865 results about "Luciferases" patented technology

Systems and methods for prophylactic measures aimed at improving wound repair. In some embodiments, laser-mediated preconditioning would enhance surgical wound healing that was correlated with hsp70 expression. Using a pulsed laser (λ=1850 nm, Tp=2 ms, 50 Hz, H=7.64 mJ / cm2) the skin of transgenic mice that contain an hsp70 promoter-driven luciferase were preconditioned 12 hours before surgical incisions were made. Laser protocols were optimized using temperature, blood flow, and hsp70-mediated bioluminescence measurements as benchmarks. Bioluminescent imaging studies in vivo indicated that an optimized laser protocol increased hsp70 expression by 15-fold. Under these conditions, healed areas from incisions that were laser-preconditioned were two times stronger than those from control wounds. Our data suggest that these methods can provide effective and improved tissue-preconditioning protocols and that mild laser-induced heat shock that correlated with an expression of Hsp70 may be a useful therapeutic intervention prior to or after surgery.

A device and methods for the rapid chemiluminescence or calorimetric assay of surfaces to detect the presence of microbial or protein contamination is disclosed. A sampling / analysis member (10) is described having a sampling wand (15) which is suitable for use by untrained personnel under the relatively harsh and variable conditions found in the field, for example in fast food restaurants and other food preparation areas. The analytical signal in the disclosed device and methods can be based on luciferase / luciferin systems or a protein assay systems utilizing bicinchoninic acid.

A rapid detection of ATP using dried luciferase / luciferin reagent. A liquid that may include an extraction reagent is applied to the testing surface, such as a counter top. A sampling device having a handle and an absorbent tip is wiped across the testing surface. The absorbent tip absorbs the sample. The sample is then placed in a counting tube. Luciferase / luciferin reagent is immobilized on the absorbent tip of the sampling device or in the bottom of a counting tube. In either case, the liquid rehydrates the luciferase / luciferin reagent allowing ATP present in the sample to react with the luciferase. Light produced by the reaction is measured with a luminometer.

Luciferase enzymes with greatly increased thermostability, e.g., at least half lives of 2 hours at 50° C., cDNAs encoding the novel luciferases, and hosts transformed to express the luciferases, are disclosed. Methods of producing the luciferases include recursive mutagenesis. The luciferases are used in conventional methods, some employing kits.

The invention discloses a method for targeted knockout of a specific gene of a Suqin yellow chicken embryonic stem cell. The method comprises the following steps: firstly, checking out an exon sequence of a gene, cloning the gene, sequencing to obtain a complete exon sequence, designing a CRISPR / Cas9 knockout target site on the sequence as gRNA, and constructing a CRISPR / Cas9 dual-promoter knockout vector; performing SSA activity detection on the constructed Cas9 vector, transfecting a control group with an empty vector, detecting a luciferase signal, and obtaining a result that the more the luciferase activity is increased relative to the control group, the higher the gRNA shearing activity is shown; transfecting the CRISPR / Cas9 vector which is high in SSA activity with ESCs, using flow cytometry to screen a GFP-positive cell, extracting genome DNA, and designing a primer.

Luciferase enzymes with greatly increased thermostability, e.g., at least half lives of 2 hours at 50° C., cDNAs encoding the novel luciferases, and hosts transformed to express the luciferases, are disclosed. Methods of producing the luciferases include recursive mutagenesis. The luciferases are used in conventional methods, some employing kits.

A method for evaluating sterilization effect of disinfectant by means of ATP bioluminescence includes preparing standard bacterial suspension, processing it with cell ATP release agent Ec and adding luciferase - luciferin reagent to detect out luminous value or filtering it with microhole film in combination with ATP scavenger and detecting out luminous value of filtering film, then carrying out equation calculation and utilizing conversion coefficient K to derive out live bacteria number in suspension for evaluation.

Provided are methods and compositions useful in detecting protease activity in a sample, as well as methods of identifying agents that modulate protease activity. The methods and compositions provide a modified luciferase polynucleotide sequence and a luciferase polypeptide containing protease recognition sequences, wherein cleavage of the recognition sequence by a protease inhibits luciferase activity. Further provided are methods and compositions for detecting and modulating caspase activity and apoptosis.

The invention provides a protein synthesis system for in-vitro protein synthesis, a kit and a preparation method for a protein through in-vitro synthesis. Specifically, the in-vitro cell-free expression system provided by the invention is capable of extremely efficiently synthesizing proteins and synthesizing complex proteins. Moreover, due to the in-vitro cell-free expression system disclosed bythe invention, the relative light unit value of the activity of the synthesized luciferase is higher than that of the conventional commercial system (such as a rabbit reticulocyte in-vitro expressionsystem) by at least one order of magnitude (more than or equal to 10 times or higher).

