Patent Application
20250230420
The genetic components described herein are referred to by sequence identifier numbers (SEQ ID NO). The SEQ ID NOs correspond numerically to the sequence identifiers <400>1, <400>2, etc. The sequence listing in written computer readable format (CRF) as a text file named “930201-8120_Sequence_Listing.xml” created on Dec. 11, 2023, and having a size of 33,987 bytes, is incorporated by reference in its entirety.
As many as 29.5 million people in the United States experienced alcohol use disorder in the year 2022. Excessive alcohol use can lead to injuries, violence, alcohol poisoning, risky behaviors, miscarriage and/or stillbirth, and death, as well as chronic health conditions including various cancers, cardiovascular diseases, gastrointestinal disorders, and diseases and disorders of the central nervous system (e.g. dementia, learning and memory problems, depression, anxiety, and more).
Although opioids are commonly prescribed pain medications, over 600,000 people have died from opioid overdoses in the past 20 years, including from prescription opioids, street drugs such as heroin, and synthetic opioids including fentanyl. Opioid abuse can lead to changes in mood or personality, financial hardship, lost jobs and/or penalties for missed school, legal troubles, and the like, and illegally manufactured synthetic opioids can be contaminated or deliberately mixed with other dangerous substances, leading to potentially lethal health effects.
Healthcare professionals have a need to assess patients fully, quickly, and correctly, including in emergency situations, such as an overdose, when the patients may not be able to communicate, in order to offer effective treatment. In some situations, that may mean administering emergency medications to metabolize or block the effects of opioids (as with naloxone) or alcohol on the brain and body.
It would thus be desirable to develop an inexpensive, safe, and easy-to-use system for identifying alcohol, opioids, and/or their metabolites in a biological sample from a patient. It would further be desirable to develop such a system for the detoxification of alcohol and/or opioids. These and other needs are satisfied by the present disclosure.
Described herein are biological devices and extracts useful for detecting alcohol and/or opioids in a biological sample from a subject; the biological devices and extracts can also be useful for detoxifying alcohol and/or opioids in a subject in need thereof. The biological devices include microbial cells transformed with a DNA construct containing genes for producing suIfotransferase, UDP-glucuronosyltransferase, O-linked N-acetylglucosamine transferase, alcohol dehydrogenase, and cytochrome P450. In some instances, the biological devices also include a gene for enhanced green fluorescent protein. Methods for using the devices are also provided herein.
The advantages of the invention will be set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by practice of the aspects described below. The advantages described below will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive.
Many aspects of the present disclosure can be better understood with reference to the following drawings, which are incorporated in and constitute a part of this specification. The components in the drawings are not necessarily to scale, emphasis instead being placed upon clearly illustrating the principles of the present disclosure.
Additional advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or can be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.
Disclosed herein are DNA constructs containing the following genetic components:
The DNA constructs may variously encode genes encoding reporter proteins, genes encoding resistance to one or more antibiotics, and the like, and may include regulatory sequences including promoters, terminators, ribosomal binding sites, LAC operons, or other components necessary for the replication of and expression of the genes encoded by the DNA constructs inside microbial hosts such as, for example, Saccharomyces cerevisiae, Escherichia coli, and other microorganisms. Also disclosed are vectors including the DNA constructs and biological devices consisting of host cells that include one or more copies of the vectors.
Also disclosed herein are methods for producing a composition useful for the detection of alcohol and/or opioids in a biological sample from a subject, and methods for detoxifying excess amounts of alcohol and/or opioids in a subject. Exemplary methods for producing the compositions are disclosed in the Examples.
Before the present compounds, compositions, articles, devices, and/or methods are disclosed and described, it is to be understood that the aspects described below are not limited to specific compounds, synthetic methods, or uses, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.
In this specification and in the claims that follow, reference will be made to a number of terms that shall defined to have the following meanings:
It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a metabolite” includes mixtures of two or more such metabolites, and the like.
“Optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, the phrase “a microorganism is optionally genetically modified” means that the microorganism may or may not be genetically modified.
An “opioid” as used herein refers to a drug that interacts with opioid receptors. In one aspect, the opioid can be a chemically synthesized narcotic including, but not limited to, acetylpropionylmorphine, desomorphine, dextromethorphan, dextropropoxyphene, diacetyldihydromorphine, dibenzoylmorphine, dipropanoylmorphine, ethylmorphine, loperamide, hydrocodone, hydromorphone, oxycodone, oxymorphone, meperidine, methadone, fentanyl, carfentanyl, pethidine, levorphanol, tramadol, tapentadol, buprenorphine, or any combination thereof. In some aspects, the term “opioid” is used herein to refer to naturally extracted opiates from Papaver somniferum such as, for example, opium, morphine, codeine, or heroin.
Throughout this specification, unless the context dictates otherwise, the word “comprise,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated element, integer, step, or group of elements, integers, or steps, but not the exclusion of any other element, integer, step, or group of elements, integers, or steps.
As used herein, the term “about” is used to provide flexibility to a numerical range endpoint by providing that a given numerical value may be “a little above” or “a little below” the endpoint without affecting the desired result. For purposes of the parent disclosure, “about” refers to a range extending from 10% below the numerical value to 10% above the numerical value. For example, if the numerical value is 10, “about 10” means between 9 and 11, inclusive of the endpoints 9 and 11.
When a range is expressed, a further aspect includes from the one particular value and/or to the other particular value. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g. the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y.’ The range can also be expressed as an upper limit, e.g. ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x,’ ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y’, and ‘less than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x,’ ‘about y,’ and ‘about z’ as well as the ranges of ‘greater than x,’ greater than y,’ and ‘greater than z.’ In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.
It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.
Disclosed are materials and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed compositions and methods. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc., of these materials are disclosed that while specific reference to each various individual and collective combination and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a UDP-glucuronosyltransferase (UGT) is disclosed and discussed and a number of different substrates on which the UGT can act are discussed, each and every combination and permutation of UGT and substrate that is possible is specifically contemplated unless specifically indicated to the contrary. For example, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F, and an example of a combination molecule, A-D, is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, in this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the subgroup of A-E, B-F, and C-E is specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. This concept applies to all aspects of this disclosure including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed, it is understood that each of these additional steps can be performed with any specific embodiment or combination of elements of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.
References in the specification and concluding claims to parts by weight, of a particular element or component in a composition or article, denote the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed. Thus, in a composition containing 2 parts by weight of component X and 5 parts by weight of component Y, X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.
A weight percent of a component, unless specifically stated to the contrary, is based on the total weight of the formulation or composition in which the component is included.
In one aspect, cells transformed with a DNA construct can be used in the methods described herein. It is understood that one way to define the variants and derivatives of the genetic components and DNA constructs described herein is in terms of homology/identity to specific known sequences. Those of skill in the art readily understand how to determine the homology of two nucleic acids. For example, the homology can be calculated after aligning two sequences so that the homology is at its highest level. Another way of calculating homology can be performed according to published algorithms (see Zuker, M., Science, 244:48-52, 1989; Jaeger et al, Proc. Natl. Acad. Sci. USA, 86:7706-7710, 1989; Jaeger et al, Methods Enzymol., 183:281-306, 1989, which are herein incorporated by reference for at least material related to nucleic acid alignment).
As used herein, “conservative” mutations are mutations that result in an amino acid change in the protein produced from a sequence of DNA. When a conservative mutation occurs, the new amino acid has similar properties as the wild type amino acid and generally does not drastically change the function or folding of the protein (e.g., switching isoleucine for valine is a conservative mutation since both are small, branched, hydrophobic amino acids). “Silent mutations,” meanwhile, change the nucleic acid sequence of a gene encoding a protein but do not change the amino acid sequence of the protein.
It is understood that the description of mutations and homology can be combined together in any combination, such as embodiments that have at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% homology to a particular sequence wherein the variants are conservative or silent mutations. It is understood that any of the sequences described herein can be a variant or derivative having the homology values listed above.
In some aspects, genes of interest can be spliced into suitable vectors using restriction enzymes and/or other techniques known in the art. Further in this aspect, synthesis and/or isolation of the genes of interest prior to inclusion in the disclosed constructs may result in the addition of C-terminal and/or N-terminal sequence data including, but not limited to, restriction enzyme recognition sites, linking bases, short segments of chromosomal DNA (including introns or portions of introns if the sequences originate from eukaryotic cells), transposons, nucleotide repeats, regulatory sequences, and/or other material that do not contribute to the known structure of the expressed protein, or are not part of the expressed protein's active site. In one aspect, presence of these remnants may lead to somewhat reduced homology with respect to gene sequence, but the DNA constructs encoding the same can still produce proteins having the desired sequence, active site, and function.
In another aspect, many eukaryotic genes include introns and mRNAs produced during transcription of the same can be spliced differently, producing several transcript variants from the same gene but having slightly different sequences (i.e., reduced levels of homology). In one aspect, different transcript variants can produce proteins having the same active site but differing in another way (e.g. in C-terminal or N-terminal sequence, affecting assembly of protein subunits or other folding processes, cellular localization of the peptides or proteins, or activity level of the peptides or proteins produced due to differential regulation, or the like.
In one aspect, a database such as, for example, GenBank, can be used to determine the sequences of genes and/or regulatory regions of interest, the species from which these elements originate, and related homologous sequences.
