20 results about "Prokaryote organisms" patented technology

The invention discloses base-modified mRNA (messenger Ribonucleic Acid) and application thereof in a cell-free protein synthesis system, and belongs to the technical field of nucleic acid modification. Natural nucleoside triphosphate is completely replaced by different types of basic group modified nucleoside triphosphate, mRNA with different chemical modifications is obtained through an in-vitro transcription method and purification, and the mRNA is put into an in-vitro cell-free protein synthesis system for reaction. A reaction element recombination system-PURE system based on prokaryote Escherichia coli purified protein and an eukaryote CFPS system taking rabbit reticulated red lysate as a material are mainly selected to detect the corresponding translation efficiency of mRNA modified by different bases in two cell-free protein synthesis systems respectively. Different regulatory activities may be shown in different cell-free protein synthesis systems when different modified nucleotides are doped into mRNA, base modified triphosphoric acid capable of improving the translation efficiency of an in-vitro protein synthesis system is screened out, and the stability of mRNA in the cell-free protein synthesis system and the corresponding protein expression level are improved.

The invention belongs to the technical field of biology, and particularly relates to an enzyme co-localization system and a construction method and application thereof. The invention relates to an enzyme co-localization system. The system contains a protein of a WS / DGAT family for constructing a lipid droplet organelle and a target enzyme fragment connected with an anchoring protein specifically targeting the lipid droplet organelle. The protein of the WS / DGAT family is an enzyme tDGAT of the WS / DGAT family, the base sequence of the enzyme tDGAT is as shown in SEQ ID NO.1, and the coding amino acid sequence of the enzyme tDGAT is as shown in SEQ ID NO.2. According to the invention, through the characteristics that lipid droplets (LD) have ubiquitous cellular structures and proteins can be positioned on LDs of different organisms in a cross-species manner, plasmids are utilized to heterologously express an enzyme tDGAT belonging to a WS / DGAT family in escherichia coli C43 (DE3), so that more stable lipid droplet organelles can be formed in prokaryotic escherichia coli; meanwhile, plasmids are used for expressing protein capable of being specifically combined with the LD surface, the N end of the protein is fused with biosynthetic pathway enzyme to form recombinant protein, then expression conditions are optimized, and local enzyme concentration is improved.

The application discloses an application of a crenarchaeota-derived Argonaute protein or a coding gene thereof in RNA interference. The Argonaute protein is named as HrAgo1, and the amino acid sequence is shown as SEQ ID No. 1. The application finds a new crenarchaeota-derived Argonaute protein, which is named as HrAgo1. Different from known prokaryote Argonaute proteins, the HrAgo1 has two characteristics. First, the HrAgo1 can use short-chain RNA as a guide chain to cut mRNA paired with the sequence. Therefore, a gene interference method based on the HrAgo1 is obviously different from other applications based on prokaryote Argonaute proteins. Second, the HrAgo1 can exert the function at 9-71.1 DEG C, and can be applied to cells growing at different temperatures.

The application discloses a method for efficiently labeling proteins with selenium, which comprises the following steps: adjusting the ratio of selenium and sulfur in the culture medium during the expression of a plasmid containing a target protein by a prokaryotic expression system, and realizing the efficient replacement of selenium atoms for the cysteine sulfur atoms in the target protein; under the premise of maintaining the good growth state of cells, the selenium labeling efficiency can reach 86%, and the protein expression amount is stably about 1 mg / g (protein / biomass wet weight); structure analysis shows that the obtained selenium labeled protein is basically consistent with the natural protein in the secondary structure and does not change obviously. The method is simple in operation, high in labeling efficiency and protein yield, good in repeatability, and suitable for structural biology research such as nuclear magnetic resonance, provides an effective means for studying the function mechanism of sulfur atoms in biological macromolecules (including small peptides, polypeptides, peptide segments, proteins, and complexes of proteins or peptides) by using selenium as a probe, and has good scientific research application prospect and industrialization popularization value.

The production of substituted 2-hydroxyacyl-CoA molecules by a novel reaction is described. The reaction involves the condensation of formyl-CoA with a carbonyl-containing molecule. Such carbonyl-containing molecules include a substituted aldehyde and a ketone. The reaction is catalyzed by enzymes using a TPP-dependent mechanism. Also described is the production of unsubstituted and substituted 2-hydroxyacyl-CoA molecules comprising the condensation of formyl-CoA with a carbonyl-containing molecule, wherein the condensation is catalyzed by a prokaryotic HACL. The 2-hydroxyacyl-CoA can be converted to chemical products having broad applications by using enzyme catalysts. The combination of enzyme catalysts comprises novel biochemical reaction pathways that can be deployed either as polypeptides in a reaction buffer or genetically encoded in recombinant microorganisms.