The invention provides a nucleic acid construct for endogenously expressing RNA polymerase in cells, and particularly discovers a novel nucleic acid construct capable of greatly enhancing protein translation efficiency. The nucleic acid construct consists of a promoter with specific promotion intensity (such as RNR2, ADH1, GAPDH, TEF1, PGK1 and SED1) and coding sequences of proteins of RNAP (suchas T7RNAP, T3RNAP, T4RNAP and T5RNAP), if the nucleic acid construct of the invention is applied into a yeast extracorporeal protein synthesis system, the activity of synthesized luciferase is quite high relative to light unit value (RLU), and the effect can be the same as the effect of T7RNAP added exogenously. The effect can reach up to 6.6* 107.

The present invention discloses a method for establishing KI-T2A-luciferase cell line based on CRISPR / Cas9 targeted genome modification technology. A T2A-luciferase reporter gene is integrated in the3<rd> end of the mmp 12 gene in a genome by using CRISPR / Cas9 technology. A knock-in cell line of MMP12-T2A-luciferase is established, the site-specific integration of the source gene in the cell lineon the genome is verified. Meanwhile, a reported transcription factor, STAT3, with activation effect on mpp12-T2A is used to transcribe and active the MMP12-T2A-luciferase cell line. The results showthat the expression level of luciferase in MMP12-T2A-luciferase cell line can accurately and sensitively reflect the expression level of MMP12 protein in the cell line. The establishment of the cellline will contribute to the study of the gene function of mmp12 and the screening of small molecule chemical drugs affecting the expression of mmp12, which provides a new experimental thinking and solution for the migration of cancer cells and related researches thereof.

A method and kit is provided for enhancing the tolerance of an assay reagent to compounds in an assay sample, the assay reagent including a luciferase enzyme. The method includes contacting the luciferase with a tolerance enhancement agent in an amount sufficient to substantially protect luciferase enzyme activity from interference of the compound and minimize interference by at least about 10% relative to an assay not having tolerance enhancement agent.

The invention provides a protein synthesis efficiency enhancing RNA element and particularly discloses a nucleic acid structure formed by coding sequences of optional promoters, yeast-derived IRES enhancers (such as ScGPR1, ScFLO8, ScNCE102, ScMSN1, KlFLO8, KlNCE102 and KlMSN1) and heterologous proteins. By application of the nucleic acid structure to a yeast in-vitro protein synthesis system, thesynthesized luciferase activity RLU (relative light unit) is extremely high.

A compound represented by the following general formula (I) or a salt thereof: [wherein R1 and R2 represent hydrogen atom, a C1-6 alkyl group, or a group represented by the following formula (A): [wherein X1 and X2 represent hydrogen atom, or a group represented as —N(R3)(R4) (R3 and R4 represent hydrogen atom, a C1-6 alkyl group, a C1-6 alkylcarbonyl group, or a C1-6 alkyloxycarbonyl group); and n represents an integer of 1 to 6], provided that R1 and R2 do not simultaneously represent hydrogen atom), which is a novel luciferin derivative that serves as a luciferase substrate.

Isolated and purified nucleic acids encoding green fluorescent proteins from Renilla reniformis and the green fluorescent protein encoded thereby are also provided. Mutants of the nucleic acid molecules and the modified encoded proteins are also provided. Compositions and combinations comprising the green fluorescent proteins and / or the luciferase are further provided.

The invention relates to a method for establishing luciferase knock-in cell line based on CRISPR-targeted genome modification technology. The knock-in cell line of SREBP1- T2A-Luciferase was established by in situ integration of the 3'end of SREBP1 gene into the T2A-Luciferase reporter gene using CRISPR / Cas9 technique, the fixed point of the Chinese and foreign source genes of the cell line on thegenome is verified. The transcriptional activation of the SREBP1-T2A-Lucifasse cell line was performed using the reported transcription factor LXR Alpha, which has an active effect on the SREBP1. Theresults show that the expression level of the Luciferase in the SREBP1-T2A-Lucifasse cell line can accurately and sensitively reflect the expression level of the SREBP1 in the cell line. The establishment of the cell line will help to study the function of the SREBP1 gene and to screen the small molecular chemical drugs affecting the expression of SREBP1, and provide a new experimental thought and solution for lipid metabolism and related research.

The present invention relates to bacterial luciferase expression cassettes suitable for conferring bioluminescence properties on Gram-positive bacteria, cells transformed with such cassettes, and methods of making and using such cassettes.

A method of measuring the enzymatic activity of a luciferase includes contacting a luminogenic protein, such as a luciferase, with a protected luminophore to form a composition; and detecting light produced from the composition. The protected luminophore provides increased stability and improved signal-to-background ratios relative to the corresponding unmodified coelenterazine.

Disclosed is a device and methods for the rapid chemiluminescence assay of surfaces to detect the presence of microbial contamination. The device and methods are suitable for use by untrained personnel under the relatively harsh and variable conditions found in the field, for example in fast food restaurants and other food preparation areas. The chemiluminescence reaction that is the source of the analytical signal in the disclosed assay device and method is preferably based on a luciferase / luciferin system.