In one aspect, the nucleic acids used in the DNA constructs described herein can be amplified using polymerase chain reaction (PCR) prior to being ligated into a plasmid or other vector. Typically, PCR-amplification techniques make use of primers, or short, chemically-synthesized oligonucleotides that are complementary to regions on each respective strand flanking the DNA or nucleotide sequence to be amplified. A person having ordinary skill in the art will be able to design or choose primers based on the desired experimental conditions. In general, primers should be designed to provide for both efficient and faithful replication of the target nucleic acids. Two primers are required for the amplification of each gene, one for the sense strand (that is, the strand containing the gene of interest) and one for the antisense strand (that is, the strand complementary to the gene of interest). Pairs of primers should have similar melting temperatures that are close to the PCR reaction's annealing temperature. In order to facilitate the PCR reaction, the following features should be avoided in primers: mononucleotide repeats, complementarity with other primers in the mixture, self-complementarity, and internal hairpins and/or loops. Methods of primer design are known in the art; additionally, computer programs exist that can assist the skilled practitioner with primer design. Primers can optionally incorporate restriction enzyme recognition sites at their 5′ ends to assist in later ligation into plasmids or other vectors.
PCR can be carried out using purified DNA, unpurified DNA that is integrated into a vector, or unpurified genomic DNA. The process for amplifying target DNA using PCR consists of introducing an excess of two primers having the characteristics described above to a mixture containing the sequence to be amplified, followed by a series of thermal cycles in the presence of a heat-tolerant or thermophilic DNA polymerase, such as, for example, any of Taq, Pfu, Pwo, Tfl, rTth, Tli, or Tma polymerases. A PCR “cycle” involves denaturation of the DNA through heating, followed by annealing of the primers to the target DNA, followed by extension of the primers using the thermophilic DNA polymerase and a supply of deoxynucleotide triphosphates (i.e., dCTP, dATP, dGTP, and TTP), along with buffers, salts, and other reagents as needed. In one aspect, the DNA segments created by primer extension during the PCR process can serve as templates for additional PCR cycles. Many PCR cycles can be performed to generate a large concentration of target DNA or genes. PCR can optionally be performed in a device or machine with programmable temperature cycles for denaturation, annealing, and extension steps. Further, PCR can be performed on multiple genes simultaneously in the same reaction vessel or microcentrifuge tube since the primers chosen will be specific to selected genes. PCR products can be purified by techniques known in the art such as, for example, gel electrophoresis followed by extraction from the gel using commercial kits and reagents.
In a further aspect, the plasmid can include an origin of replication, allowing it to use the host cell's replication machinery to create copies of itself.
As used herein, “operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one affects the function of another. For example, if sequences for multiple genes are inserted into a single plasmid, their expression may be operably linked. Alternatively, a promoter is said to be operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence.
As used herein, “expression” refers to transcription and/or accumulation of an mRNA derived from a gene or DNA fragment. Expression may also be used to refer to translation of mRNA into a peptide, polypeptide, or protein.
In one aspect, provided herein are DNA constructs having at least the following genetic components:
Each component of the DNA constructs is described in detail below.
In one aspect, the DNA constructs disclosed herein incorporate a gene that encodes sulfotransferase. In a further aspect, sulfotransferase is an enzyme that catalyzes the transfer of a sulfo group from a donor molecule to an alcohol or amine, wherein the molecule to be modified can be part of numerous different biomolecules including proteins, lipids, and/or carbohydrates. Sulfotransferase enzymes are involved in drug metabolism and may be cytosolic or membrane-associated.
In one aspect, the gene that encodes sulfotransferase is isolated from a mammal such as, for example, human, bonobo, Western lowland gorilla, chimpanzee, Bornean orangutan, Northern white-cheeked gibbon, Sumatran orangutan, silvery gibbon, siamang, golden snub-nosed monkey, Indochinese rhesus macaque, crab-eating macaque, Tibetan macaque, Southern pig-tailed macaque, drill, green monkey, Ugandan red colobus, Angola colobus, Francois' langur, olive baboon, black-and-white snub-nosed monkey, sooty mangabey, black-capped squirrel monkey, common marmoset, gelada, Panamanian white-faced capuchin, South-central black rhinoceros, plains zebra, Philippine tarsier, Southern white rhinoceros, African wild ass, horse, Przewalski's horse, Sunda slow loris, Northern greater galago, elk, Coquerel's sifaka, dwarf musk deer, red deer, Chinese tree shrew, tufted capuchin, goat, ring-tailed lemur, sheep, scimitar oryx, carabao, takin, Eastern gray squirrel, creeping vole, lesser Egyptian jerboa, hybrid cattle, or cattle. In a further aspect, the gene that encodes sulfotransferase has SEQ ID NO. 1 or at least 70% homology thereto, at least 75% homology thereto, at least 80% homology thereto, at least 85% homology thereto, at least 90% homology thereto, at least 95% homology thereto, or at least 99% homology thereto.
Other sequences encoding sulfotransferase or related or homologous genes can be identified in a database such as, for example, GenBank. In one aspect, the gene that encodes sulfotransferase is isolated from Homo sapiens and can be identified by the GI number DQ891165.2 in the GenBankdatabase. In another aspect, sequences useful herein include those with GI numbers listed in Table 1:
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens
Pan paniscus
synthetic construct
Gorilla gorilla gorilla
Pan troglodytes
Pongo pygmaeus
Nomascus leucogenys
Pongo abelii
Hylobates moloch
Symphalangus syndactylus
Rhinopithecus roxellana
Macaca mulatta
Macaca fascicularis
Macaca thibetana thibetana
Macaca fascicularis
Macaca nemestrina
Mandrillus leucophaeus
Chlorocebus sabaeus
Macaca fascicularis
Piliocolobus tephrosceles
Colobus angolensis palliatus
Trachypithecus francoisi
Homo sapiens
Papio anubis
Macaca mulatta
Rhinopithecus bieti
Chlorocebus sabaeus
Cercocebus atys
Saimiri boliviensis boliviensis
Callithrix jacchus
Callithrix jacchus
Theropithecus gelada
Cebus imitator
Diceros bicornis minor
Equus quagga
Carlito syrichta
Ceratotherium simum simum
Equus quagga
Equus asinus
Equus asinus
Diceros bicornis minor
Equus caballus
Equus przewalskii
Nycticebus coucang
Otolemur garnettii
Carlito syrichta
Cervus canadensis
Propithecus coquereli
Saimiri boliviensis boliviensis
Equus caballus
Nycticebus coucang
Otolemur garnettii
Moschus berezovskii
Cervus elaphus
Tupaia chinensis
Sapajus apella
Equus asinus
Sapajus apella
Nycticebus coucang
Nycticebus coucang
Nycticebus coucang
Otolemur garnettii
Capra hircus
Equus przewalskii
Lemur catta
Ovis aries
Oryx dammah
Otolemur garnettii
Bubalus carabanensis
Nycticebus coucang
Nycticebus coucang
Nycticebus coucang
Budorcas taxicolor
Otolemur garnettii
Diceros bicornis minor
Nycticebus coucang
Nycticebus coucang
Nycticebus coucang
Sciurus carolinensis
Microtus oregoni
Cervus elaphus
Cervus elaphus
Jaculus jaculus
Bos indicus × Bos taurus
Bos taurus
Equus asinus
In one aspect, the DNA constructs disclosed herein incorporate a gene that encodes UDP glucuronosyltransferase. In a further aspect, UDP glucuronosyltransferase is an enzyme found in microsomes that is involved in the elimination of common prescription drugs as well as dietary chemicals, environmental chemicals, toxic substances, and the like. UDP glucuronosyltransferase catalyzes the transfer of a glucuronosyl group from UDP to a substrate containing oxygen, nitrogen, sulfur, or a carboxyl group, producing a polar molecule that can be easily excreted such as, for example, by the kidneys.
In one aspect, the gene that encodes UDP glucuronosyltransferase is isolated from a mammal such as, for example, Human, Northern white-cheeked gibbon, silvery gibbon, Sumatran orangutan, Western lowland gorilla, bonobo, chimpanzee, crab-eating macaque, Southern pig-tailed macaque, Bornean orangutan, gelada, Tibetan macaque, Angola colobus, siamang, olive baboon, black-and-white snub-nosed monkey, Francois' langur, drill, sooty mangabey, Indochinese rhesus macaque, Nancy Ma's night monkey, Panamanian white-faced capuchin, Ugandan red colobus, golden snub-nosed monkey, common marmoset, black-capped squirrel monkey, Philippine tarsier, Daubenton's bat, Southern white rhinoceros, brown bear, polar bear, ferret, European mink, African wild ass, Pacific walrus, Eurasian otter, black-footed ferret, horse, Coquerel's sifaka, Alpine marmot, or domestic water buffalo. In a further aspect, the gene that encodes UDP glucuronosyltransferase has SEQ ID NO. 2 or at least 70% homology thereto, at least 75% homology thereto, at least 80% homology thereto, at least 85% homology thereto, at least 90% homology thereto, at least 95% homology thereto, or at least 99% homology thereto.
