Disclosed in the present invention is an application of a reducing agent in a heme peroxidase catalytic reaction. The reducing agent is selected from ascorbic acid, dehydroascorbic acid, gallic acid and pyrogallic acid. The reducing agent has a significant promotion effect on oxidation reaction and hydroxylation reaction catalyzed by heme peroxidase.
An efficient large DNA fragment synthesis and assembly method based on a novel programmable nuclease Argonaute. The method specifically comprises: constructing a resistance gene reconstruction vector and using Argonaute to perform linearization processing on the resistance gene reconstruction vector, fragmenting a target DNA into a plurality of small DNA fragments and synthesizing the small DNA fragments, loading the small DNA fragments into the resistance gene reconstruction vector, and using SLIC cloning and resistance gene reconstruction to implement assembly of the target DNA. SLIC cloning and a resistance gene reconstruction strategy are integrated, and 5-6 small fragments can be assembled by reconstructing a resistance gene once, thereby achieving higher efficiency and shorter cycle; and the frequency of reconstructing the resistance gene can be flexibly selected on the basis of the length of fragments of a DNA, mutation caused by PCR is not introduced, and the reconstructed large fragment does not need to undergo secondary sequencing, thereby saving time and reducing costs, and providing new ideas for the synthesis and assembly technology of large DNA fragments.
C12N 15/10 - Processes for the isolation, preparation or purification of DNA or RNA
C12N 15/70 - Vectors or expression systems specially adapted for E. coli
C12N 15/66 - General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligationUse of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
3.
PFU DNA POLYMERASE MUTANT HAVING REVERSE TRANSCRIPTASE ACTIVITY AND USE THEREOF
Provided are a Pfu DNA polymerase mutant having a reverse transcriptase activity and the use thereof. The Pfu DNA polymerase mutant comprises K467R/F588L/W769R, R382H/R385H/V390I, I38L/R97M, E665K/E735K and K118I/N713V, wherein the K118I/N713V has the strongest reverse transcription activity. In addition, the Pfu DNA polymerase mutant also retains the polymerase activity of a wild-type enzyme. The mutant has a high fidelity due to the proofreading activity thereof compared with the lower fidelity of a conventional reverse transcriptase, and the mutant has a high heat resistance, so that the mutant can generate cDNA with a high efficiency and a high fidelity by means of using RNA as a substrate, and then the cDNA is amplified under standard PCR reaction conditions. In the whole process, additional addition of the reverse transcriptase is not required, and a single-enzyme "one-step" RT-PCR is realized.
H01L 29/04 - Semiconductor bodies characterised by their crystalline structure, e.g. polycrystalline, cubic or particular orientation of crystalline planes
5.
GROUP OF HRV 3C PROTEASE MUTANTS ALTERED FOR SUBSTRATE P1' SITE SPECIFICITY
Disclosed is a group of HRV 3C protease mutants altered for substrate P1' site specificity, which belongs to the technical field of genetic engineering and enzyme engineering. The molecular modification is carried out on a wild-type HRV 3C protease by means of a saturated mutation and random mutation technique, so that the resulting protease will recognize a polypeptide sequence LEVLFQ↓M rather than an original polypeptide sequence LEVLFQ↓G, and a series of HRV 3C protease mutants are obtained. Compared with wt-HRV 3C-P, the provided mutants have better specificity and cleavage activity with respect to the substrate LEVLFQ↓M, and can achieve the effect of seamless excision of a protein fusion tag, thereby widening the practical application range of the HRV 3C protease.
Provided are a PfAgo mutant protein having target nucleic acid cleavage activity at medium temperatures and a use thereof. The PfAgo mutant protein has an amino acid mutation at position 617 and/or position 618 relative to the sequence as shown in SEQ ID NO. 1, specifically, K617E/G and/or L618Y/F/W/G. Compared with a wild-type PfAgo protein, the provided PfAgo mutant protein has activity in a temperature range of 30-95°C, and thus, the scope of use of the pAgo protein is effectively expanded.
Disclosed is use of a prokaryotic Argonaute protein only having RNA target cleavage activity in RNA editing. The Argonaute protein is derived from a mesophilic prokaryote Verrucomicrobia bacterium, the amino acid sequence thereof is set forth in SEQ ID NO: 1, or the Argonaute protein is a protein that is extremely high in similarity to SEQ ID NO: 1 and has the same function; the protein has binding activity to a single-stranded guide DNA, and only has nuclease activity on an RNA target that is complementarily paired with the single-stranded guide DNA. Therefore, the protein can be used for in-vivo and in-vitro targeted RNA editing, thereby providing a brand-new powerful tool for RNA editing.
