111 results about "A-site" patented technology

Methods and compositions for gene disruption, gene editing or gene stacking within a FAD2 loci by cleaving, in a site directed manner, a location in a FAD2 gene in a soybean cell, to generate a break in the FAD2 gene and then optionally integrating into the break a nucleic acid molecule of interest is disclosed.

A method of gene editing or gene stacking within a FAD2 loci by cleaving, in a site directed manner, a location in a FAD2 gene in a cell, to generate a break in the FAD2 gene and then ligating into the break a nucleic acid molecule associated with one or more traits of interest is disclosed.

The invention discloses a method for preparing a DNA coding compound library containing two or more pharmacophores. A novel initial head fragment used by the method has two or more chemically-modifiedlinking groups and comprises a nucleotide chain, 5' and 3' of the nucleotide chain respectively have a group R1 and a group R2 capable of performing chemical reaction, n connecting heads are arrangedon the nucleotide chain, each of the connecting heads is a long chain extended after a nucleotide monomer on the nucleotide chain is modified, and the long chain has a site G capable of performing chemical reaction. The site G of the initial head fragment respectively reacts with a fragment compound to generate a fragment compound residue B connecting the site G with the fragment compound together, a DNA coding compound is formed, and finally, the DNA coding compound library containing the two or more pharmacophores can be obtained. Through the method disclosed by the invention and the initial head fragment, the DNA coding compound library containing the two or more pharmacophores can be efficiently, simply and quickly obtained.

The invention discloses a D-amino acid oxidase mutant and application thereof. The mutant is obtained by carrying out single mutagenesis or multiple mutagenesis on a 52nd site, a 54th site, a 58th site, a 213rd site and a 335th site of amino acid with an amino acid sequence shown in SEQ ID NO.1, wherein glycine at the 52nd site is mutated into leucine, asparagine at the 54th site is mutated into valine, phenylalanine at the 58th site is mutated into glutamine, methionine at the 213rd site is mutated into serine, and serine at the 335th site is mutated into glycine. According to the D-amino acid oxidase mutant and the application thereof, a D-amino acid oxidase gene with an amino acid sequence as shown in SEQ ID NO.2 is mutated by utilizing a site-saturation mutagenesis technology, so thatthe enzyme activity and the product conversion rate are far higher than those of a wild type, and therefore the product yield in a 4-(hydroxymethylphosphoryl)-2-carbonyl-butanoic acid production process is increased.

The invention discloses a site-directed mutation modified algin lyase mutant and application thereof, and belongs to the technical field of enzyme engineering. The algin lyase mutant provided by the invention is obtained by replacing glutamic acid at position 212 of the an algin lyase with an amino acid sequence as shown in SEQ ID NO.1 by histidine and/or replacing arginine at position 222 by lysine. According to the invention, the active center amino acid of the algin lyase is determined through molecular docking simulation, and site-directed saturated mutation is carried out to change aminoacid residues near active sites of protein molecules, so that catalytic efficiency of the algin lyase is improved, and the yield of the algin lyase is further improved. According to the invention, constructed recombinant escherichia coli with enhanced secretion capacity of algin lyase can improve enzyme activity of the algin lyase by 2.83 times compared with an original strain. The improved genetic engineering bacteria have obviously improved enzyme production capacity, and activity of algin lyase produced by shake flask fermentation reaches 15000U/mL or above, so that the improved genetic engineering bacteria are more suitable for industrial application, production cost can be reduced, and production efficiency is improved.

The invention discloses recombined alkaline pectinase with good pH stability and high specific enzyme activity and a construction method thereof. A Site-directed mutagenesis technique is adopted for mutagenesis of an alkaline pectinase coding gene pel168 of bacillus subtilis 168 and 457-bit guanine G is transformed into thymine T so that the 132-bit valine V of the mature peptide of the coded pectinase is mutated into phenylalanine F, a mutant gene pel168V132F is expressed in colon bacillus Rosset Blue (DE3) and subjected to inducible expression, bacterium breaking and purification, the purified enzyme is subjected to enzyme activity measurement, and the relative activity of the obtained pectinase mutant PEL168V132F is much higher than that of the original pectinase PEL168 when the pH is 3, 5 and 6; and meanwhile, the specific enzyme activity is 2.25 times that of the original pectinase, thus good foundation is laid for wide application of the alkaline pectinase.