Other sequences encoding UDP glucuronosyltransferase or related or homologous genes can be identified in a database such as, for example, GenBank. In one aspect, the gene that encodes UDP glucuronosyl transferase (UGT) is isolated from Homo sapiens and can be identified by the GI number KJ899004.1 in the GenBank database. In another aspect, sequences useful herein include those with GI numbers listed in Table 2:
Human ORFeome Gateway entry
vector
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens
Nomascus leucogenys
Nomascus leucogenys
Hylobates moloch
Pongo abelii
Hylobates moloch
Pongo abelii
Pongo abelii
Homo sapiens
Homo sapiens
Gorilla gorilla gorilla
Pan paniscus
Nomascus leucogenys
Pan troglodytes
Macaca fascicularis
Macaca fascicularis
Pongo abelii
Homo sapiens
Homo sapiens
Macaca fascicularis
Macaca nemestrina
Pongo pygmaeus
Theropithecus gelada
Macaca thibetana thibetana
Colobus angolensis palliatus
Symphalangus syndactylus
Papio anubis
Papio anubis
Rhinopithecus bieti
Pan paniscus
Gorilla gorilla gorilla
Pan troglodytes
Trachypithecus francoisi
Homo sapiens
Macaca fascicularis
Macaca fascicularis
Papio anubis
Mandrillus leucophaeus
Papio anubis
Macaca fascicularis
Cercocebus atys
Macaca fascicularis
Macaca mulatta
Papio anubis
Theropithecus gelada
Homo sapiens
Macaca thibetana thibetana
Trachypithecus francoisi
Colobus angolensis palliatus
Papio anubis
Aotus nancymaae
Cebus imitator
Piliocolobus tephrosceles
Rhinopithecus roxellana
Cebus imitator
Macaca thibetana thibetana
Rhinopithecus bieti
Cebus imitator
Aotus nancymaae
Callithrix jacchus
Callithrix jacchus
Callithrix jacchus
Rhinopithecus roxellana
Callithrix jacchus
Saimiri boliviensis boliviensis
Carlito syrichta
Gorilla gorilla gorilla
Theropithecus gelada
Nomascus leucogenys
Mandrillus leucophaeus
Mandrillus leucophaeus
Rhinopithecus roxellana
Theropithecus gelada
Cercocebus atys
Myotis daubentonii
Ceratotherium simum simum
Piliocolobus tephrosceles
Ursus arctos
Ursus maritimus
Mustela putorius furo
Mustela lutreola
Equus asinus
Odobenus rosmarus divergens
Lutra lutra
Homo sapiens
Mustela nigripes
Equus caballus
Propithecus coquereli
Marmota marmota marmota
Bubalus bubalis
In one aspect, the DNA constructs disclosed herein incorporate a gene that encodes 0-linked N-acetylglucosamine transferase (OGT). In a further aspect, OGT is an enzyme that catalyzes the addition of one N-acetylglucosamine involved in an O-glycosidic linkage to a serine or threonine. OGT may, in some aspects, compete for serine and threonine residues with protein kinases or may have substrate specificity. OGT is found in both cytoplasmic and mitochondrial isoforms.
In one aspect, the gene that encodes OGT is isolated from a mammal such as, for example, an Arctic ground squirrel, groundhog, yellow-bellied marmot, thirteen-lined ground squirrel, dromedary camel, carabao, Eastern gray squirrel, wild yak, domestic water buffalo, alpaca, hybrid cattle, cattle, brown bear, Bactrian camel, polar bear, olive baboon, gelada, sooty mangabey, American black bear, Etruscan shrew, drill, red deer, elk, giant panda, Tibetan macaque, striped hyena, Damara mole rat, siamang, crab-eating macaque, Indochinese rhesus macaque, cougar, horse, dog, clouded leopard, takin, Southern pig-tailed macaque, dingo, golden snub-nosed monkey, Angola colobus, goat, dwarf musk deer, common raccoon dog, bobcat, leopard cat, sheep, scimitar oryx, Canada lynx, red fox, white-tailed deer, cheetah, European rabbit, snow leopard, fishing cat, plains zebra, cat, common degu, green monkey, Alpine marmot, silvery gibbon, Bornean orangutan, common marmoset, or leopard. In a further aspect, the gene that encodes OGT has SEQ ID NO. 4 or at least 70% homology thereto, at least 75% homology thereto, at least 80% homology thereto, at least 85% homology thereto, at least 90% homology thereto, at least 95% homology thereto, or at least 99% homology thereto.
Other sequences encoding OGT or related or homologous genes can be identified in a database such as, for example, GenBank. In one aspect, the gene that encodes O-linked N-acetylglucosamine transferase (OGT) is isolated from Urocitellus parryii and can be identified by the GI number XM_026412627.1 in the GenBank database. In another aspect, sequences useful herein include those with GI numbers listed in Table 3:
Urocitellus parryii
Marmota monax
Marmota flaviventris
Ictidomys tridecemlineatus
Ictidomys tridecemlineatus
Urocitellus parryii
Camelus dromedarius
Marmota monax
Bubalus carabanensis
Sciurus carolinensis
Marmota flaviventris
Bos mutus
Ictidomys tridecemlineatus
Ictidomys tridecemlineatus
Bubalus bubalis
Vicugna pacos
Bos indicus × Bos taurus
Bos taurus
Ursus arctos
Camelus bactrianus
Camelus bactrianus
Ursus maritimus
Bubalus carabanensis
Papio anubis
Theropithecus gelada
Cercocebus atys
Ursus americanus
Marmota flaviventris
Suncus etruscus
Mandrillus leucophaeus
Cervus elaphus
Cervus canadensis
Marmota monax
Ailuropoda melanoleuca
Camelus dromedarius
Bubalus carabanensis
Macaca thibetana thibetana
Sciurus carolinensis
Bubalus bubalis
Hyaena hyaena
Fukomys damarensis
Symphalangus syndactylus
Bos mutus
Macaca fascicularis
Vicugna pacos
Macaca mulatta
Bos indicus × Bos taurus
Puma concolor
Equus caballus
Canis lupus familiaris
Neofelis nebulosa
Ursus arctos
Budorcas taxicolor
Macaca nemestrina
Canis lupus dingo
Camelus bactrianus
Ursus maritimus
Bos taurus
Bos taurus
Rhinopithecus roxellana
Colobus angolensis palliatus
Papio anubis
Theropithecus gelada
Capra hircus
Moschus berezovskii
Nyctereutes procyonoides
Cercocebus atys
Lynx rufus
Ursus americanus
Prionailurus bengalensis
Ovis aries
Oryx dammah
Canis lupus familiaris
Canis lupus familiaris
Suncus etruscus
Lynx canadensis
Vulpes vulpes
Odocoileus virginianus texanus
Odocoileus virginianus texanus
Acinonyx jubatus
Oryctolagus cuniculus
Mandrillus leucophaeus
Panthera uncia
Prionailurus viverrinus
Equus quagga
Equus quagga
Felis catus
Cervus elaphus
Cervus canadensis
Octodon degus
Chlorocebus sabaeus
Marmota marmota marmota
Panthera uncia
Ailuropoda melanoleuca
Hylobates moloch
Pongo pygmaeus
Callithrix jacchus
Panthera pardus
Macaca thibetana thibetana
Hyaena hyaena
In one aspect, the DNA constructs disclosed herein incorporate a gene that encodes alcohol dehydrogenase, such as, for example, alcohol dehydrogenase II (ADHII). In one aspect, the gene that expresses alcohol dehydrogenase catalyzes the conversion of ethanol to acetaldehyde. In another aspect, alcohol dehydrogenase can act with any one of a number of primary unbranched aliphatic alcohols. In some aspects, alcohol dehydrogenase II requires at least two Zn2+ ions per subunit to function. In other aspects, one molecule of NAD+ is required to convert an alcohol to an aldehyde or ketone using alcohol dehydrogenase.
In one aspect, the gene that encodes alcohol dehydrogenase is isolated from a fungus such as, for example, a Saccharomyces cerevisiae strain including, but not limited to, N85, S288C, YJM1083, YJM1129, YJM1133, YJM1190, YJM1208, YJM1244, YJM1250, YJM1252, YJM1307, YJM1336, YJM1356, YJM1381, YJM1383, YJM1385, YJM1386, YJM1387, YJM1388, YJM1389, YJM1415, YJM1417, YJM1419, YJM1433, YJM1460, YJM1478, YJM1526, YJM1527, YJM1592, YJM1615, YJM271, YJM450, YJM451, YJM453, YJM456, YJM470, YJM541, YJM554, YJM555, YJM681, YJM682, YJM689, YJM969, YJM972, YJM975, YJM978, YJM981, YJM984, YJM987, YJM990, YJM993, or YJM996. In a further aspect, the gene that encodes alcohol dehydrogenase has SEQ ID NO. 3 or at least 70% homology thereto, at least 75% homology thereto, at least 80% homology thereto, at least 85% homology thereto, at least 90% homology thereto, at least 95% homology thereto, or at least 99% homology thereto.
Other sequences encoding alcohol dehydrogenase or related or homologous genes can be identified in a database such as, for example, GenBank. In one aspect, the gene that encodes alcohol dehydrogenase is isolated from Saccharomyces cerevisiae and can be identified by the GI number CP033482.1 in the GenBank database. In another aspect, sequences useful herein include those with GI numbers listed in Table 4:
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae YJM1386
Saccharomyces cerevisiae YJM1385
Saccharomyces cerevisiae YJM1381
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae S288C
Saccharomyces cerevisiae S288C
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae YJM1419
Saccharomyces cerevisiae YJM1615
Saccharomyces cerevisiae YJM1208
Saccharomyces cerevisiae
Saccharomyces cerevisiae YJM1592
Saccharomyces cerevisiae N85
Saccharomyces cerevisiae YJM1389
Saccharomyces cerevisiae YJM1388
Saccharomyces cerevisiae YJM1307
Saccharomyces cerevisiae S288C
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae YJM451
Saccharomyces cerevisiae YJM1460
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae YJM1433
Saccharomyces cerevisiae YJM456
Saccharomyces cerevisiae YJM689
Saccharomyces cerevisiae YJM682
Saccharomyces cerevisiae YJM1383
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae YJM1250
Saccharomyces cerevisiae YJM1083
Saccharomyces cerevisiae YJM470
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae YJM1356
Saccharomyces cerevisiae YJM1190
Saccharomyces cerevisiae YJM978
Saccharomyces cerevisiae YJM555
Saccharomyces cerevisiae YJM1133
Saccharomyces cerevisiae YJM975
Saccharomyces cerevisiae YJM554
Saccharomyces cerevisiae YJM1526
Saccharomyces cerevisiae YJM972
Saccharomyces cerevisiae YJM996
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae YJM1336
Saccharomyces cerevisiae YJM1244
Saccharomyces cerevisiae YJM993
Saccharomyces cerevisiae YJM453
Saccharomyces cerevisiae YJM990
Saccharomyces cerevisiae YJM987
Saccharomyces cerevisiae YJM450
Saccharomyces cerevisiae YJM984
Saccharomyces cerevisiae YJM681
Saccharomyces cerevisiae YJM981
Saccharomyces cerevisiae YJM1129
Saccharomyces cerevisiae YJM969
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae YJM1478
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae YJM271
Saccharomyces cerevisiae YJM1527
Saccharomyces cerevisiae YJM1252
Saccharomyces cerevisiae YJM541
Saccharomyces cerevisiae YJM1387
Saccharomyces cerevisiae YJM1417
Saccharomyces cerevisiae YJM1415
In one aspect, the DNA constructs disclosed herein incorporate a gene that encodes cytochrome P450. In a further aspect, cytochrome P450 is an enzyme that oxidizes steroids, fatty acids, and external compounds such as drugs. In an aspect, cytochrome P450 is involved in the clearance of certain drugs from the body. Cytochrome P450 enzymes typically include a heme-iron center which is used in catalysis.
