Novel synthesis-regulating srna and method for preparing same

A technology of regulators and transcriptional regulators, applied in the fields of biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., which can solve the problems of difficult gene repair and few or no reports of synthetic sRNA.

Active Publication Date: 2014-12-31
KOREA ADVANCED INST OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Third, genes deleted from chromosomes by traditional gene deletion methods are difficult to repair
[0010] The basic functions of E. coli sRNAs have been continuously studied, but there are few or no reports on the use of the above mechanisms to construct synthetic sRNAs

Method used

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  • Novel synthesis-regulating srna and method for preparing same
  • Novel synthesis-regulating srna and method for preparing same
  • Novel synthesis-regulating srna and method for preparing same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0118] Example 1: Performance testing of synthetic sRNA

[0119] 1-1: Construction of synthetic sRNA and construction of pWAS plasmid for regulating expression

[0120] Construct Figure 18 Basic plasmids are shown in , where the expression of synthetic sRNA is suppressed, but can be efficiently expressed if desired. exist Figure 18 In the pWAS plasmid, the target gene mRNA binding region can be inserted into the promoter by site-directed mutagenesis (P R ) and MicC Hfq binding region to construct synthetic sRNA.

[0121] Restriction enzymes used in this and the following examples were purchased from New England Labs (USA), and PCR polymerase was purchased from Invitrogen (USA). Other reagents are indicated separately.

[0122] First, PCR was performed using the pKD46 plasmid (GenBank AY048746.1 (Datsenko et al, PNAS, 97(12): 6640-6645, 2000)) as a template and primers of SEQ ID NO: 2 and SEQ ID NO: 3, and using SacI / EcoRI cloned the araC-PBAD promoter into the pWA pl...

Embodiment 2

[0167] Example 2: Screening of mRNA positions effective for the function of synthetic sRNA

[0168] In order to determine the binding positions of mRNAs effective for the stabilizing function of synthetic sRNAs, synthetic rRNAs were constructed using the pWAS plasmid as a template to bind to various sites of DsRed2.

[0169] In the same manner as described in Example 1-3, the region introduced into each synthetic sRNA and the region into base pairing with DsRed2 mRNA, which was introduced including the Hfq binding site of MicC, were amplified by PCR using the following primers in the pWAS plasmid.

[0170] exist Figure 4 Among them, sequence A utilizes the primer of SEQ ID NO:27 and SEQ ID NO:28 to obtain; Sequence B1 utilizes the primer of SEQ ID NO:33 and SEQ ID NO:34 to obtain; Sequence B2 utilizes the primer of SEQ ID NO: 35 and the primers of SEQ ID NO:36 are obtained; Sequence C1 is obtained by utilizing the primers of SEQ ID NO:37 and SEQ ID NO:38; Sequence C2 is o...

Embodiment 3

[0198] Example 3: Construction and cross-reactivity determination of sRNA for other target mRNAs

[0199] In order to test whether the sRNA according to the present invention can inhibit the expression of mRNAs of genes other than DsRed2, a sequence complementary to the RBS of each mRNA of the LuxR, AraC and KanR genes was inserted into the MicC structure by site-directed mutagenesis in order to bind to the RBS . exist Figure 6 In , the sequence complementary to the RBS of each target gene is underlined and shown in red.

[0200] Synthetic sRNA anti-LuxR was constructed by site-directed mutagenesis using the pWAS plasmid (containing the MicC-based construct) as a template and primers of SEQ ID NO:57 and SEQ ID NO:58, anti-AraC was constructed using SEQ ID NO :59 and the primers of SEQ ID NO:60 were constructed, and anti-KanR was constructed using the primers of SEQ ID NO:61 and SEQ ID NO:62. In order to detect the expression level of each gene, the fusion protein of each...

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Abstract

The present invention relates to a customized synthesized sRNA for reducing gene expression in a prokaryotic cell having a new structure, to a method for preparing the synthesized sRNA and to a use of the synthesized sRNA. More particularly, the present invention relates to a synthesized sRNA which contains an Hfg binding site derived from any one sRNA of MicC, SgrS and MicF, and a zone for forming complementary binding with target gene mRNA. The present invention also relates to a method for preparing the synthesized sRNA and to a use of the synthesized sRNA.; The synthesized sRNA according to the present invention has the advantages of regulating the degree of inhibition by regulating the binding force to mRNA of the target gene, and can be effectively produced without deleting an existing gene through synthesized sRNA for regulating gene expression and may reduce an expression of the target gene, thus enabling the synthesized sRNA of the present invention to be valuably used in the production of recombinant microorganisms. The synthesized sRNA of the present invention may be quickly applied to various strains, and therefore, is very suitable for measuring the metabolizability of each strain and selecting an optimum strain.; In addition, recombinant microorganisms for producing tyrosine or cadaverine at a high efficiency according to the present invention, obtained by regulating metabolic flux of microorganisms by means of synthesized sRNA can be valuably used as microorganisms for drugs and industrial solvents. That is, selecting an expression inhibitory target gene for high efficiency production of metabolites can be easily performed using the sRNA of the present invention. Thus, the sRNA of the present invention can be used in producing recombinant strains for the efficient production of various metabolites and in establishing methods for producing efficient production, and thus is very useful.

Description

technical field [0001] The present invention relates to a novel custom-made sRNA that reduces gene expression in prokaryotes, its preparation method and its application, and in particular to a synthetic sRNA and its preparation method and application. The synthetic sRNA includes MicC, SgrS and The Hfq binding site of any of the sRNAs in MicF and the region that base-pairs with the mRNA of the target gene. Background technique [0002] In recent years, there has been increasing global concern about environmental issues and the depletion of limited resources. As an alternative method to solve such problems, the construction of production systems based on environmentally friendly and renewable organisms has attracted increasing interest. These systems can be constructed by regulating the metabolic pathways of organisms and optimizing the metabolic flux of desired metabolites, and various molecular biology techniques are required in the process of regulating metabolic pathways....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/63C12N1/21
CPCC12P7/22C12P13/225C12N15/67C12N15/111C12N2310/13C12N2310/18C12N2310/3519C12N2310/11C12P13/001C12N15/113C12N1/20C12N15/63C12N15/1138C12N15/70C12N2310/531
Inventor 李相烨罗度钧柳承珉
Owner KOREA ADVANCED INST OF SCI & TECH
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