The application relates to the technical field of hydrogel preparations, and discloses a hydrogel preparation, a preparation method and application in repairing sperm damage. The method comprises the following steps: step one, constructing a recombinant plasmid; step two, plasmid transformation; step three, protein purification; step four, preparing mEXOs wrapped SKAP2 proteins; and step five, preparing a SKAP2 hydrogel preparation. In the application, SKAP2 proteins are expressed by means of a prokaryote Escherichia coli, milk exosomes are extracted, the SKAP2 proteins are then wrapped into the milk exosomes, and the exosomes containing the SKAP2 proteins are then fused into a hydrogel preparation. The sperm activity of asthenospermia mice can be significantly improved through skin smearing, including improving the sperm activity reduction effect of asthenospermia caused by heavy metal lead, organic endocrine disruptors phthalate, high temperature and aging, and the problem that there is no effective repair preparation for asthenospermia in the prior art is solved.

The application provides an RNA framework for gene editing and a gene editing method, the RNA framework comprising a sequence upstream of a target site, a sequence to be inserted, and a sequence downstream of the target site in the 5' to 3' direction. The gene editing method is based on the inherent mechanism of eukaryotes, uses RNP or RNA (which can be prepared in vitro) and related proteins as carriers, and is transferred into the cytoplasm and nucleus to realize gene editing on a specific sequence or site on the genome of a target system, such as insertion, deletion, replacement and site replacement of a specific sequence, and has high targeting accuracy. The application is more suitable for further clinical application than other prior art because no exogenous system or substance such as protein derived from prokaryotes is introduced, and no double-strand break is generated.

This invention discloses overexpression cysK The application of genes in increasing the soluble protein content of leguminous forage grasses belongs to the field of biotechnology. cysK The CDS sequence of the gene is shown in SEQ ID NO. 2. A plant overexpression vector containing the nucleotide sequence shown in SEQ ID NO. 2 was constructed, and the plant overexpression vector was introduced into leguminous forage plant cells to obtain overexpressed genes. cysK Transgenic leguminous forage lines with overexpressed genes in alfalfa. cysK The gene significantly increases the soluble protein content in its leaves by approximately 28%. This invention is the first to utilize Escherichia coli derived from prokaryotes. cysK The successful application of genes to the quality improvement of leguminous forage grasses has overcome the technical barrier of distant species kinship, providing new genetic resources and efficient technical approaches for breeding new varieties of leguminous forage grasses with high protein and high nutritional value, which is of great significance for promoting the sustainable development of the grass animal husbandry industry.

The application provides a renaturation tag for purifying inclusion body proteins, and a gene-derived product and application thereof, and belongs to the technical field of protein purification. The application provides a renaturation tag P67, and the amino acid sequence is at least one of SEQ ID NO:1-SEQ ID NO:3, wherein the 33th amino acid is lysine, glutamic acid or proline. The renaturation tag P67 is fused with target proteins for expression in prokaryotes, so that the recombinant expressed inclusion body (insoluble inclusion body) is successfully renatured and has biological activity. The renaturation tag P67 establishes a foundation for subsequent protein function research and application transformation.

The application discloses a non-coding sRNA for regulating gene expression and application thereof, and belongs to the technical field of biology. The application firstly finds that the non-coding sRNA formed by the combination of any one of the transcription terminator (or endogenous terminator) independent of the rho factor and a specific mRNA complementary pairing region of a target gene can play a gene regulation function. The discovery is taken as a basic and universal principle to design the non-coding sRNA, so that the purpose of specifically regulating the expression of any gene can be achieved. Taking the gfp, ftsZ gene and the synthesis bypass pathway of the ergothioneine synthesis substrate as examples, it is verified that the non-coding sRNA designed under the design principle can effectively regulate the expression of the gfp and ftsZ, or improve the ergothioneine yield. The method of the application can provide more effective and convenient regulation technology for the gene regulation of prokaryotes, and can realize the rapid regulation of any gene.

To provide a method for producing a population of high titer recombinant adeno-associated virus (AAV) lacking prokaryotic sequences.SOLUTION: Culturing a human embryo cell strain in suspension, transfecting the human embryo cell strain with (a) nucleic acids sequences encoding helper proteins sufficient for rAAV replication, (b) nucleic acids sequences encoding AAVrep and AAVcap genes, and (c) a closed ended linear duplex rAAV vector nucleic acids comprising at least one inverted terminal repeats (ITR) sequence and a heterologous transgene operably linked to one or more regulatory elements, incubating the transfected human cell strain for about 40 to 400 hours, and optionally, B. lysing the transfected human cell line to purify the nucleic acid sequence encoding the rAAV, thereby producing the rAAV.SELECTED DRAWING: None

Provided is a technique or the like that enables genome editing in prokaryotes. A CRISPR-Cas vector system for use in prokaryotes according to the present disclosure includes (a) a polynucleotide for encoding guide RNA and a terminator sequence that include a base sequence capable of hybridizing to target DNA in a prokaryote, and (b) a polynucleotide for encoding a Cas protein. The aforementioned (a) and (b) are located on the same or different vectors. The guide RNA and the Cas protein can form a complex. In the aforementioned (a), the terminator sequence is located on the 3'-end side of the guide RNA.