In one aspect, the gene that encodes cytochrome P450 is isolated from a mammal such as, for example, a human, bonobo, chimpanzee, Western lowland gorilla, siamang, Northern white-cheeked gibbon, olive baboon, Sumatran orangutan, grivet, gelada, Bornean orangutan, Indochinese rhesus macaque, Japanese macaque, green monkey, Angola colobus, Southern pig-tailed macaque, Tibetan macaque, sooty mangabey, drill, crab-eating macaque, golden snub-nosed monkey, Francois' langur, Ugandan red colobus, tufted capuchin, black-and-white snub-nosed monkey, Panamanian white-faced capuchin, black-capped squirrel monkey, common marmoset, Nancy Ma's night monkey, or silvery gibbon. In a further aspect, the gene that encodes cytochrome P450 has SEQ ID NO. 5 or at least 70% homology thereto, at least 75% homology thereto, at least 80% homology thereto, at least 85% homology thereto, at least 90% homology thereto, at least 95% homology thereto, or at least 99% homology thereto.
Other sequences encoding cytochrome P450 or related or homologous genes can be identified in a database such as, for example, GenBank. In one aspect, the gene that encodes cytochrome P450 family 3 subfamily A member 4 is isolated from Homo sapiens and can be identified by the GI number DQ924960.1 in the GenBank database. In another aspect, sequences useful herein include those with GI numbers listed in Table 5:
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens
Human ORFeome Gateway entry vector
Homo sapiens
Homo sapiens
Pan paniscus
Pan troglodytes
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens
Gorilla gorilla gorilla
Symphalangus syndactylus
Symphalangus syndactylus
Nomascus leucogenys
Papio anubis
Pongo abelii
Nomascus leucogenys
Chlorocebus aethiops
Theropithecus gelada
Pongo pygmaeus
Theropithecus gelada
Pongo pygmaeus
Chlorocebus aethiops
Theropithecus gelada
Macaca mulatta
Macaca fuscata
Chlorocebus sabaeus
Colobus angolensis palliatus
Macaca nemestrina
Colobus angolensis palliatus
Macaca thibetana thibetana
Macaca thibetana thibetana
Macaca thibetana thibetana
Macaca thibetana thibetana
Macaca mulatta
Cercocebus atys
Mandrillus leucophaeus
Macaca fascicularis
Macaca fascicularis
Mandrillus leucophaeus
Macaca thibetana thibetana
Macaca thibetana thibetana
Colobus angolensis palliatus
Rhinopithecus roxellana
Macaca thibetana thibetana
Trachypithecus francoisi
Homo sapiens
Piliocolobus tephrosceles
Mandrillus leucophaeus
Sapajus apella
Rhinopithecus bieti
Trachypithecus francoisi
Cebus imitator
Saimiri boliviensis boliviensis
Saimiri boliviensis boliviensis
Symphalangus syndactylus
Callithrix jacchus
Callithrix jacchus
Symphalangus syndactylus
Symphalangus syndactylus
Chlorocebus sabaeus
Aotus nancymaae
Nomascus leucogenys
Mandrillus leucophaeus
Papio anubis
Nomascus leucogenys
Nomascus leucogenys
Theropithecus gelada
Chlorocebus aethiops
Chlorocebus sabaeus
Hylobates moloch
Symphalangus syndactylus
Symphalangus syndactylus
Cercocebus atys
Cercocebus atys
Pan troglodytes
Pongo abelii
Pongo pygmaeus
Pongo pygmaeus
Pan paniscus
Papio anubis
Mandrillus leucophaeus
Macaca mulatta
Chlorocebus sabaeus
Macaca thibetana thibetana
Symphalangus syndactylus
Pan troglodytes
In one aspect, the DNA construct has the following genetic components: a) a gene that encodes a sulfotransferase, b) a gene that encodes UGT, c) a gene that encodes OGT, d) a gene that encodes an alcohol dehydrogenase, and e) a gene that encodes cytochrome P450 family 3 subfamily A member 4.
In another aspect, said construct further includes a) a promoter, b) a terminator or stop sequence, c) a gene that confers resistance to an antibiotic (a “selective marker”), d) a reporter protein, or any combination thereof. Each of these elements is described in further detail below.
In one aspect, the construct includes from 5′ to 3′ the following genetic components in the following order: (1) a gene that encodes sulfotransferase, (2) a gene that encodes UGT, (3) a gene that encodes OGT, (4) a gene that encodes an alcohol dehydrogenase, and (5) a gene that encodes cytochrome P450 family 3 subfamily A member 4.
In one aspect, the construct includes from 5′ to 3′ the following genetic components in the following order: a gene that encodes sulfotransferase having SEQ ID NO. 1 or at least 70% homology thereto, a gene that encodes UGT having SEQ ID NO. 2 or at least 70% homology thereto, a gene that encodes OGT having SEQ ID NO. 4 or at least 70% homology thereto, a gene that encodes an alcohol dehydrogenase having SEQ ID NO. 3 or at least 70% homology thereto, and a gene that encodes a cytochrome P450 having SEQ ID NO. 5 or at least 70% homology thereto. In some aspects, the construct optionally includes a gene that encodes enhanced green fluorescent protein having SEQ ID NO. 6 or at least 70% homology thereto.
In another aspect, the construct includes from 5′ to 3′ the following genetic components in the following order: (1) a gene that encodes sulfotransferase, (2) a CYC1 terminator, (3) a GAL1 promoter, (4) a gene that encodes UGT, (5) a CYC1 terminator, (6) a GAL1 promoter, (7) a gene that encodes OGT, (8) a CYC1 terminator, (9) a GAL1 promoter, (10) a gene that encodes alcohol dehydrogenase, (11) a CYC1 terminator, (12) a GAL1 promoter, and (13) a gene that encodes cytochrome P450.
In another aspect, the construct includes from 5′ to 3′ the following genetic components in the following order: (1) a gene that encodes sulfotransferase having SEQ ID NO. 1 or at least 90% homology thereto, (2) a CYC1 terminator, (3) a GAL1 promoter, (4) a gene that encodes UGT having SEQ ID NO. 2 or at least 90% homology thereto, (5) a CYC1 terminator, (6) a GAL1 promoter, (7) a gene that encodes OGT having SEQ ID NO. 4 or at least 90% homology thereto, (8) a CYC1 terminator, (9) a GAL1 promoter, (10) a gene that encodes alcohol dehydrogenase having SEQ ID NO. 3 or at least 90% homology thereto, (11) a CYC1 terminator, (12) a GAL1 promoter, and (13) a gene that encodes cytochrome P450 having SEQ ID NO. 5 or at least 90% homology thereto.
In still another aspect, the construct is a pYES2 plasmid having from 5′ to 3′ the following genetic components in the following order: (1) a gene that encodes sulfotransferase having SEQ ID NO. 1 or at least 70% homology thereto, (2) a CYC1 terminator, (3) a GAL1 promoter, (4) a gene that encodes UGT having SEQ ID NO. 2 or at least 70% homology thereto, (5) a CYC1 terminator, (6) a GAL1 promoter, (7) a gene that encodes OGT having SEQ ID NO. 4 or at least 70% homology thereto, (8) a CYC1 terminator, (9) a GAL1 promoter, (10) a gene that encodes alcohol dehydrogenase having SEQ ID NO. 3 or at least 70% homology thereto, (11) a CYC1 terminator, (12) a GAL1 promoter, and (13) a gene that encodes cytochrome P450 having SEQ ID NO. 5 or at least 70% homology thereto. In some embodiments, the construct further includes a gene that encodes enhanced green fluorescent protein having SEQ ID NO. 6 or at least 70% homology thereto, placed in a position 3′ to the gene that encodes cytochrome P450.
In another aspect, the DNA construct has SEQ ID NO. 7 or at least 70% homology thereto, at least 75% homology thereto, at least 80% homology thereto, at least 85% homology thereto, at least 90% homology thereto, at least 95% homology thereto, or at least 99% homology thereto.
In another aspect, said construct further includes a) a promoter, b) a terminator or stop sequence, c) a gene that confers resistance to an antibiotic (a “selective marker”), d) a reporter protein, or any combination thereof.
In one aspect, the construct includes a regulatory sequence. In a further aspect, the regulatory sequence is already incorporated into a vector such as, for example, a plasmid, prior to genetic manipulation of the vector. In another aspect, the regulatory sequence can be incorporated into the vector through the use of restriction enzymes or any other technique known in the art.
In one aspect, the regulatory sequence is a promoter. The term “promoter” refers to a DNA sequence capable of controlling the expression of a coding sequence. In another aspect, the coding sequence to be controlled is located 3′ to the promoter. In still another aspect, the promoter is derived from a native gene. In an alternative aspect, the promoter is composed of multiple elements derived from different genes and/or promoters. A promoter can be assembled from elements found in nature, from artificial and/or synthetic elements, or from a combination thereof. It is understood by those skilled in the art that different promoters can direct the expression of a gene in different tissues or cell types, at different stages of development, in response to different environmental or physiological conditions, and/or in different species. In one aspect, the promoter functions as a switch to activate the expression of a gene.