Provided are a eukaryote-derived Argonaute protein and the use thereof. The Argonaute protein has an amino acid sequence as shown in SEQ ID NO. 1, or has at least 50% sequence identity with the sequence as shown in SEQ ID NO. 1; the specific cleavage activity of a eukaryotic Argonaute protein on DNA has been proved for the first time; and experimental proof is provided for the study of the interaction between eukaryotic Argonaute and DNA. In addition, a polypeptide, nucleic acid, expression vector, composition, kit and method used can perform a site-specific operation on genetic material inside and outside a cell, and can be effectively applied to many fields of biotechnology, thus providing a new tool for gene editing, modification and molecular detection of an Argonaute polypeptide based on a eukaryotic biological source.
Escherichia coli and an application thereof in artificial protein scaffolds are provided. The protein complex includes one or more of interaction pairs formed by a CL2 protein and an Im2 protein, a CL7 protein and an Im7 protein, a CL8 protein and an Im8 protein, or a CL9 protein and an Im9 protein. By protein engineering of a carboxyl terminus DNase domain of the DNA enzymes CE2, CE7, CE8 and CE9, mutants that lose DNA enzyme activity but still retain the ultra-high affinity with the corresponding Im protein are obtained, and protein interaction pairs CL2/Im2, CL7/Im7, CL8/Im8 and CL9/Im9 are constructed. These protein interaction pairs have properties of heat resistance, high affinity, high specificity, small molecular weight, fast assembly speed, etc. Based on this, an artificial protein scaffold is constructed for the construction of artificial multienzyme complexes.
Disclosed in the present invention are a protein complex based on DNases of a colicin E family and an application thereof in an artificial protein scaffold. The protein complex comprises any one or more of an interaction pair formed by a CL2 protein and an Im2 protein, an interaction pair formed by a CL7 protein and an Im7 protein, an interaction pair formed by a CL8 protein and an Im8 protein, or an interaction pair formed by a CL9 protein and an Im9 protein. According to the present invention, protein engineering transformation is performed on carboxyl terminal DNase domains of DNases CE2, CE7, CE8, and CE9 of a CE family to obtain a mutant which loses DNase activity but still retains ultrahigh affinity to a corresponding immune protein Im, and CL2/Im2, CL7/Im7, CL8/Im8 and CL9/Im9 protein interaction pairs are constructed. Research finds that the protein interaction pairs have properties such as heat resistance, high affinity, high specificity, small molecular weight, and high assembly speed; on this basis, an artificial protein scaffold is constructed, and a novel platform is established for construction of an artificial multi-enzyme complex.
Disclosed in the present invention are a prokaryote-derived Mbp_Argonaute protein and the use thereof, wherein the Mbp_Argonaute protein has an amino acid sequence as shown in SEQ ID NO: 1 or as shown in a sequence having at least 50% or at least 80% homology to SEQ ID NO: 1. According to the present invention, an Argonaute protein gene derived from a cold-resistant prokaryote Mucilaginibacter paludis is synthesized, and the protein is named as MbpAgo. It is found in the study that the MbpAgo has a binding activity to single-stranded guide DNA, and has a nuclease activity to the target RNA and/or target DNA that is complementarily paired with the single-stranded guide DNA. Therefore, the MbpAgo can be used for targeted RNA editing in vivo and in vitro, and then for specific site modification of genetic materials. The MbpAgo not only can modify RNA having an advanced structure, but also does not affect an endogenous RNAi pathway of animal and plant cells, provides a brand new powerful tool for RNA editing, and has a high cutting activity and a good specificity.
Provided are a method for inducing the differentiation of extended pluripotent stem cells (EPSCs) into cardiomyocytes and an application thereof, which belong to the technical field of biomedicine. A reagent used for inducing differentiation is a culture medium having clear chemical components, and cardiomyocytes having high purity and stable inter-batch differentiation efficiency can be obtained. Compared with cardiomyocytes differentiated from existing pluripotent stem cells, the obtained cardiomyocytes have strong early proliferation ability, and more cardiac muscle can be obtained. After prolonged culture and construction into myocardial micro-tissue, maturity is high, the arrangement structure is more organized, and functional contractility is enhanced. Therefore, the present invention is suitable for various applications such as heart disease mechanism research, drug screening, and cell therapy, and thus the present invention has good practical application values.
Provided are a mesophilic prokaryote kurthia massiliensis-derived Argonaute protein KmAgo and an application thereof. Also provided are a polynucleotide encoding the KmAgo and an expression vector containing the polynucleotide, a kit containing the KmAgo, a method for in vitro and intracellular cleavage of target RNA by using the KmAgo, and a method for site-specific modification of a genetic material of a cell.