The invention provides a method and kit for detecting RNA N6-methyladenosine modification at single-base resolution in a range of a whole transcriptome. According to the method, on the basis of an N6-allyl label of in-vivo ribonucleic acid (RNA) adenine and chemical treatment, base mutation of the in-vivo ribonucleic acid (RNA) adenine in a process of reverse transcription into DNA is induced, andthen a mutation site is recognized by means of nucleic acid sequencing, so that an a6A site is obtained, and the a6A site is a site originally modified by m6A in cell RNA. By means of the method, thespecific label of N6-allyladenine in a cell is achieved for the first time, and the label not only can be used for replacing an N6-methyladenosine site in the cell, but also can be positioned by means of mutation sequencing. Compared with existing gene sequencing technologies applied to m6A detection, the method for detecting RNA N6-methyladenosine modification at single-base resolution in the range of the whole transcriptome has the advantage that due to the fact that the mutation site can be accurate down to single-base resolution, the precision of m6A site detection based on m6A antibody immunoprecipitation and a massively parallel sequencing method which are currently and generally adopted is improved, so that the method for detecting RNA N6-methyladenosine modification at single-baseresolution in the range of the whole transcriptome is a direct high-throughput single-base identification method.

The invention provides a method for porcine H11 site-specific insertion by using a site specific cleavage system. The method comprises the following steps: 1) designing5'-terminal homologous arm and 3'-terminal homologous arm of knocked-out gene and corresponding universal primers; 2) introducing the above homologous arms, the universal primers and gene to be inserted into a vector in order to a targeting vector; 3) transferring into cells; and 4) carrying out PCR amplification, and identifying the insertion result. The targeting vector is designed mainly relying on the porcine H11 site cleavage system, and the vector can accurately introduce exogenous gene into the porcine the H11 site in order to solve the problems of low efficiency of traditional targeting techniques, inconvenient designing of PCR detection primers and large detection difficulty. The site-specific insertion method provided by the invention allows the exogenous gene to be stably expressed in H11, and builds a stable platform for pig transgene.

The problem to be solved in the present invention is to provide a simplified and efficiently improved DNA recombination method.

The above problem can be solved with the present method for preparing a recombinant DNA by inserting a DNA fragment of interest into a vector DNA, the method comprising the step of carrying out the following reactions at the same reacting location at the substantially simultaneouse time: (a) a reaction for simultaneously cleaving a site of the vector for inserting the fragment and a DNA containing the fragment in the presence of a restriction enzyme whose DNA recognition site and DNA cleavage site are discrete; and (b) a reaction for inserting the fragment into the vector in the presence of a DNA ligase.

The present invention provides a Candida rugosa lipase mutant, wherein Candida rugosa (ATCC NO:14830) lipase LIP1 (CRL1) is subjected to thermal stability modification by using a site-directed mutation technology so as to optimize the practicality in the high temperature industrial environment. According to the present invention, the CRL1 gene is used as the template, the protein rational design technology is used, and the amino acid Asp at 4578 site of the CRL1 lipase gene sequence is substituted with Phe so as to obtain the lipase mutant with the improved thermal stability, wherein the application range and the catalytic efficiency of the enzyme are improved; and the CRL1 is subjected to site-directed mutation by using the molecular biology method to obtain the lipase mutant, wherein theTm value of the lipase mutant is increased by 9.4 DEG C, the optimum temperature is increased by 10 DEG C, and the T1/2 (50 DEG C) is 6.5 times the T1/2 (50 DEG C) of the parent lipase.

The invention discloses a molecular binding site detection method and device, electronic equipment and a storage medium, and belongs to the technical field of computers. The method comprises the following steps: acquiring three-dimensional coordinates of each site in a target molecule; determining a first target point and a second target point corresponding to each site, extracting position features with rotation invariant characteristics in the three-dimensional coordinates of each site, calling a site detection model to predict the extracted position features, obtaining a prediction probability that each site belongs to a binding site or not, and determining a binding site based on the prediction probability. The first target point and the second target point are related to each site andhave certain spatial representativeness; the method is beneficial to constructing the position characteristics which can comprehensively reflect the detail structure of the target molecule and have the rotation invariant characteristic, detail loss caused by designing voxel characteristics for the target molecule is avoided, and the accuracy of the molecular binding site detection process is improved.

