBRMAS),tetrahydrocannibinolic acid (THCA), olivetolic acid, polyketidesynthase, and cannabigerolic acid synthase. Also disclosed is the unique and novel application of the TTAs in the function of a compost toilet additive and for the low-impact, sustainable, macrobioticcontrol of pests including Alabama argillacea (Riley 1885), Pierisbrassicae (Beling 1932), Melolontha melolontha (Mateeva 1995), and Aphelenchoides composticola, (Grewal 1989).
[0034] First the transgenically enhanced vectors (TEVs)as necessary and leading up to the cannabigerolic acid phase of biosynthesis (Figure 1) are added into the growth medium and let torest for anywhere from 12 hours to several days with a temperature range of 25-35 degrees centigrade, and also depending on the volumeof waste, the thickness of the muck, and the general nature of the festering filth. If time is of the essence one may speed up growthtimes by dispersing units of TEVs around the afflicted region throughartificial or assisted means. If precision in timing is desired itmay be convenient to include a bioluminescent protein operably linkeda functional promoter to the TEVs similar in methods to (USPTO6,544,729) and created such as to reflect the activity of the TEVs.
[0035] Next the transgenic plant vector is placed atopthe festering sludge. The transgenic plant vector releasescannabinoids into the sludge, and as it appropriates greater theproduct of transgenic E. Coli(s) so shall it release cannabinoids -all the while eradicating both the transgenic E. Coli vector as wellas the numerous pathogens, microbes, insects, fungi etc... that are defenseless against the cannabinoids.
[0036]We do hereby claim that enzymatically biosynthesizeddelta-9-tetrahydrocannabinol and delta-9-tetrahydrocannabinolic-acidprovides a novel and unique technique for ridding sludge and sewage containing bacterium, insects and their larvae, fungi, and other non-human organisms. This claim extends to all enzymatically generated cannabinols grown in any non-human organism specificallyfor the purpose of pestilence killing or minimization, with especial claim to the techniques illustrated in the summary of the inventionand Table 1, wherein one skilled in the arts may clearly review the methods for said endeavor.
[0037]We do hereby claim that the methods of enzymatically generating delta-9-tetrahydrocannabinol and delta-9-tetrahydrocannabinolic-acid are both unique and novel and of substantial benefit to humanity as alow cost strategy for generating industrial quantities of said compounds.
[0040] Table 1 Enzyme Reagents Product Time Temp Vector Key References Accession # Polyketide STCSL / CHS 3 MalonylCo-A & 1 n-Hexanoyl-CoA OSCoA OSCoA Olivetolic Acid 1-24 hrs 25-35 C E. Coli M15 Raharjo, 2004 AY082343 CBDAs Cannabigerolic Acid Cannabichromenic Acid 24-48 hrs 25-35 C Tobacco Root Hairs Morimoto, 1999 Prenylase Olivetolic Acid + GPP Cannabigerolic Acid 1-24 hrs 25-35 C E. Coli M15 Fellermeier, 1998 CBCAs Cannabigerolic Acid Cannabidiolic Acid 24-48 hrs 25-35 C Tobacco Root Hairs Morimoto, 1999 THCAs Cannabigerolic Acid Tetrahydrocannabinolic Acid 24-48 hrs 25-35 C Tobacco Root Hairs Taura, 1995 & Sirikantaramas 2004 AB057805
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