In one aspect, the promoter is “constitutive.” A constitutive promoter is a promoter that causes a gene to be expressed in most cell types at most times. In another aspect, the promoter is “regulated.” A regulated promoter is a promoter that becomes active in response to a specific stimulus. A promoter may be regulated chemically, such as, for example, in response to the presence or absence of a particular metabolite (e.g., lactose or tryptophan), a metal ion, a molecule secreted by a pathogen, or the like. A promoter also may be regulated physically, such as, for example, in response to heat, cold, water stress, salt stress, oxygen concentration, illumination, wounding, or the like.
Promoters that are useful to drive expression of the nucleotide sequences described herein are numerous and familiar to those skilled in the art. Suitable promoters include, but are not limited to, the following: T3 promoter, T7 promoter, an iron promoter, araBAD promoter, and GAL1 promoter. In a further aspect, the promoter is a native part of the vector used herein. Variants of these promoters are also contemplated. The skilled artisan will be able to use site-directed mutagenesis and/or other mutagenesis techniques to modify the promoters to promote more efficient function. The promoter may be positioned, for example, from 10-100 nucleotides from a ribosomal binding site.
In one aspect, the promoter is a GAL1 promoter. In another aspect, the GAL1 promoter is native to the plasmid used to create the vector. In another aspect, a GAL1 promoter is positioned before the gene that encodes sulfotransferase, the gene that encodes UGT, the gene that encodes OGT, the gene that encodes alcohol dehydrogenase, the gene that encodes cytochrome P450, or any combination thereof. In another aspect, the promoter is a GAL1 promoter obtained from or native to the pYES2 plasmid.
In one aspect, the regulatory sequence is an operon such as, for example, the LAC operon or LAC operator. As used herein, an “operon” is a segment of DNA containing a group of genes wherein the group is controlled by a single promoter. Genes included in an operon are all transcribed together. In a further aspect, the operon is a LAC operon and can be induced when lactose crosses the cell membrane of the biological device.
In another aspect, the regulatory sequence is a terminator or stop sequence. As used herein, a terminator is a sequence of DNA that marks the end of a gene or operon to be transcribed. In a further aspect, the terminator is an intrinsic terminator or a Rho-dependent transcription terminator. As used herein, an intrinsic terminator is a sequence wherein a hairpin structure can form in the nascent transcript that disrupts the mRNA/DNA/RNA polymerase complex. As used herein, a Rho-dependent transcription terminator requires a Rho factor protein complex to disrupt the mRNA/DNA/RNA polymerase complex. In one aspect, the terminator is an rrnB terminator obtained from or native to the pBAD plasmid. In an alternative aspect, the terminator is a CYC1 terminator obtained from or native to the pYES2 plasmid.
In a further aspect, the regulatory sequence includes both a promoter and a terminator or stop sequence. In a still further aspect, the regulatory sequence can include multiple promoters or terminators. Other regulatory elements, such as enhancers, are also contemplated. Enhancers may be located from about 1 to about 2000 nucleotides in the 5′ direction from the start codon of the DNA to be transcribed, or may be located 3′ to the DNA to be transcribed. Enhancers may be “cis-acting,” that is, located on the same molecule of DNA as the gene whose expression they affect.
In another aspect, the vector contains one or more ribosomal binding sites. As used herein, a “ribosomal binding site” or “rbs” is a sequence of nucleotides located 5′ to the start codon of an mRNA that recruits a ribosome to initiate protein translation. In one aspect, the ribosomal binding site can be positioned before one or more or all genes in the DNA construct, or a before a subset of genes in a DNA construct.
In one aspect, when the vector is a plasmid, the plasmid can also contain a multiple cloning site or polylinker. In a further aspect, the polylinker contains recognition sites for multiple restriction enzymes. The polylinker can contain up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 recognition sites for restriction enzymes. Further, restriction sites may be added, disabled, or removed as required, using techniques known in the art. In one aspect, the plasmid contains restriction sites for any known restriction enzyme such as, for example, HindIII, KpnI, SacI, BamHI, BstXI, EcoRI, BasBI, NotI, XhoI, XphI, XbaI, ApaI, SalI, ClaI, EcoRV, PstI, SmaI, XmaI, SpeI, EagI, SacII, or any combination thereof. In a further aspect, the plasmid contains more than one recognition site for the same restriction enzyme.
In one aspect, the restriction enzyme can cleave DNA at a palindromic or an asymmetrical restriction site. In a further aspect, the restriction enzyme cleaves DNA to leave blunt ends; in an alternative aspect, the restriction enzyme cleaves DNA to leave “sticky” or overhanging ends. In another aspect, the enzyme can cleave DNA at a distance of from 20 bases to over 1000 bases away from the restriction site. A variety of restriction enzymes are commercially available and their recognition sequences, as well as instructions for use (e.g., amount of DNA needed, precise volumes of reagents, purification techniques, as well as information about salt concentration, pH, optimum temperature, incubation time, and the like) are provided by enzyme manufacturers.
In one aspect, a plasmid with a polylinker containing one or more restriction sites can be digested with one restriction enzyme and a nucleotide sequence of interest can be ligated into the plasmid using a commercially-available DNA ligase enzyme. Several such enzymes are available, often as kits containing all reagents and instructions required for use. In another aspect, a plasmid with a polylinker containing two or more restriction sites can be simultaneously digested with two restriction enzymes and a nucleotide sequence of interest can be ligated into the plasmid using a DNA ligase enzyme. Using two restriction enzymes provides an asymmetric cut in the DNA, allowing for insertion of a nucleotide sequence of interest in a particular direction and/or on a particular strand of the double-stranded plasmid. Since RNA synthesis from a DNA template proceeds from 5′ to 3′, usually starting just after a promoter, the order and direction of elements inserted into a plasmid can be especially important. If a plasmid is to be simultaneously digested with multiple restriction enzymes, these enzymes must be compatible in terms of buffer, salt concentration, and other incubation parameters.
In some aspects, prior to ligation using a ligase enzyme, a plasmid that has been digested with a restriction enzyme is treated with an alkaline phosphatase enzyme to remove 5′ terminal phosphate groups. This prevents self-ligation of the plasmid and thus facilitates ligation of heterologous nucleotide fragments into the plasmid.
In one aspect, different genes can be ligated into a plasmid in one pot. In this aspect, the genes will first be digested with restriction enzymes. In certain aspects, the digestion of genes with restriction enzymes provides multiple pairs of matching 5′ and 3′ overhangs that will spontaneously assemble the genes in the desired order. In another aspect, the genes and components to be incorporated into a plasmid can be assembled into a single insert sequence prior insertion into the plasmid. In a further aspect, a DNA ligase enzyme can be used to assist in the ligation process.
In another aspect, the ligation mix may be incubated in an electromagnetic chamber. In one aspect, the incubation lasts for about 1 minute, about 2 minutes, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, or about 1 hour.
The DNA construct described herein can be part of a vector. In general, plasmid vectors containing replicon and control sequences that are derived from species compatible with the host cell are used in connection with the hosts. The vector ordinarily carries a replication site as well as marking sequences that are capable of performing phenotypic selection in transformed cells. Plasmid vectors are well known and commercially available. Such vectors include, but are not limited to, pWLneo, pSV2cat, pOG44, pXT1, pSG, pSVK3, pBSK, pYES, pYES2, pBSKII, pET, pUC, pUC19, pBAD, and pETDuet-1 vectors.
Plasmids are double-stranded, autonomously-replicating, genetic elements that are not integrated into host cell chromosomes. Further, these genetic elements are usually not part of the host cell's central metabolism. In bacteria, plasmids may range from 1 kilobase (kb) to over 200 kb. Plasmids can be engineered to encode a number of useful traits including the production of secondary metabolites, antibiotic resistance, the production of useful proteins, degradation of complex molecules and/or environmental toxins, and others. Plasmids have been the subject of much research in the field of genetic engineering, as plasmids are convenient expression vectors for foreign DNA in, for example, microorganisms. Plasmids generally contain regulatory elements such as promoters and terminators and also usually have independent replication origins. Ideally, plasmids will be present in multiple copier per host cell and will contain selectable markers (such as genes for antibiotic resistance) to show the skilled artisan to select host eels that have been successfully transfected with the plasmids (for example, by growing the host cells in a medium containing the antibiotic).
In one aspect, the vector encodes a selection marker. In a further aspect, the selection marker is a gene that confers resistance to an antibiotic. In certain aspects, during fermentation of host cells transformed with the vector, the cells are contacted with the antibiotic. For example, the antibiotic may be included in the culture medium. Cells that have not been successfully transformed cannot survive in the presence of the antibiotic; only cells containing the vector, which confers antibiotic resistance, can survive. Optimally, only cells containing the vector to be expressed will be cultured, as this will result in the highest production efficiency of the desired gene products (e.g., peptides). Cells that do not contain the vector would otherwise compete with transformed cells for resources. In one aspect, the antibiotic is tetracycline, neomycin, kanamycin, ampicillin, hygromycin, chloramphenicol, amphotericin B, bacitracin, carbapenam, cephalosporin, ethambutol, fluoroquinolones, isonizid, methicillin, oxacillin, vancomycin, streptomycin, quinolines, rifampin, rifampicin, sulfonamides, cephalothin, erythromycin, streptomycin, gentamycin, penicillin, other commonly-used antibiotics, or a combination thereof.
In certain aspects, the DNA construct can include a gene that encodes a reporter protein. The selection of the reporter protein can vary. For example, the reporter protein can be a yellow fluorescent protein, a red fluorescent protein, a green fluorescent protein, or a cyan fluorescent protein. In one aspect, the reporter protein is a green fluorescent protein and the gene that encodes the reporter protein has SEQ ID NO. 6 or at least 70% homology thereto. The amount of fluorescence that is produced can be correlated to the amount of DNA incorporated into the transfected cells. The fluorescence produced can be detected and quantified using techniques known in the art. For example, spectrofluorometers are typically used to measure fluorescence.