The invention discloses a site for stably expressing protein in a CHO (Chinese hamster ovary) cell gene NW_003613781.1 and application of the site. The obtained stable expression site is positioned on a basic group in a 100bp range of the upstream and downstream of the 1497187 basic group of the CHO cell gene NW_003613781.1, namely the 1497087-1497287 basic group, and an exogenous protein gene can be integrated and stably expressed. According to the invention, the target gene is subjected to site-specific integration to the stable expression region in a site-specific integration manner, so that the problem of unclear integration sites caused by random integration is solved. According to the invention, by site-specific integration of an exogenous gene in the range of 1497087-1497287 bases upstream and downstream at the 1497187 base of a stable expression site NW_003613781.1 in a CHO genome, the expression instability and repeated and tedious cell strain screening process caused by a position effect are overcome.

The invention relates to a recombinant lactate dehydrogenase gene. The nucleotide sequence of the gene is represented by SEQ ID NO:1, and tyrosine is mutated into any one of alanine, valine and leucine in the 52 position of the sequence. The invention further relates to recombinant Escherichia coli containing the recombinant lactate dehydrogenase gene and a method for synthetizing D-phenyllactic acid through the recombinant Escherichia coli. According to the method, the tyrosine on a substrate recognition site of D-LDH is replaced by smaller hydrophobic amino acid residue-valine with a site-directed mutagenesis technology, so that the affinity and the catalytic efficiency of the D-LDH to substrate phenylpyruvic acid are improved, after conversion of an expression host, the yield of phenyllactic acid is significantly increased when being compared with that of un-mutated strains, and after whole-cell conversion for 6 h, the yield of the phenyllactic acid is increased from 18 g/L to 24.6 g/L.

The invention provides a mutant of cyclodextrin glycosyltransferase (CGT enzyme for short) with capability for high-yield of beta-cyclodextrin and mutating method thereof, and belongs to the fields of gene engineering and enzyme engineering. The invention employs a site-directed mutagenesis method for improving beta-cyclodextrin production capability of CGT enzyme, and provides a mutating scheme for improving beta-cyclodextrin production capability of Bacillus circulans STB01CGT, that is alanine at the 315th position of the calcium ion binding site in CGT enzyme is changed into arginine (Arg) or aspartic acid (Asp), and the mutants A315R and A315D are obtained. Compared with the wild CGT enzyme, the two mutants have higher beta-cyclodextrin production capability, and are suitable for industrial production of beta-cyclodextrin.

The invention discloses a site-directed mutagenesis escherichia coli DNA photolyase and a construction method thereof. The photolyase has an amino acid sequence shown by SEQ ID NO:1. The method comprises the following steps: obtaining a DNA photolyase gene from the existing WT escherichia coli, designing a mutation primer to obtain a mutated gene fragment, and connecting the mutated gene fragment with a pET22b plasmid to construct a recombinant expression plasmid pET22b-A377N; and converting the recombinant expression plasmid into an escherichia coli competent cell to construct a genetic engineering bacterium BL21(DE3)/pET22b-A377N for mutant enzyme expression. The site-directed mutagenesis escherichia coli DNA photolyase obtains better expression under a 1mM ITPG condition at 18 DEG C. The expressed photolyase is more stable in activity, and the antioxidation effect of the photolyase is significantly improved.

The invention discloses a site for stably expressing protein in the CHO cell gene NW_003614092.1 and application of the site. The obtained stable expression site is positioned on a base in a 200-bp base range with the 1159467 base of the CHO cell gene NW_003614092.1 as a central point, namely, the 1159367 to 1159567 base, and can integrate an exogenous protein gene and perform stable expression. According to the invention, a target gene is subjected to site-specific integration to a stable expression region in a site-specific integration manner, so the problem of unclear integration sites caused by random integration is solved; and through site-specific integration of an exogenous gene in the range of 1159367 to 1159567bases, relative to the stable expression site at the 1159467 base in the CHO cell gene NW_003614092.1, expression instability and a repeated and tedious cell strain screening process caused by a position effect are overcome.

The invention discloses a CRICPR-Cas9-based method for in-vitro modifying adenovirus vectors and further provides a molecular cloning method suitable for modifying large plasmid vectors. The CRICPR-Cas9-based method includes: designing proper sgRNA according to a to-be-modified site of a starting plasmid; adopting Cas9 protein and the sgRNA to in-vitro cut the starting plasmid; connecting the starting plasmid which is cut with a target gene on the basis of a homologous recombination method to complete modifying. By using the CRICPR-Cas9-based method, the problem that proper restriction enzyme cutting sites are difficult to find for modifying the large plasmid vectors through conventional molecular biologic means is solved effectively. The method is an improvement of a site mutation kit which generally includes site mutation of target protein and building of truncation with deleted structural domain. By the method, the technical problem that secondary mutation (random mutation caused by PCR enzyme fidelity) is easy to be caused when the site mutation kit is used for mutation of large vectors exceeding 10kb is solved effectively.