The invention provides a DNA element for high-throughput in vitro protein synthesis, and specifically discloses a novel DNA element capable of greatly enhancing protein translation efficiency. According to the present invention, with the application of the DNA element in a yeast in-vitro protein synthesis system, the relative light unit value of the activity of the synthesized luciferase is enhanced by about 22.3 times compared to the DNA element containing only the omega sequence.

The present invention provides methods, compositions, substrates, and kits useful for analyzing the metabolic activity in cells, tissue, and animals and for screening test compounds for their effect on cytochrome P450 activity. In particular, a one-step and two-step methods using luminogenic molecules, e.g. luciferin or coelenterazines, that are cytochrome P450 substrates and that are also bioluminescent enzyme, e.g., luciferase, pro-substrates are provided. Upon addition of the luciferin derivative or other luminogenic molecule into a P450 reaction, the P450 enzyme metabolizes the molecule into a bioluminescent enzyme substrate, e.g., luciferin and / or luciferin derivative metabolite, in a P450 reaction. The resulting metabolite(s) serves as a substrate of the bioluminescent enzyme, e.g., luciferase, in a second light-generating reaction. Luminescent cytochrome P450 assays with low background signals and high sensitivity are disclosed and isoform selectivity is demonstrated. The present invention also provides an improved method for performing luciferase reactions which employs added pyrophosphatase to remove inorganic pyrophosphate, a luciferase inhibitor which may be present in the reaction mixture as a contaminant or may be generated during the reaction. The present method further provides a method for stabilizing and prolonging the luminescent signal in a luciferase-based assay using luciferase stabilizing agents such as reversible luciferase inhibitors.

The invention discloses a method for detecting promoter activity by utilizing double luciferase reporter genes. The method for detecting the promoter activity by utilizing the double luciferase reporter genes comprises step 1, building a pGL3-basic-MSTNpro recombinant which contains 7 sections of MSTN gene 5' control region fragments with different lengths; step 2, performing cultivation and planking on target cells, configuring a mixture of the pGL3-basic-MSTNpro of the step 1 and a lipidosome and enabling a renilla luciferase carrier pRL-TK to be served as an internal reference to perform cell co-transfection; step 3, performing detection on luciferase activity through the double luciferase reporter genes.

Methods, devices and systems are provided for detection of adenosine triphosphate (ATP) in samples using the luciferin-luciferase reaction. An aspect of the invention includes a low molarity and low pH composition for use in detecting the presence of ATP. The low molarity and low pH composition can be used in combination with methods for reading, calculating and interpreting luminescence generated by the ATP-luciferin-luciferase reaction. Both the low molarity, low pH composition and the methods for reading, calculating and interpreting luminescence can be used with a single service hygiene monitoring format.

Provided are luciferyl peptide substrates that are produced by attaching specifically prepared peptide conjugates to luciferin, and / or its analogs and derivatives. The luciferyl peptide substrates are incapable of penetrating cell membranes and tissue barriers. Cleavage of the peptide conjugates from the luciferyl peptide substrates releases the luciferin, which upon contact with luciferase emits photons for easy detection. The luciferyl peptide substrates may be used in assays to detect pathogens, test protease inhibitors, probe cell physiology, assess protease activity in oncogenesis, and improve specific and regulated drug delivery.

Disclosed is a process for releasing ATP from living cells in aqueous mixtures of polymers or pigments. The aqueous mixture of a polymer or pigment is agitated in the presence of a particulate disruption agent to cause rupturing of the living cells and release of the ATP contained therein. Also disclosed is a process for detecting living cells in a aqueous mixtures of polymer or pigments by detecting the ATP released by the disruption process by a luciferin / luciferase assay. A kit for the detection of living cells in aqueous mixtures of polymers or pigments also is described.

Isolated and purified nucleic acids encoding green fluorescent proteins from Renilla reniformis and the green fluorescent protein encoded thereby are also provided. Mutants of the nucleic acid molecules and the modified encoded proteins are also provided. Compositions and combinations comprising the green fluorescent proteins and / or the luciferase are further provided.

The NF-E2-related factor 2 (Nrf2) is a key transcriptional regulator of antioxidant defense and detoxification. To directly monitor stabilization of Nrf2 we fused its Neh2 domain, responsible for the interaction with its nucleocytoplasmic regulator, Keap1, to firefly luciferase (Neh2-luciferase). It is shown herein that Neh2 domain is sufficient for recognition, ubiquitination and proteasomal degradation of Neh2-luciferase fusion protein. The novel Neh2-luc reporter system allows direct monitoring of the adaptive response to redox stress and classification of drugs based on the time-course of reporter activation. The novel reporter was used to screen a library of compounds to identify activators of Nrf2. The most robust and yet non toxic Nrf2 activators found—nordihydroguaiaretic acid, fisetin, and gedunin-induced astrocyte-dependent neuroprotection from oxidative stress via an Nrf2-dependent mechanism.