The DNA construct described herein can be part of a vector. In one aspect, the vector is a plasmid, a phagemid, a cosmid, a yeast artificial chromosome, a bacterial artificial chromosome, a virus, a phage, or a transposon.
Exemplary methods for producing the DNA constructs described herein are provided in the Examples. Restriction enzymes and purification techniques known in the art can be used to assemble the DNA constructs. Backbone plasmids and synthetic inserts can be mixed together for ligation purposes at different ratios ranging from 1:1, 1:2, 1:3, 1:4, and up to 1:5. In one aspect, the ratio of backbone plasmid to synthetic insert is 1:4. After the vector comprising the DNA construct has been produced, the resulting vector can be incorporated into the host cells using the methods described below.
A variety of different types of cells can be used in the methods described herein. In one aspect, the cells can be wild-type cells (i.e., not genetically-modified). In one aspect, the cells are from an animal such as, for example, a mammal, bird, fish, reptile, amphibian, or invertebrate. In another aspect, the cells are from a plant such as, for example, an agricultural crop, a decorative plant, a woody plant, a medicinal plant, or a combination thereof. In another aspect, the cells are from a multicellular fungus such as, for example, a mushroom, a mycorrhizal fungus, or a commercially-important mold.
In another aspect, the cells include a biological device. A “biological device” is formed when a microbial cell is transfected with a DNA construct. The biological devices are generally composed of microbial host cells, where the host cells are transformed (i.e., genetically-modified) with a DNA construct.
In one aspect, the DNA construct is carried by the expression vector into the cell and is separate from the host cell's genome. In another aspect, the DNA construct is incorporated into the host cell's genome. In still another aspect, incorporation of the DNA construct into the host cell enables the host cell to produce an extract or composition that can remove metals and/or other contaminants from water or petroleum, such as, for example, those disclosed herein. “Heterologous” genes and proteins are genes and proteins that have been experimentally inserted into a cell that are not normally expressed by the cell. A heterologous gene may be cloned or derived from a different cell type or species than the recipient cell or organism. Heterologous genes may be introduced into cells by transduction or transformation.
An “isolated” nucleic acid is one that has been separated from other nucleic acid molecules and/or cellular material (peptides, proteins, lipids, saccharides, and the like) normally present in the natural source of the nucleic acid. An “isolated” nucleic acid may optionally be free of the flanking sequences found on either side of the nucleic acid as it naturally occurs. An isolated nucleic acid can be naturally occurring, can be chemically synthesized, or can be a cDNA molecule (i.e., is synthesized from an mRNA template using reverse transcriptase and DNA polymerase enzymes).
“Transformation” or “transfection” as used herein refers to a process for introducing heterologous DNA into a host cell. Transformation can occur under natural conditions or may be induced using various methods known in the art. Many methods for transformation are known in the art and the skilled practitioner will know how to choose the best transformation method based on the type of cells being transformed. Methods for transformation include, for example, viral infection, electroporation, lipofection, chemical transformation, and particle bombardment. Cells may be stably transformed (i.e., the heterologous DNA is capable of replicating as an autonomous plasmid or as part of the host chromosome) or may be transiently transformed (i.e., the heterologous DNA is expressed only for a limited period of time).
“Competent cells” refers to microbial cells capable of taking up heterologous DNA. Competent cells can be purchased from a commercial source, or cells can be made competent using procedures known in the art. Exemplary procedures for producing competent cells are provided in the Examples.
The host cells as referred to herein include their progeny, which are any and all subsequent generations formed by cell division. It is understood that not all progeny may be identical due to deliberate or inadvertent mutations. A host cell may be “transfected” or “transformed,” which refers to a process by which an exogenous nucleic acid is transferred or introduced into the host cell.
A transformed cell includes the primary subject cell and its progeny. The host cells can be naturally-occurring cells or “recombinant” cells. Recombinant cells are distinguishable from naturally-occurring cells in that naturally-occurring cells do not contain heterologous DNA introduced through molecular cloning procedures. In one aspect, the host cell is a prokaryotic cell such as, for example, Escherichia coli. In other aspects, the host cell is a eukaryotic cell such as, for example, the yeast Saccharomyces cerevisiae. Host cells transformed with the DNA construct described herein are referred to as “biological devices.”
The DNA construct is first delivered into the host cell. In one aspect, the host cells are naturally competent (i.e., able to take up exogenous DNA from the surrounding environment). In another aspect, cells must be treated to induce artificial competence. This delivery may be accomplished in vitro, using well-developed laboratory procedures for transforming cell lines. Transformation of bacterial cell lines can be achieved using a variety of techniques. One method involves calcium chloride. The exposure to the calcium ions renders the cells able to take up the DNA construct. Another method is electroporation. In this technique, a high-voltage electric field is applied briefly to cells, producing transient holes in the membranes of the cells through which the vector containing the DNA construct enters. Another method involves exposing intact yeast cells to alkali cations such as, for example, lithium. In one aspect, this method includes exposing yeast to lithium acetate, polyethylene glycol, and single-stranded DNA such as, for example, salmon sperm DNA. Without wishing to be bound by theory, the single-stranded DNA is thought to bind to the cell wall of the yeast, thereby blocking plasmids from binding. The plasmids are then free to enter the yeast cell. Enzymatic and/or electromagnetic techniques can also be used alone, or in combination with other methods, to transform microbial cells. Exemplary procedures for transforming yeast and bacteria with specific DNA constructs are provided in the Examples. In certain aspects, two or more types of DNA can be incorporated into the host cells. Thus, different metabolites can be produced from the same host cells at enhanced rates.
A satisfactory microbiological culture contains available sources of hydrogen donors and acceptors, carbon, nitrogen, sulfur, phosphorus, inorganic salts and, in certain cases, vitamins or other growth-promoting substances. For example, the addition of peptone provides a readily-available source of nitrogen and carbon. Furthermore, the use of different types of media results in different growth rates and different stationary phase densities. A rich media results in a short doubling time and higher cell density at stationary phase. Minimal media results in slow growth and low final cell densities. Efficient agitation and aeration increase final cell densities.
Culturing or fermenting of host cells can be accomplished by any technique known in the art. In one aspect, batch fermentation can be conducted. In batch fermentation, the composition of the culture medium is set at the beginning and the system is closed to future alterations. In some aspects, a limited form of batch fermentation may be carried out, wherein factors such as oxygen concentration and pH are manipulated, but additional carbon is not added. Continuous fermentation methods are also contemplated. In continuous fermentation, equal amounts of a defined medium are continuously added to and removed from a bioreactor. In other aspects, microbial cells are immobilized on a substrate. Fermentation may be carried out on any scale and may include methods in which literal “fermentation” is carried out as well as other culture methods that are non-fermentative.
In one aspect, the microorganisms can be cultured for a period of from 2 days to 2 weeks, or for about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, or about 14 days, where any value can be the lower or upper endpoint of a range (e.g., about 3 days to about 13 days, about 8 days to about 12 days, etc.). In one aspect, the microorganisms are cultured for about 10 days.
In another aspect, the microorganisms can be cultured at any temperature appropriate for the microorganisms, with the understanding that the temperature may vary according to the microorganism (for example, a thermophilic microorganism may require a higher culture temperature than a mesophile). In one aspect, the microorganisms are cultured at a temperature of from about 20 to about 37° C., or are cultured at about 20° C., about 21° C., about 22° C., about 23° C., about 24° C., about 25° C., about 26° C., about 27° C., about 28° C., about 29° C., about 30° C., about 31° C., about 32° C., about 33° C., about 34° C., about 35° C., about 36° C., or about 37° C., where any value can be the lower or upper endpoint of a range, where any value can be the lower or upper endpoint of a range (e.g., about 21° C. to about 36° C., about 25° C. to about 30° C., etc.).
In certain aspects, after culturing the microorganisms for a sufficient time, the microbial cells can be lysed with one or more enzymes. For example, when the microbial cells are fungal, the fungal cells can be lysed with lyticase. In one aspect, the lyticase concentration can be about 500 μL, about 600 μL, about 700 μL, about 800 μL, about 900 μL, or about 1,000 μL per liter of culture, where any value can be the lower or upper endpoint of a range, where any value can be the lower or upper endpoint of a range (e.g., about 500 μL to about 900 μL, about 600 μL to about 800 μL, etc.).
In addition to or in place of enzymes, other components can be used to facilitate lysis of the microbial cells. In one aspect, chitosan can be used in combination with an enzyme to lyse the microbial cells. Chitosan is generally composed of glucosamine units and N-acetylglucosamine units and can be chemically or enzymatically extracted from chitin, which is a component of arthropod exoskeletons and fungal and microbial cell walls. In certain aspects, the chitosan can be acetylated to a specific degree of acetylation. In one aspect, the chitosan is from about 60% to about 100% acetylated, or about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% acetylated, where any value can be the lower or upper endpoint of a range, where any value can be the lower or upper endpoint of a range (e.g., about 60% to about 90%, about 70% to about 80%, etc.).
The molecular weight of the chitosan can vary, as well. For example, the chitosan can comprise about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 glucosamine units and/or N-acetylglucosamine units, where any value can be the lower or upper endpoint of a range, where any value can be the lower or upper endpoint of a range (e.g., 2 to 19, 3 to 10, 5 to 7, etc.). In one aspect, chitosan can be added until a concentration of about 0.0015%, about 0.0025%, about 0.005%, about 0.0075%, about 0.01%, about 0.015%, about 0.02%, about 0.03%, about 0.04%, or about 0.05%, where any value can be an upper or lower endpoint of a range (e.g., 0.002% to 0.04%, 0.05% to 0.015%, etc.).