The invention discloses a site for stably expressing protein in a CHO (Chinese hamster ovary) cell gene NW-003613756.1 and application of the site. The stable expression site obtained by the invention is positioned on a basic group in the upstream and downstream 150bp range of the 101424th basic group of the CHO cell gene NW-003613756.1, namely the 101274th to 101574th basic groups, and an exogenous protein gene can be integrated and stably expressed. According to the site, the target gene is subjected to site-specific integration to the stable expression region in a site-specific integration manner, so that the problem of unclear integration sites caused by random integration is solved; and by site-specific integration of an exogenous gene in the basic group range of 101274-101574 upstream and downstream of the 101424th basic group of the stable expression site NW-003613756.1 in the CHO genome, the expression instability and repeated and tedious cell strain screening process caused by a position effect are overcome.

The invention discloses a site specific integration vector capable of removing random integration, a construction method thereof and an application thereof. The vector comprises a pUC ori, an attB sequence and a multiple cloning site (MCS). The upstream and downstream of the attB sequence is respectively provided with a first loxP sequence and a first rox sequence. A CMV promoter is connected to the upstream of the first loxP sequence. A negative screening gene is connected to the first rox sequence. The downstream of the negative screening gene is connected to an internal ribosome binding site IRES2. The downstream of the IRES2 is a fluorescence labeling gene. The downstream of the fluorescence labeling gene is the MCS. The upstream and downstream of the MCS is respectively provided with a second rox sequence and a second loxP sequence. The direction of the second rox sequence and the direction of the first rox sequence are same, and the direction of the second loxP sequence and the direction of the first loxP sequence are same. The downstream of the MCS is a positive screening element. The application relates to rejection of random integration recombinant cells so as to directly screen false attP site specific integration recombinant cells.

The invention discloses a site-specific integration method of exogenous genes, and particularly discloses a method for integrating the exogenous genes into second exons of bovine beta-casein genes in a site-specific manner. The method comprises the following steps: expressing TALE (Transcription Activator Like Effector) protein-I and TALE protein-II in isolated bovine cells so as to obtain cells, the target sequences of second exons of which change suddenly; meanwhile, recombining the exogenous genes into the target sequences in a homologous manner so as to realize the site-specific integration of the exogenous genes on the second exons of the bovine beta-casein genes. The method has the advantages that human lysozyme genes or other target genes are capable of transcribing the site-specific integration of very active beta-casein gene loca in mammary gland, the location effect is overcome, the target gene expression silencing is avoided, the relatively high expression level of the human lysozyme or other recombinant protein in mammary gland can be realized, the success rate of transgenic breeding is increased, and meanwhile, the cost of producing and purifying the recombinant protein in a later period can be saved.

The invention discloses a recombinant saccharomyces cerevisiae production of ambrein and a construction method. The construction method comprises the following steps: a site-directed mutant hopene cyclase encoding gene triangle SHC and a tetraphenyl-beta-curcumene synthase encoding gene BmeTC are introduced into a Saccharomyces cerevisiaeW303-1a to obtain a recombinant strain 1; a truncated3-hydroxy-3-methylalutaryl coenzyme A reductase encoding gene tHMG1 is introduced into the recombinant strain 1 to obtain a recombinant strain 2; and the experiment proves that the recombinant saccharomycescerevisiae for production of the ambrein can be fermented to produce the ambrein, and lays a foundation for artificial synthesis of the ambrein.

The invention discloses a method for improving the structural isomerism catalytic activity of a CE enzyme and a mutant thereof, and belongs to the field of enzyme engineering. According to the invention, cellobiose epimerase (CsCE) derived from Caldicellosirutorschcharlyticus is taken as a research object, site-saturation mutagenesis is carried out on a site related to substrate binding based on a semi-rational strategy of sequence alignment and crystal structure analysis, a plurality of mutants with obviously improved structural isomerism catalytic activity are obtained, and the structural isomerism activity of the mutants is improved by about 36-232% than that of a wild type CE enzyme. The invention provides a feasible scheme for CE enzyme improvement, and has important significance for promoting enzymatic industrial synthesis of lactulose.