A zymomonas mobilis-based CRISPR-Cas system, comprising four CRISPR structured sequences and one cas gene cluster, wherein the cas gene cluster comprises the genes of cas1, cas3, csy1, csy 2, csy3 and csy4, and the gene cas3 is in the form of a fusion gene of cas2 and cas3. Further provided is a genome editing system, which is established by using zymomonas mobilis as the type strain, the CRISPR-Cas system encoded by the genome of the zymomonas mobilis, and exogenous Cas12a.
C12N 15/74 - Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
C12N 15/66 - General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligationUse of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
C12N 1/21 - BacteriaCulture media therefor modified by introduction of foreign genetic material
A method for preparing a near-infrared responsive functional coating on the surface of a cylindrical titanium nail, comprising the following steps: pretreating a titanium nail; growing a titanium dioxide nanotube on the titanium nail: placing the treated titanium nail into an electrolyte and connecting same to an anode, connecting a cathode to a custom tubular graphite electrode, reacting at room temperature for 3h at a reaction voltage of 40V, drying ethanol after ultrasonication, and calcining the mixture at 450°C for 2h to obtain a titanium nail/TNT; synthesizing gold nanoparticles and carbon quantum dots; and loading the gold nanoparticles and the carbon quantum dots onto the titanium dioxide nanotube on the surface of the titanium nail to obtain titanium nail/TNT/AU/CQDS. In using the described method, a titanium dioxide nanotube loaded with gold nanoparticles and carbon quantum dots on the surface of a cylindrical titanium nail may be prepared, and the obtained titanium nail equipped with a functional coating has a good photothermal effect and generates active oxygen after being irradiated for 15 min by infrared rays at 808 nm, has better antibacterial effects on both Staphylococcus aureus and Escherichia coli, and also has good cell compatibility and osteogenic properties.
The present invention provides a method for identifying biological elements based on a dual fluorescent reporter gene system and a biological element library constructed based on same. The method comprises: using a pEZ15Asp plasmid as a skeleton, constructing a single fluorescent reporter gene system for screening fluorescent proteins and determining fluorescent reporter genes; obtaining the pEZ15Asp skeleton; assembling fluorescent genes; obtaining a dual fluorescent reporter gene system skeleton; and constructing a recombinant plasmid, and finally transforming same into competent cells for quantitative analysis of fluorescence intensity.
C40B 40/02 - Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cellsLibraries contained in or displayed by vectors, e.g. plasmidsLibraries containing only microorganisms or vectors
G01N 33/68 - Chemical analysis of biological material, e.g. blood, urineTesting involving biospecific ligand binding methodsImmunological testing involving proteins, peptides or amino acids
G01N 33/569 - ImmunoassayBiospecific binding assayMaterials therefor for microorganisms, e.g. protozoa, bacteria, viruses
43.452.34.34.3 heterogeneous structural phase polyhedron nanoparticle, a preparation method therefor, and an application of serving as an efficient fuel battery oxygen reduction catalyst. The heterogeneous structural phase polyhedron nanoparticle consists of Fe, Pt, and Cu and is provided with a face-centered tetragonal phase shell layer and a face-centered cubic phase nuclear structure that have a high crystal face index, 1-2 Pt-rich atomic layers are provided on the surface, and the particle diameter is 8.4 nm. The present invention is obtained by adding oleylamine and oleic acid after evenly mixing hexadecylamine, ferric acetylacetonate, copper acetylacetonate, platinum acetylacetonate, and 1,2-hexadecanediol, and performing a condensation backflow reaction under the condition of 320-330°C. The synthesized nanoparticle of the present invention has an excellent ORR property, the half-wave potential is higher by 50 mV compared with Pt/C, the quality activity is 10.9 times that of Pt/C under the half-wave potential, and the nanoparticle has the potential application value in the fields such as electro-catalysis and high density magnetic recording.
B01J 23/89 - Catalysts comprising metals or metal oxides or hydroxides, not provided for in group of the iron group metals or copper combined with noble metals
18.
2 ALLOY SEMICONDUCTOR EPITAXIAL THIN FILM MATERIAL AND PREPARATION METHOD THEREFOR, APPLICATION THEREOF AND DEVICE THEREOF
x1-x2222, wherein: x is greater than 0 and less than 1; Me is Zr or Hf or Si or a combination of any two or three among the foregoing. Also disclosed are a preparation method for and application of the described thin film material, as well as a deep ultraviolet light detector using the described thin film material as a matrix material layer.
x3-xx3-xx3-xx3-xx3-xx3-xx3-x3-x thin film of the present invention is prepared by using a low temperature anti-solvent method, and the prepared LED device can realize the integration of self-powered visible detection and visible illumination, and can be used as an emission terminal or a receiving end in visible light wireless communication to solve the problem of reverse communication in visible light wireless communication technologies.