This invention belongs to the field of genetic engineering and relates to a method for preparing amylase derived from halophilic archaea and its application; based on halophilic archaea... Haloarcula sp. ZJK10-12、 Haladaptatus The genes for α-amylase, maltose amylase, and glucoamylase were obtained from the whole genome sequence of sp. ZJK29-3. Recombinant plasmids were constructed using primer design and molecular cloning, and transformed into prokaryotic host bacteria to obtain the corresponding recombinant strains. The bacteria were enriched and cultured in Hv-YPC liquid medium, and the cells were collected and lysed by sonication. α-amylase, maltose amylase, and glucoamylase were obtained using nickel column affinity chromatography. The amylases exhibit excellent enzymatic properties and outstanding salt tolerance. Their application in the fermentation of high-salt, dilute soy sauce can improve raw material utilization and product quality, demonstrating good industrial applicability and promotional value.

The present application belongs to the field of synthetic biology, and relates to a kind of unstable domain mutant based on prokaryotic transcription factor TtgR and its application.The present application constructs protein level reversible control switch by directional modification of prokaryotic transcription factor, and upgrades traditional transcription level regulation to protein level precise regulation.The regulation system (TtgR regulation system) has modular structure and wide adaptability: in specific embodiments, the coding sequence of reporter gene EGFP has been connected and modified into a fusion protein, and can precisely regulate the expression of EGFP under the induction of various small molecule ligands.The system can be applied to the operable connection of the coding sequences of various target proteins, including but not limited to CAR-T, CAR-NK related functional proteins, IL-2 and other cytokines, and other therapeutic proteins, functional proteins, etc.

The present invention relates to the field of prokaryotic products with improved stability, in particular fermentative bacteria such as lactic acid bacteria, a method for preparing products with improved stability and compositions which may be prepared by said method.

Disclosed in the present invention are a xylose-inducible transcription factor component and a use thereof. The xylose-inducible transcription factor component comprises: a xylose-inducible transcription factor XlnR derived from a filamentous fungus and a binding consensus sequence of the xylose-inducible transcription factor XlnR. By combining the transcription factor XlnR with a prokaryotic xylose-inducible transcriptional repressor XylR, a dual-regulated inducible system obtained exhibits an extremely low leakage level, but maintains a maximum activation level comparable to that of an XlnR single-regulated xylose-inducible system. The present invention solves the problems of low induction strength and slow response in transcription factor-dependent xylose transcriptional induction systems in the field of synthetic biology.

The present invention is related to a nucleic acid molecule, which is also referred to as third nucleic acid molecule, wherein the third nucleic acid molecule comprises(1) a nucleic acid molecule comprising the following elements:(a) optionally, a first part of a genome of a virus;(b) a nucleotide sequence, preferably a genomic nucleotide sequence, or a transcription unit;(c) a regulatory nucleic acid sequence which has a regulatory activity in a prokaryote;(d) exactly one site-specific recombination site;(e) a nucleotide sequence providing for a negative selection marker;(f) a bacterial nucleotide sequence unit comprising (i) bacterial nucleotide sequences for conditional replication and (ii) a nucleotide sequence providing for a positive selection marker;(g) optionally a first restriction site; or(2) a nucleic acid molecule comprising a nucleotide sequence according to SEQ ID NO: 6; or(3) a nucleic acid molecule identical or similar to the nucleic acid molecule contained in the organism deposited with the DSMZ under the Budapest treaty under accession number DSM 23754, wherein preferably the nucleic acid molecule contained in the organism is a heterologous nucleic acid molecule;wherein the third nucleic acid molecule is either a linear or a circular molecule.

The invention belongs to the field of synthetic biology, and relates to an unstable structural domain mutant based on a prokaryote transcription factor TtgR and application thereof. The protein level reversible regulation switch is constructed by directionally modifying a prokaryote transcription factor, and traditional transcription level regulation is upgraded to protein level precise regulation. The regulation and control system (TtgR regulation and control system) has a modular structure and wide adaptability, in the specific embodiment, the TtgR regulation and control system is connected with a coding sequence of a reporter gene EGFP to be transformed into fusion protein, and expression of the EGFP can be precisely regulated and controlled through induction of various small molecule ligands. The system can be popularized and applied to operable connection of coding sequences of various target proteins, including but not limited to CAR-T, CAR-NK related functional proteins, cell factors such as IL-2 and the like, and other therapeutic proteins, functional proteins and the like.

The invention belongs to the technical field of microorganisms, and discloses an NTase protein, a screening method thereof and application of the NTase protein in preparation of an anti-phage signal system antagonist and / or an antibacterial preparation. The NTase protein provided by the invention specifically comprises an amino acid fragment with a sequence as shown in SEQ ID NO: 1 or a variant fragment with at least 70% sequence identity with the sequence as shown in SEQ ID NO: 1, the NTase protein has an excellent antagonistic effect on a CBASS system of prokaryotes, and the infection efficiency of viruses on prokaryotic cells can be improved by inhibiting the CBASS system; therefore, a better antibacterial effect is achieved, and the compound has a very excellent application prospect in research and development of novel antibacterial drugs.