82 results about "Cell-free system" patented technology

The invention is based on the reaction of Duplex Mutational Vector in a cell-free system containing a cytoplasmic cell extract and a test plasmid. The reaction specifically converts a mutant kanr gene to recover the resistant phenotype in transformed MutS, RecA deficient bacteria. Using this system a type of Duplex Mutational Vector termed a Non-Chimeric Mutational Vector, having no RNA:DNA hybrid-duplex is shown to be an effective substrate for eukaryotic enzymes. The invention concerns the use of Non-Chimeric Mutational Vectors protected from 3' exonuclease attack in eukaryotic cells. Such protection can be conferred by replacement of a tetrathymidine linker by a nuclease resistant oligonucleotide, such as tetra-2'-O-methyl-uridine, to link the two strands of the recombinagenic oligonucleobase.

The present invention relates, in general, to thermography and, in particular, to a method of using infrared thermography to monitor physiological and molecular events that elicit a thermogenic response in animals (including humans), plants, tissues, cells and cell-free systems. The present method can be used for screening, identifying, and ranking drug candidates for multiple diseases, disorders and conditions.

The invention relates to the field of molecular biology and aims to provide an ARMS-qPCR (Allele Refractory Mutation System-quantitative Polymerase Chain Reaction) detection kit for KRAS (Kirsten Rat Sarcoma Viral Oncogene Homolog) gene mutation subtype and a detection method. The kit comprises a qPCR hybrid reaction solution, a locked nucleic acid retardant probe, a reference primer, an ARMS primer and a positive control sample, wherein the qPCR hybrid reaction solution comprises a PCR buffer solution, dNTPs (Deoxynucleotide Triphosphates), MgCl2, GoldStarbest Taq enzyme, a universal PCR reverse primer and a universal TaqMan probe. The kit provided by the invention can be used for rapidly and accurately detecting specific locus mutation of KRAS genes in various cancer tissues with high sensitivity, has high sensitivity, and can be used for detecting genome DNA with various tissue origins, specially free DNA segments adopting cell-free systems, such as blood serum and blood plasma, orother body fluid origins, wherein the genome DNA is derived from cell systems. Compared with direct sequencing and other mutation detection technologies, the kit and the detection method thereof havethe advantages of strong specificity, high sensitivity, simplicity and rapidness in operation, high throughput, safety, definiteness and objectivity in result identification and the like for detecting the KRAS gene mutation.

The present invention relates, in general, to thermography and, in particular, to a method of using infrared thermography to monitor physiological and molecular events that elicit a thermogenic response in animals (including humans), plants, tissues, cells and cell-free systems. The present method can be used for screening, identifying, and ranking drug candidates for multiple diseases, disorders and conditions.

There is provided a method for producing a soluble protein domain comprising: (a) preparing two or more DNA fragments by partially digesting a DNA coding for a protein; (b) expressing the protein which is coded on each of said DNA fragments, as a fusion protein with a functional protein; (c) selecting the fusion protein exhibiting said function among two or more fusion proteins synthesized in step (b); and, (d) synthesizing the soluble protein domain which is coded on said DNA fragment in a cell-free system, wherein said soluble protein domain is included in said fusion protein selected in step (c). By using this method, it can be easy and efficient to analyze the three dimensional structure of proteins of many clones.

The present invention relates to methods for RNA and/or protein synthesis using in vitro or in vivo expression systems. More specifically, the present invention provides a method for RNA and/or protein synthesis characterized in that the concentration of alpha subunit of RNA polymerase, but not of other subunits, is increased in the cellular or cell-free system, comparing to its natural concentration existing in the cellular or cell-free system.

Provided herein are cell-free systems for generating carbapenems, e.g., a compound of the Formula (I):

or salts thereof; wherein , R¹, R², R³, R⁴, R⁵, and R⁶ are defined herein. Also provided are pharmaceutical compositions comprising a compound generated by the inventive cell-free system, and use of these compounds and compositions for the treatment of bacterial infections.

Methods are provided for controlling metabolic flux rate in a cell-free system comprising a complex set of enzymes, to produce a desired product of a pathway of interest. In the methods of the invention, measurements of metabolic performance parameters are taken by continuous monitoring or intermittent monitoring. Based on the metabolic performance parameters, the system is modified by one or more steps comprising: (i) altering enzyme levels in the cell-free system; (ii) altering feed rate of a substrate that controls redox flux or carbon flux to the cell-free system; (iii) altering O₂ addition to the cell-free system; (iv) controlling efficiency of electron transport system by altering leakage across a membrane; wherein enzymes present in the pathway of interest catalyze production of a desired product.

The invention relates to compositions, specifically novel nucleic acid constructs encoding a cardiovirus 2A polypeptide operably linked to suitable promoters. Also, disclosed are methods whereby the nucleic acid constructs are introduced into cells or cell free systems to regulate cellular mRNA transcription and cap-dependent or internal ribosomal entry site (IRES)-dependent mRNA translation.

Prepared is an extract composition having an improved protein synthetic activity in a cell-free protein synthesis system using a mammalian cultured cell extract. An eukaryotic translation initiation factor and/or translational regulator are added to a cell-free protein synthesis system comprising an extract prepared from cultured mammalian cells and a template mRNA. These factors are one or more selected from the group consisting of eukaryotic translation initiation factors 4E (eIF4E), 2 (eIF2) and 2B (eIF2B), and eukaryotic translational regulator p97.

Methods and kits for generating circular nucleic acids in a cell-free system, and uses for the generated circular nucleic acids are provided. The methods comprise in vitro amplification of a nucleic acid template comprising a recombination site to produce tandem repeat nucleic acid sequence, and employ a recombination protein to generate the circular nucleic acids from the tandem repeat nucleic acid sequence.

