12 results about "T7 RNA polymerase" patented technology

The present application relates to the field of genetic engineering and bioengineering, and particularly relates to a genetically engineered surfactin-producing bacterium and application thereof.The genome of the genetically engineered bacterium comprises the following: an expression frame of T7 RNA polymerase; an expression frame of a surfactin synthesis gene cluster srfAABCD; and an expression frame of a long-chain fatty acid-coenzyme A ligase gene lcfA; the expression frame of the surfactin synthesis gene cluster srfAABCD and the expression frame of the long-chain fatty acid-coenzyme A ligase gene lcfA further comprise a T7 promoter.The present application realizes efficient synthesis of surfactin by Bacillus subtilis, and the final shake flask yield reaches more than 19.3 g / L, which has great industrial application potential.

The application discloses a method for synchronously marking germ cells and interstitial cells in the testis tissue of Paralichthys olivaceus, which comprises the following steps: cloning MIR17HG-a (long non-coding RNA) of Paralichthys olivaceus by using a probe primer, obtaining a target plasmid after plasmid extraction, carrying out double enzyme digestion and purification to obtain an enzyme-digested plasmid as a probe template, synthesizing Paralichthys olivaceus MIR17HG-a probe by using an SP6 / T7 RNA polymerase, preparing a tissue slice from the testis tissue of Paralichthys olivaceus, and carrying out fluorescent in situ hybridization on the tissue slice by using the Paralichthys olivaceus MIR17HG-a probe, so that the Paralichthys olivaceus MIR17HG-a probe can synchronously mark the germ cells and interstitial cells in the testis tissue of Paralichthys olivaceus. The method solves the defects of traditional marking technology, such as the need for combined marking or multiple step-by-step marking, and fills the blank of synchronous marking of germ cells and interstitial cells in the testis tissue of Paralichthys olivaceus.

The present application relates to the technical field of biology, and in particular to a T7 RNA polymerase mutant and application thereof. The present application provides a T7 RNA polymerase mutant and application thereof in IVT reaction. The mutant can significantly reduce the content of dsRNA by-products in in vitro transcription of T7 RNA polymerase, has higher transcription activity, can improve the integrity of products, and has good thermal stability.

The application provides a preparation method and application of an RNA cell-free synthesis preparation, and belongs to the technical field of molecular biology. The preparation method of the RNA cell-free synthesis preparation is mainly as follows: taking Escherichia coli as a chassis strain, using a pET-28a vector to construct a recombinant strain for expressing T7 RNA polymerase, the T7 RNA polymerase has enzymatic activity at 50-60 DEG C; the bacterial cells are collected by induction culture and are subjected to ultrasonic lysis, and the lysis solution is treated to obtain the preparation for RNA cell-free synthesis. The preparation provided by the application naturally contains various cofactors, and the RNA polymerase does not need to be extracted and purified, so that the production cost is reduced.

The present application relates to the technical field of biology, and discloses a mutant T7 RNA polymerase with reduced by-products in transcription process, and a preparation method and application thereof. The mutant T7 RNA polymerase of the present application is a protein as described in A1) or A2): A1) the mutant T7 RNA polymerase comprises amino acid substitution sites A124E / A258D, A124E / M190D or A124E / S192P relative to a wild-type T7 RNA polymerase; the amino acid sequence of the wild-type T7 RNA polymerase is shown as SEQ ID NO:1; A2) a fusion protein obtained by connecting a tag to the N terminal or / and C terminal of the amino acid sequence described in A1). Relative to the wild-type T7 RNA polymerase, the mutant T7 RNA polymerase of the present application has reduced by-products such as dsRNA in the transcription process.

The present application provides a class of RNA polymerase variants, which have improved catalytic activity relative to wild-type T7 RNA polymerase, and can improve the capping rate of mRNA products in the in vitro co-transcription capping process. In addition, the present application also provides a method for preparing RNA using such variants, and using the method for preparing RNA molecules, more capped mRNA can be obtained.

The application provides a T7 RNA polymerase mutant with a molecular tag and application thereof. In the T7 RNA polymerase mutant with the molecular tag, the molecular tag comprises a DNA binding protein or a binding fragment thereof, which is directly connected or connected to an N terminal of an amino acid sequence of the T7 RNA polymerase mutant through a linker. The T7 RNA polymerase mutant with the molecular tag has thermal stability.

The present application relates to a kind of T7 RNA polymerase mutant and its application, the T7 RNA polymerase mutant has the following mutation relative to wild type T7 RNA polymerase: P266L, S430P, N433T, S633P, Y639F, V689Q, V783M, F849I, F880Y;Or, at least one of the following mutations can also be based on the above-mentioned mutation: S43Y, G47A, S641A.The T7 RNA polymerase mutant provided by the present application can recognize and incorporate rNTPs, 2'-F-dNTPs and one or more nucleotide substrates of dNTPs, to efficiently synthesize single-stranded DNA molecules, fluorinated RNA molecules and perfluorinated DNA molecules in vitro.

The application discloses a single cell detection method, and the application realizes accurate analysis on single cells by specific recognition of target cells through antibody-DNA conjugates and release of nucleic acid markers by using photolysis technology. Through entropy-driven cyclic amplification and T7 RNA polymerase transcription amplification, the detection sensitivity is further improved, and the specific cutting amplification reaction of CRISPR / Cas12a is combined to realize high-sensitivity, low-cost and scalable single cell level detection. The electrochemiluminescence signal reading mode of the application makes the detection result intuitive and suitable for high-throughput analysis, and can be widely applied to the fields of cancer research, pathogen screening, drug development and precision medicine.

The application provides a T7 RNA polymerase mutant and application thereof. The T7 RNA polymerase mutant of the application is selected from the following: a T7 RNA polymerase mutant consisting of the amino acid sequence shown in SEQ ID No. 2; or a T7 RNA polymerase mutant having substantially equivalent functions and obtained by substitution, deletion or insertion of one or more amino acids based on the amino acid sequence shown in SEQ ID No. 2. The T7 RNA polymerase mutant of the application has thermal stability.

This invention discloses a method for constructing and fermenting a strain of Bacillus subtilis to enhance anaerobic expression and improve surfactant synthesis. First, a chassis strain 158T7P is obtained by integrating and inserting an inducible transcriptional T7 RNA polymerase expression gene into the genome of Bacillus subtilis 158. Further genetic manipulation of this Bacillus subtilis, through systematic substitution of the T7 promoter, yields strain 158T7P-srf, capable of producing surfactant in high-density microaerobic fermentation. Specifically, this involves using homologous recombination to transform the operon of Bacillus subtilis 158... resde The starter and control sfp The promoters for transcription of the gene and the srfABCD operon were replaced with T7 promoters. The obtained Bacillus subtilis 158T7P-Srf was able to achieve high production of surfactant using inexpensive carbon sources such as brown sugar as substrates. In the fermentation experiment, the surfactant production reached a maximum of 36.58 g / L.

The application provides a T7 RNA polymerase variant and its application, the variant can significantly reduce the substrate high concentration inhibition in the in vitro transcription process, and improve the mRNA yield relative to the wild type T7 RNA polymerase. In addition, the application also provides a method for preparing RNA by using the variant, and the RNA molecules prepared by using the method can be obtained economically in high concentration.