A polypoind rice photothermally-sensitive cytoplasmic male sterility line breeding method. The method comprises: a. determining a diploid rice line having photothermally-sensitive cytoplasmic male sterility and PMeS characteristics to serve as a parent; b. hybridizing the determined photothermally-sensitive cytoplasmic male sterility line with a diploid PMeS genetic line, culturing a hybridized plant seedling into a doubled hybrid tetraploid; c. backcrossing the hybridized tetraploid with a tetraploid photothermally-sensitive cytoplasmic male sterility line; d. selecting from backcrossing offsprings a tetraploid male sterile line, crossbreeding with another tetraploid rice line having PMeS genes after self-breeding during a period of low temperatures and short sun exposure; g. selecting the tetraploid male sterile line, detecting the stability of the tetraploid male sterile line after successive self-breeding; and I, determining the tetraploid rice sterile line consistent in stability as a tetraploid rice photothermally-sensitive cytoplasmis male line, which is named as PSXXX.
A two-line restorer line for polyploid rice and breeding method therefor. The method comprises: a. determining a hybrid parent of a breeding restorer line; b. breeding a restorer line of indica-japonica type, or breeding a restorer line of japonica-indica type, and performing backcrossing or composite crossing after parental hybridization; c. selecting a single strain that meets a breeding objective, performing composite crossing again, and performing a first molecular marker test; d. comparing and selecting a single strain having good traits, and performing continuous self-crossing until the strain is basically stable; e. selecting a stable strain having good traits and having a tested molecular marker; f. performing test crossing with different types of polyploid sterile lines using a preferred strain as the male parent; and g. choosing a good hybrid combination and a restorer line thereof. Good strains of restorer lines for polyploid rice are bred using a combination of a restoring ability of a photo-thermo-sensitive male sterile gene and a high fecundity of a polyploid rice of a PMeS genic strain.
A method for selectively breeding a polyploid two-line hybrid rice plant, the selective breeding method comprising: determining a tetraploid rice plant photo-thermo-sensitive male sterile line having the genetic feature of PMeS (polyploid meiosis stability) and a tetraploid rice plant restored line having the genetic feature of PMeS; performing hybridization using an Indica/Japonica-restored or Japonica/Indica-restored hybrid combination; hybridizing and generating a tetraploid rice plant hybrid using the tetraploid rice plant photo-thermo-sensitive male sterile line and the tetraploid rice plant restored line; breeding a stable tetraploid rice plant hybrid combination and determining said combination to be a polyploid two-line hybrid rice plant combination. The breeding method utilizes the powerful heterosis of a polyploid rice plant to transform existing diploid heterosis into the heterosis of a polyploid "two-line" hybrid rice plant. Using the method may breed new varieties of polyploid two-line hybrid rice plants which have large rice ears, large rice grains and a high yield.
The present invention relates to the field of artificial synthesis of genes. Disclosed is a primer-free gene synthesis method. First, disclosed is a pNew carrier plasmid, wherein the pNew carrier plasmid has a nucleotide sequence shown in SEQ ID NO.33. Also disclosed is a carrier bank that is constructed by using pNew carrier plasmids and contains 16384(47) plasmids. Further disclosed is a primer-free gene synthesis method. The method comprises the following steps: (1) grouping DNA sequences of target genes to be synthesized according to a length of 82bp per fragment; (2) finding plasmids corresponding to the grouped fragments in the constructed plasmid bank; (3) carrying out enzyme digestion on the corresponding plasmids, carrying out screening by using an antibiotic and carrying out reconstruction to obtain a plasmid containing a target gene sequence of 82bp; and (4) splicing the gene fragments by means of a golden-gate cloning reaction to obtain a complete target gene. The gene synthesis method of the present invention is free of primers, has low synthesis costs, very low mutation rate and high accuracy, can synthesize special gene sequences such as a highly repetitive sequence and Poly A, has simple operations and can realize automatic operations.
C12N 15/63 - Introduction of foreign genetic material using vectorsVectorsUse of hosts thereforRegulation of expression
C12N 15/10 - Processes for the isolation, preparation or purification of DNA or RNA
C40B 50/06 - Biochemical methods, e.g. using enzymes or whole viable microorganisms
C40B 40/02 - Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cellsLibraries contained in or displayed by vectors, e.g. plasmidsLibraries containing only microorganisms or vectors
patents
