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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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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 Image 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 novel custom-made synthetic sRNA that reduces gene expression in prokaryotes, its preparation method and application thereof, and particularly relates to a synthetic sRNA that includes the combination of Hfq derived from any sRNA in MicC, SgrS and MicF The site and the base paired region with the target gene mRNA also relate to the preparation method and application of the synthetic sRNA. The advantage of the synthetic sRNA of the present invention is that the degree of inhibition of the target gene can be controlled by regulating the binding ability of the synthetic sRNA to the mRNA of the target gene. Utilizing the synthetic sRNA that regulates the expression of the target gene can effectively construct the recombinant microorganism without the traditional gene deletion method and can reduce the expression of the target gene, so the synthetic sRNA is very useful for the production of the recombinant microorganism. In addition, synthetic sRNA can be quickly applied to various strains, making it ideal for determining the metabolic capacity of strains and selecting the best strains. In addition, recombinant microorganisms that efficiently produce tyrosine or cadaverine obtained by manipulating metabolic flux using synthetic sRNA are very useful in the fields of medicine and industry. In other words, the use of the sRNA of the present invention makes it easy to select a target gene whose expression will be suppressed for efficient production of metabolites. Therefore, synthetic sRNA can be used to construct recombinant strains that efficiently produce various metabolites and to establish efficient methods for producing various metabolites, 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....
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