In another aspect, cells can first be fermented, for example, in a biofermenter, at a temperature conducive to cell growth. In one aspect, the cells are fermented at 30° C. In a further aspect, the cells are fermented for a time period sufficient to produce the metabolite(s) of interest. In one aspect, the cells are fermented for from 6 hours to 96 hours, or for 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, or about 96 hours, or a combination of any of the foregoing values, or a range encompassing any of the foregoing values. In one aspect, during fermentation, a micro-current can be applied to the cells as described above. In some aspects, the micro-current is applied for the entire culture period. In another aspect, the micro-current is applied for only a part of the culture period, or for several non-consecutive parts of the culture period. In one aspect, the micro-current is the same throughout the entire culture period. In an alternative aspect, the micro-current is varied during the culture period.
Exemplary methods for culturing cells and/or the biological devices disclosed herein are provided in the Examples.
In some aspects, the cells are suspended in a culture medium. In another aspect, the culture medium can be Dulbecco's Modified Eagle Medium (DMEM), RPMI 1640, Minimal Essential Medium (MEM), Eagle's Minimal Essential Medium (EMEM), Iscove's Modified Dulbecco's Medium (IMDM), DMEM/F12 Medium, Murashige and Skoog (MS) medium, White's medium, Agrobacterium minimal medium, Banana AGS basal medium, Blaydes basal medium, Bold's basal medium, Chu (N6) medium, De Greef and Jacobs Medium, DKW basal medium, Economou and Read basal medium, Gamborg (B5) medium, Gresshoff and Doy medium, Heller medium, Hoagland complete medium, Jensen's medium, Kao and Michayluk medium, Litvay medium, NB basal medium, Nitsch medium. NLN medium, Quoirin and Lepoivre medium, Schenk and Hildebrandt medium, TAP medium, TM4G medium, Vacin and Went medium, wheat callus induction medium, Luria Bertani (LB) broth, terrific broth, tryptic soy broth, minimal salts (M9) medium, SOB medium, SOC medium, yeast malt medium, YPD broth, YNB broth, synthetic complete (SC) medium, YPG medium, Hartwell's complete (HC) medium, or a combination thereof. In one aspect, the culture medium is Luria Bertani (LB) broth or yeast malt medium.
In another aspect, the culture medium can contain supplemental compounds such as, for example, vitamins, nucleosides, nucleotides, amino acids, a carbohydrate, an antibiotic, or a combination thereof.
In one aspect, the culture medium can be a liquid. In another aspect, the methods disclosed herein can be performed in a biofermenter. In an alternative aspect, the cells can be distributed on a substrate. In one aspect, the substrate can be agar, a culture dish, contaminated soil, a wastewater treatment device, mineral ore, a plant organ, a tissue scaffold, or a fermentable material. When the substrate is a plant organ, in some aspects, the plant organ can be a root, leaf, stem, rhizome, tuber, flower, seed, fruit, vegetable, callus, or a combination thereof. When the substrate is a fermentable material, in some aspects, the substrate can be milk, a grain, cabbage, soybeans, fish, or a biomass feedstock. When the substrate is a biomass feedstock, in some aspects, the substrate can be forestry residue, logging residue, sawmill residue, animal manure, a recycled material, a carbohydrate waste, corn cob, corn stover, wheat straw, nut hulls, soy hulls, switchgrass, gammagrass, paper, or a combination thereof.
In one aspect, the methods disclosed herein can be used to increase the production of metabolites by cells. In some aspects, the metabolites are secreted into a culture medium and collected. In other aspects, the metabolites remain in the cells, requiring the cells to be lysed prior to collection and purification of the metabolites.
In one aspect, prior to collection of any metabolite(s) of interest, fermentation can be stopped. In some aspects, the micro-current will be withdrawn or turned off (e.g., by turning off a power supply to a biofermenter or a similar mechanism). In another aspect, an enzyme such as, for example, lyticase can optionally be used to lyse cells following fermentation. In still another aspect, the cell culture can optionally be autoclaved for a sufficient time following cell lysis in order to ensure no living cells remain in the culture. Following lysis and autoclaving, or instead of performing these two processes, centrifugation, sonication, and filtration can be performed to facilitate collection of relevant metabolites. In an alternative aspect, culture medium including an increased concentration of the desired metabolite(s) from the biofermenter can be used without further processing.
In any of the above aspects, cell cultures of biological devices such as those disclosed herein, purified metabolites, and/or extracts containing metabolites can be applied to other cells and/or tissues in order to increase metabolite production. In one aspect, the cell cultures, metabolites, and/or extracts are applied to plant tissue such as, for example, plant calluses. Following plant tissue growth, calluses can be crushed and macerated with a solvent in order to extract metabolites from the plant tissue. In one aspect, choice of solvent depends on the chemical characteristics of the metabolite being extracted. For example, lycopene would be extracted with a hydrophobic solvent.
Any recited method can be carried out in the order of events recited or in any other order that is logically possible. That is, unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.
The present disclosure can be described in accordance with the following numbered aspects, which should not be confused with the claims.
Aspect 1. A DNA construct comprising the following genetic components:
Aspect 2. The DNA construct of aspect 1, wherein the gene that encodes the sulfotransferase has SEQ ID NO. 1 or at least 70% homology thereto.
Aspect 3. The DNA construct of aspect 1, wherein the gene that encodes the UGT has SEQ ID NO. 2 or at least 70% homology thereto.
Aspect 4. The DNA construct of aspect 1, wherein the gene that encodes the OGT has SEQ ID NO. 3 or at least 70% homology thereto.
Aspect 5. The DNA construct of aspect 1, wherein the gene that encodes the alcohol dehydrogenase has SEQ ID NO. 4 or at least 70% homology thereto.
Aspect 6. The DNA construct of aspect 1, wherein the gene that encodes the cytochrome P450 has SEQ ID NO. 5 or at least 70% homology thereto.
Aspect 7. The DNA construct of aspect 1, wherein the construct further comprises at least one promoter.
Aspect 8. The DNA construct of aspect 7, wherein the at least one promoter is a T3 promoter, a T7 promoter, an iron promoter, a GAL1 promoter, or any combination thereof.
Aspect 9. The DNA construct of aspect 8, wherein the at least one promoter is a GAL1 promoter, and the GAL1 promoter is positioned before the gene that encodes the sulfotransferase, UGT, OGT, alcohol dehydrogenase, cytochrome P450, or any combination thereof.
Aspect 10. The DNA construct of aspect 1, wherein the DNA construct further comprises a gene that confers resistance to an antibiotic.
Aspect 11. The DNA construct of aspect 10, wherein the antibiotic comprises tetracycline, neomycin, kanamycin, ampicillin, hygromycin, chloramphenicol, amphotericin B, bacitracin, carbapenem, cephalosporin, ethambutol, fluoroquinolones, isoniazid, methicillin, oxacillin, vancomycin, streptomycin, quinolines, rifampin, rifampicin, sulfonamides, cephalothin, erythromycin, streptomycin, gentamycin, penicillin, other commonly-used antibiotics, or a combination thereof.
Aspect 12. The DNA construct of aspect 1, wherein the DNA construct further comprises at least one terminator.
Aspect 13. The DNA construct of aspect 12, wherein the at least one terminator is a CYC1 terminator.
Aspect 14. The DNA construct of aspect 1, wherein the DNA construct further comprises a reporter protein.
Aspect 15. The DNA construct of aspect 14, wherein the reporter protein is a fluorescent reporter protein.
Aspect 16. The DNA construct of aspect 15, wherein the fluorescent reporter protein is a red fluorescent protein, a cyan fluorescent protein, a green fluorescent protein, or a yellow fluorescent protein.
Aspect 17. The DNA construct of aspect 16, wherein the fluorescent reporter protein is a green fluorescent protein.
Aspect 18. The DNA construct of aspect 17, wherein the green fluorescent protein is SEQ ID NO. 6 or has at least 70% homology thereto.
Aspect 19. The DNA construct of aspect 1, wherein the construct comprises from 5′ to 3′ the following genetic components in the following order: (a) the gene that encodes the sulfotransferase; (b) the gene that encodes the UGT; (c) the gene that encodes the OGT; (d) the gene that encodes the alcohol dehydrogenase; and (e) the gene that encodes the cytochrome P450.
Aspect 20. The DNA construct of aspect 1, wherein the construct comprises from 5′ to 3′ the following genetic components in the following order: (a) the gene that encodes the sulfotransferase having SEQ ID NO. 1 or at least 70% homology thereto; (b) the gene that encodes the UGT having SEQ ID NO. 2 or at least 70% homology thereto; (c) the gene that encodes the OGT having SEQ ID NO. 3 or at least 70% homology thereto; (d) the gene that encodes the alcohol dehydrogenase having SEQ ID NO. 4 or at least 70% homology thereto; and (e) the gene that encodes the cytochrome P450 having SEQ ID NO. 5 of at least 70% homology thereto.
Aspect 21. The DNA construct of aspect 1, wherein the construct comprises from 5′ to 3′ the following genetic components in the following order: (a) the gene that encodes the sulfotransferase; (b) a CYC1 terminator; (c) a GAL1 promoter; (d) the gene that encodes the UGT; (e) a CYC1 terminator; (f) a GAL1 promoter; (g) the gene that encodes the OGT; (h) a CYC1 terminator; (i) a GAL1 promoter; (j) the gene that encodes the alcohol dehydrogenase; (k) a CYC1 terminator; (l) a GAL1 promoter; and (m) the gene that encodes the cytochrome P450.