The present invention relates to enzymes, compositions and methods for catalyzing asymmetric recombination of non-palindromic recombination sites in a cell free system, in isolated cells or in living organisms. The enzymes and methods of the invention are suitable for mediating specific recombinations between DNA sequences comprising specific recombination sites without being limited to strict palindromic symmetry within each recombination site.

The invention discloses a method catalytically synthesizing (S)-N, N-dimethyl-3-hydroxy-(2-thiofuran)-1-propylamine((S)-DHTP) by aldehyde ketone reductase recombinant strain crude enzyme system, and belongs to the technical field of biological catalysis asymmetric conversion. An auxiliary substrate and original bulk coenzyme NADP<+> are added in the recombinant strain crude enzyme system to accelerate regeneration cycle of coenzyme NADPH in the system; the reaction substrate is N, N-dimethyl-3-ketone-(2-thiophene)-1-propylamine (DTKP); the recombinant strain is E. coliBL21(DE3)(pETCPAR4); aldehyde ketone reducing enzyme gene cpar4 comes from Candida parapsilosis; the gene coded aldehyde ketone reducing enzyme gene CPAR4 catalytically and asymmetrically reduces the DKTP into (S)-DHTP. The method utilizes the cell-free system to conduct catalytic reaction; coupling enzyme required by regeneration of the coenzyme is not extra added in the reaction system; direct acting efficiency of the enzyme and the substrate is improved; reaction time is shortened; the conversion effect is relatively good.

The present disclosure pertains to novel cell cultivation and cell and/or cell-derived product production processes that have advantages over currently existing fermentation strategies. The processes and methods according to the present disclosure may be used for an efficient supply of highly viable and metabolically active eukaryotic cells for transient production platforms, as an alternative production process with advantages over currently applied processes (batch, fed-batch or perfusion strategies) and for generating metabolically highly active biomass for subsequent use for transient expression systems or infection by a virus or pseudovirus or in cell-free systems.

The present invention is directed to a cell-free system for producing a glycosylated protein. This system comprises an isolated oligosaccharyltransferase capable of transferring a glycan from a lipid carrier molecule to a glycoprotein target, one or more isolated glycans, where each glycan is linked to a lipid carrier molecule, and a glycoprotein target comprising one or more glycan acceptor amino acid residues or a nucleic acid molecule encoding said glycoprotein target. The present invention further relates to kits and methods for producing a glycosylated protein in this cell-free system.

Methods for the determination of the rate of synthesis of biopolymer synthesis and degradation in cells, tissues, or cell-free systems using monomer which has been labeled with a stable isotope are provided. Further, the present invention provides methods for the determination or identification of an unknown biopolymer and for the identification of an unknown cell type, a physiological state of a cell or tissue. Also, the present invention provides a database of descriptors which can be used to define an organism, tissue type, cell type, and the like, and which database can be used in conjunction with other public and private databases to identify or characterize an organism, tissue type, cell type, state of differentiation, or physiologic state of an organism, or tissue or cell sample.

A stable linkage between a genotype and a phenotype in a cell-free system was successfully achieved by using interaction between a RNA-binding protein and RNA, between a DNA-binding protein and DNA, or by using a protein that inactivates a ribosome. Furthermore, it was found that functional proteins could be selected by using these stable linkages.

The invention discloses a method of synthesizing CdSe quantum dots with Amino acid and polypeptide, comprising the following steps: A, preparing cell-free system of selenium-containing amino acid; B, preparing Cd-glutathione; C, synthesizing CdSe quantum dots. The invention is provided with a simple technology and mild reaction conditions. As no organometallic compounds are included in the preparation, the protection from the inert gas is not needed. The production is safe and low in cost, helpful to the industrial synthesis of the nano-particles (including quantum dots).

Provided is a method capable of replicating or amplifying circular DNA, and particularly, long-chain circular DNA, in a cell-free system. Specifically, provided is a method for suppressing generation of a DNA multimer as a by-product, when circular DNA having a replication origin sequence (origin of chromosome (oriC)) is replicated or amplified by using the following enzyme groups:

• • (1) a first enzyme group that catalyzes replication of circular DNA;
  • (2) a second enzyme group that catalyzes an Okazaki fragment maturation and synthesizes two sister circular DNAs constituting a catenane; and
  • (3) a third enzyme group that catalyzes a separation of two sister circular DNAs.

Moreover, also provided is a method comprising introducing oriC into circular DNA by using a transposon.

The invention discloses a method for carrying out gene mutation detection on a cell-free system by an HRM technology. The invention is characterized in that the method comprises the following steps: (1) designing and selecting a plurality of primer pairs containing continuous nucleotide sequences formed by at least 15 continuous nucleotides in the targeted region of a target gene; (2) extracting DNA from the cell-free system, and utilizing the plurality of primer pairs screened in advance to carry out specific amplification on the targeted sequence of the target gene; and (3) carrying out mutational site detection on the amplified gene sequence according to a high-resolution fusion curve method. The method provides more extensive selectable samples for the detection and the analysis on related diseases by the cell-free system application technical research and obviously reduces invasive harm of the diseases on patients.

The present invention provides a preparation method of an insect cell extract solution for cell-free protein synthesis, the insect cell extract solution, a protein synthesis method in a cell-free system, which uses the insect cell extract solution, and a kit for cell-free protein synthesis containing the insect cell extract solution. The extract solution is easily prepared by the method of the present invention and can synthesize a higher amount of protein than by extract solutions prepared by conventional methods.

The invention relates to a method or a cell free system of detecting dioxin-like compounds in a test sample using a whole cell lysate derived from a cell transfected to express a fusion protein comprising an AHR fused to a reporter peptide. The method in combination with bioluminescence resonance energy transfer (BRET) technique is also provided so as to improve the sensitivity.