Method for knocking out cytidine deaminase (cdd) gene in escherichia coli by utilizing CRISPR-Cas9 technology and application

A technology of cytidine deaminase and Escherichia coli, which is applied in the direction of microorganism-based methods, other methods of inserting foreign genetic materials, applications, etc., can solve adverse effects on human health and industrial production, affect efficient utilization of substrates, and separation operations Complicated problems, to achieve the effect of improving knockout efficiency, low cost, and high recognition efficiency

Inactive Publication Date: 2020-06-23
NANJING UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although the first method can obtain four products at one time, except for cytidylic acid, the other three become by-products, and the source of RNA is more restricted, and the separation operation in the production process is complicated and the yield is low; At present, chemical methods are mainly used to synthesize 5'-cytidylic acid in industry. The main disadvantage of this method is that there are many wastes, which corrode equipment and are not conducive to the health of personnel and industrial production.
From the perspective of the demand of the 5'-cytidine acid industry, although the output has increased steadily, the production of its by-products has affected the efficient utilization of substrates

Method used

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  • Method for knocking out cytidine deaminase (cdd) gene in escherichia coli by utilizing CRISPR-Cas9 technology and application
  • Method for knocking out cytidine deaminase (cdd) gene in escherichia coli by utilizing CRISPR-Cas9 technology and application
  • Method for knocking out cytidine deaminase (cdd) gene in escherichia coli by utilizing CRISPR-Cas9 technology and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Making Escherichia coli Competent Containing the Cas9 Plasmid Target Strain and Containing the cdd Gene

[0035] 1. The plasmid pCas9 was electrotransformed into Escherichia coli BL21(DE3) competent, and the target strain was screened out by kana resistance

[0036] Add 2-5 μg of plasmid pCas9 stored in the laboratory to 20-30 μL of Escherichia coli BL21(DE3) competent in the laboratory, pre-cool on ice for 2-5 minutes; add the pre-cooled electroporation cup, the conditions are: 200Ω, 2.0 kv electric shock; after electric shock, add 800-1000 μL LB medium immediately (the formula of LB medium is: peptone 10g / L, yeast powder 5g / L, sodium chloride 5g / L), 30℃, 2h culture; take 100μL medium The bacterial solution was coated with LB-resistant plate (kanamycin) and cultivated overnight at 30°C; single colony was picked, colony PCR was used to verify whether E. coli BL21(DE3) contained pCas9 plasmid, and the successfully constructed strain was named BL21(DE3)- cas9, ...

Embodiment 2

[0040] Example 2 Design and synthesis of mutant sgRNA

[0041] 1. Selection of N20 target site of cdd cytidine deaminase gene

[0042] The 399th position of the cdd PAM region of the cytidine deaminase gene was selected as the N20 target site by using the chop chop website, and the nucleotide sequence of the N20 target site is shown in SEQ ID NO.1.

[0043] 2. Design and synthesize mutant sgRNA

[0044] Use the snap gene software to open the sgRNA plasmid map purchased in the laboratory, replace the original N20 sequence with the selected N20 sequence, and design two primer sequences cdd-F1 (SEQ ID NO.2) and cdd-R1 (SEQ ID NO.3) Send it to Qingke Company for synthesis; use the sgRNA plasmid as a template and primers cdd-F1 and cdd-R1 for PCR amplification.

[0045] The PCR system is: primer cdd-F1 (10 μM) 2 μL, cdd-R1 (10 μM) 2 μL, 5×Buffer 10 μL, dNTPs (2.5mM) 5 μL, Fastpfu polymerase: 1 μL, sgRNA plasmid (100ng / μL) 2 μL, ddH 2 O to make up to 50 μL.

[0046] The reaction...

Embodiment 3

[0048] Example 3 Construction of cytidine deaminase gene cdd Donor DNA fragment

[0049] 1. Construction of upper and lower homology arms

[0050] Use the NCBI website to find out the specific base sequence of the cytidine deaminase gene cdd, and import it using the snap gene software. Select the first 314bp of the gene as the upper homology arm, and the last 394bp as the lower homology arm, and design primers cdd-f-f (SEQ ID NO .5), cdd-f-r (SEQ ID NO .6), cdd-r-f (SEQ ID NO . NO.7), cdd-r-r (SEQ ID NO.8) were sent to Qingke Company for synthesis, using the Escherichia coli K12 series genome as a template, primers cdd-f-f and cdd-f-r, cdd-r-f and cdd-r-r were amplified by PCR respectively Add upper and lower homology arms M1, M2.

[0051] The PCR system is primer cdd-f-f / cdd-r-f (10μM) 2μL, cdd-f-r / cdd-r-r (10μM) 2μL, 5×Buffer 10μL, dNTPs (2.5mM) 5μL, Fastpfu polymerase: 1μL, Escherichia coli K12 series Genome (100ng / μL) 2 μL, ddH 2 O to make up to 50 μL.

[0052] The re...

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Abstract

The invention discloses a method for knocking out a cytidine deaminase (cdd) gene in escherichia coli by utilizing a CRISPR-Cas9 technology and application. The method comprises the following steps: 1, making escherichia coli BL21 (DE3) competence containing a Cas9 plasmid; 2, designing and synthesizing a mutant sgRNA; 3, constructing Donor DNA; 4, transforming the sgRNA plasmid and the Donor DNAinto the escherichia coli competence carrying the Cas9 plasmid by employing an electro transformation method, and knocking out the cdd gene; and 5, introducing a target plasmid pET28a-UCK into a knock-out strain to produce cytidylic acid. The method has the advantages that operation is simple, the knockout success rate of the cdd gene is high, and the conversion rate of the knock-out strain on a substrate is improved, and is suitable for industrial production of the cytidine acid and the like. Compared with producing the cytidylic acid without strain knockout, producing the cytidylic acid withstrain knockout improves the conversion rate of the substrate cytidine and adenosine triphosphate (ATP) by 15%, and the conversion rate reaches 99%.

Description

technical field [0001] The invention belongs to the technical field of microbial genetic engineering, and specifically relates to a method and application for knocking out the cytidine deaminase gene cdd in Escherichia coli by using CRISPR-Cas9 technology. Background technique [0002] CRISPR-Cas system is a kind of immune defense system of bacteria, which is formed by the evolution of bacteria in the process of resisting foreign DNA for a long time. It can degrade invading foreign DNA (viruses or phages, etc.), and widely exists in bacteria and archaea. . CRISPR-Cas systems are divided into three types: type I, type II, and type III, and their core proteins are Cas3, Cas9, and Cas10, respectively. Compared with type I and type III systems that require multiple Cas proteins to participate in the reaction and form a relatively complex complex, type II system is the most thoroughly studied, and only one Cas9 protein can cut the DNA of the target gene. The type II CRISPR-Cas9...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/90C12N15/70C12N15/55C12R1/19
CPCC12N9/78C12N15/70C12N15/902C12Y305/04005
Inventor 王昕王静陈可泉马琛秦航欧阳平凯
Owner NANJING UNIV OF TECH
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