Aspect 22. The DNA construct of aspect 1, wherein the construct comprises from 5′ to 3′ the following genetic components in the following order: (a) the gene that encodes the sulfotransferase having SEQ ID NO. 1 or at least 70% homology thereto; (b) a CYC1 terminator; (c) a GAL1 promoter; (d) the gene that encodes the UGT having SEQ ID NO. 2 or at least 70% homology thereto; (e) a CYC1 terminator; (f) a GAL1 promoter; (g) the gene that encodes the OGT having SEQ ID NO. 3 or at least 70% homology thereto; (h) a CYC1 terminator; (i) a GAL1 promoter; (j) the gene that encodes the alcohol dehydrogenase having SEQ ID NO. 4 or at least 70% homology thereto; (k) a CYC1 terminator; (l) a GAL1 promoter; and (m) the gene that encodes the cytochrome P450 having SEQ ID NO. 5 or at least 70% homology thereto.
Aspect 23. The DNA construct of aspect 1, wherein the DNA construct has SEQ ID NO. 7.
Aspect 24. A vector comprising the DNA construct of aspect 1.
Aspect 25. The vector of aspect 24, wherein the vector is a plasmid.
Aspect 26. The vector of aspect 25, wherein the plasmid is pWLneo, pSV2cat, pOG44, pXT1, pSG, pSVK3, pBSK, pBSKII, pYES, pYES2, pET, pUC, or pUC19.
Aspect 27. The vector of aspect 26, wherein the vector is pYES2.
Aspect 28. A biological device comprising host cells transformed with the DNA construct in any one of aspects 1-23.
Aspect 29. The device of aspect 28, wherein the host cells comprise fungi or bacteria.
Aspect 30. The device of aspect 29, wherein the fungi comprise Saccharomyces cerevisiae.
Aspect 31. A method for producing a composition for detecting or detoxifying alcohol or opioids, the method comprising growing the biological device of any one of aspects 28-30 for a time sufficient to produce the composition.
Aspect 32. The method of aspect 31, wherein after growing the biological device to produce the composition, the method further comprises the step of lysing the host cells in the composition to produce a lysed composition.
Aspect 33. A composition produced by the method of aspect 31 or 32.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary of the disclosure and are not intended to limit the scope of what the inventors regard as their disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in ° C. or is at ambient temperature, and pressure is at or near atmospheric.
The DNA construct was composed of the genetic components described herein and assembled in plasmid vectors (e.g., pYES2, pBAD). Sequences of genes and/or proteins with desired properties were identified in GenBank; these included a gene that encodes sulfotransferase, a gene that encodes UDP-glucuronosyltransferase (UGT), a gene that encodes OGT, a gene that encodes alcohol dehydrogenase, and a gene that encodes cytochrome P450 family 3 subfamily A member 4 (CYP3A4). These sequences were synthesized by CloneTex Systems, Inc. (Austin, TX). Other genetic parts were also obtained for inclusion in the DNA constructs including, for example, promoter genes (e.g., GAL1 promoter), reporter genes (e.g., enhanced green fluorescent reporter protein), and terminator sequences (e.g., CYC1 terminator). These genetic parts included restriction sites for ease of insertion into plasmid vectors.
The cloning of the DNA construct into the biological devices was performed as follows. Sequences of individual genes were amplified by polymerase chain reaction using primers that incorporated restriction sites at their 5′ ends to facilitate construction of the full sequence to be inserted into the plasmid. Genes were then ligated using standard protocols to form an insert. The plasmid was then digested with restriction enzymes according to directions and using reagents provided by the enzymes' supplier (Promega). The complete insert, containing restriction sites on each end, was then ligated into the plasmid. Successful construction of the insert and ligation of the insert into the plasmid were confirmed by gel electrophoresis.
In some experiments, each gene was PCR amplified using gene-specific overlap primers and assembled sequences were sub-cloned into a pYES2 vector. PCR amplified pieces of all fragments were combined using homologous recombination technology (Gibson Assembly). Clones obtained after transformation were sequenced and analyzed for DNA sequence accuracy.
From 5′ to 3′, one version of the construct for producing an alcohol and opioid detection and/or detoxifying DNA composition or extract includes (a) a gene that encodes sulfotransferase, (b) a gene that encodes UDP-glucuronosyltransferase, (c) a gene that encodes OGT, (d) a gene that encodes alcohol dehydrogenase, (e) a gene that encodes cytochrome P450 family 3 subfamily A member 4 and, (f) a gene that encodes an enhanced green fluorescent protein (
PCR was used to enhance DNA concentration using a Mastercycler Personal 5332 ThermoCycler (Eppendorf North America) with specific sequence primers and the standard method for amplification (Sambrook, J., E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, 2nd ed., Vol. 1, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY). Digestion and ligation were used to ensure assembly of DNA synthesized parts using restriction enzymes and reagents (PCR master mix of restriction enzymes: XhoI, KpnI, XbaI, EcoRI, BamHI, and HindIII, with alkaline phosphatase and quick ligation kit, all from Promega). DNA was quantified using a NanoVue spectrophotometer (GE Life Sciences) and a standard UV/Visible spectrophotometer using the ratio of absorbances at 260 nm and 280 nm. In order to verify final ligations, DNA was visualized and purified via electrophoresis using a Thermo EC-150 power supply.
The DNA construct was made with gene parts fundamental for expression of sequences such as, for example, native and constitutive promoters, reporter genes, and transcriptional terminators or stops. Backbone plasmids and synthetic inserts can be mixed together for ligation purposes at different ratios ranging from 1:1, 1:2, 1:3, 1:4, and up to 1:5. In one aspect, the ratio of backbone plasmid to synthetic insert is 1:4. After the vector comprising the DNA construct has been produced, the resulting vector can be incorporated into the host cells using the method described below.
Some constructs were produced using transfected yeasts (Saccharomyces cerevisiae, ATCC® 200892™). Yeast cells were made competent by subjecting them to an electrochemical process adapted from Gietz and Schiestl (Nature Protocols, 2007, 2:35-37). Briefly, a single yeast colony was inoculated into 100 mL YPD (yeast extract peptone dextrose) growth media. Yeast was grown overnight on a shaker at 30° C. to OD600=1.0. (Acceptable results were obtained with OD600 values ranging from 0.6 to 1.8.) Cells were centrifuged at 2000 rpm in a tabletop centrifuge and resuspended in 10 mL TEL buffer (10 mM Tris-HCl, 1 mM EDTA, 0.1 M LiAc, pH=7.5) and shaken vigorously overnight at room temperature. Alternatively, INVSc1 cells were prepared to be competent using a kit from Sigma-Aldrich, Inc. Cells were again centrifuged and resuspended in 1 mL TEL buffer. Cells prepared in this manner could be stored in the refrigerator for up to one month.
Alternatively, bacterial devices were constructed with one of the following strains of cells: Escherichia coli, ONESHOT® Top10 competent cells from Life Technologies™, BL21 (DE3) E. coli from Novagen, Inc., or DH5α™ E. coli from Thermo Fisher Scientific.
Competent cells were stored in the freezer until needed. Cells were thawed on ice and 100 μL of competent cells in TEL buffer were placed in a sterile 1.5 mL microcentrifuge tube. To this was added 5 μL of a 10 mg/mL solution of salmon sperm DNA (carrier DNA). Transforming DNA was added in various amounts. From 1 to 5 μg was sufficient for plasmids from commercial sources, but more DNA was required when transforming yeast with artificial DNA constructs. 10 μL of the DNA device were added to the microcentrifuge tube containing the competent yeast cells and the contents of the tube were mixed. The DNA-yeast suspension was incubated for 30 min at room temperature.
A PLATE solution (consisting of 40% PEG-3350 in 1×TEL buffer) was prepared. 0.7 mL of PLATE solution was added to the DNA-yeast suspension and the contents were mixed thoroughly and incubated for 1 h at room temperature. The mixture was placed in an electromagnetic chamber for 30 minutes. Cells were then heated at 42° C. for 5-10 minutes and 250 μL aliquots were plated on yeast malt agar to which selective growth compounds had been added. Plates were incubated overnight at 30° C.
DNA expression and effectiveness of transformation were determined by fluorescence of the transformed cells expressed in fluorescence units (FSUs) using a 20/20 Luminometer (Promega) according to a protocol provided by the manufacturer. Plasmid DNA extraction, purification, PCR, and gel electrophoresis were also used to confirm transformation. Different transformed devices were obtained. Different types of fluorescent reporter proteins were used (e.g., yellow, red, green, and cyan) for all transformed cells and/or constructs. However, the yellow fluorescent protein was preferred. When no fluorescent reporter protein was assembled, no fluorescence was observed.
S. cerevisiae cells were subjected to transformation with the modified pYES2 plasmids for producing metal- and contaminant-binding components as described above. Transformed yeast cells were incubated for 30 min at 28-30° C. Colonies of transformed yeast cells were selected, their DNA isolated and subjected to PCR amplification. Two control treatments were also carried out: (1) a negative control involving competent yeast and nuclease free water instead of a plasmid and (2) a positive control involving competent yeast with unmodified pYES2 plasmid.
Four clones were selected from a transformed plate and processed for full-length DNA sequencing. A clone with 100% DNA sequence accuracy was selected for further processing and was used to obtain a high concentration of plasmid construct at a mid-scale plasmid purification level. Yeast competent cells were transformed with the recombinant plasmid and selected on synthetic complete (SC) dropout plate deficient in uracil. Well isolated clones were isolated and preserved in YPD medium containing 15% glycerol for storage at −80° C.
Microbial Extracts Containing Alcohol and Opioid Detecting and/or Detoxifying Metabolites
The following non-limiting procedure was used to produce the disclosed extracts:
Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the compounds, compositions, and methods described herein.
Various modifications and variations can be made to the compounds, compositions, and methods described herein. Other aspects of the compounds, compositions, and methods described herein will be apparent from consideration of the specification and practice of the compounds, compositions, and methods disclosed herein. It is intended that the specification and examples be exemplary.
This application claims the benefit of U.S. Provisional Application Ser. No. 63/619,814, filed Jan. 11, 2024, which is incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63619814 | Jan 2024 | US |
