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High-throughput screening tool for enabling escherichia coli to obtain effective NHEJ system and application of high-throughput screening tool

An Escherichia coli, high-throughput technology, applied in the field of gene editing, to achieve the effect of short time-consuming and high screening efficiency

Pending Publication Date: 2022-03-11
GENEWIZ INC SZ
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although Tianyuan Su, Xuan Zheng et al. successfully introduced the NHEJ system into E. coli by expressing Ku and ligD proteins from other microorganisms in E. coli, there is still no efficient method for screening the effective NHEJ system for E. coli

Method used

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  • High-throughput screening tool for enabling escherichia coli to obtain effective NHEJ system and application of high-throughput screening tool
  • High-throughput screening tool for enabling escherichia coli to obtain effective NHEJ system and application of high-throughput screening tool
  • High-throughput screening tool for enabling escherichia coli to obtain effective NHEJ system and application of high-throughput screening tool

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] Example 1 Construction of a high-throughput screening tool that enables Escherichia coli to obtain an effective NHEJ system

[0092] This embodiment provides a high-throughput screening tool for obtaining an effective NHEJ system for Escherichia coli, which includes:

[0093] pDual-Cas9-Parental plasmid vector: Rep101 gene, pSC101 replicon, kanamycin resistance gene, Cas9 gene, araC gene, 2 arabinose promoters, BsaI restriction enzyme recognition for cloning Ku gene connected in sequence site and the BbsI restriction enzyme recognition site for cloning the ligD gene;

[0094] And pDual-sgRNA-lacZ plasmid vector: the sgRNA sequence targeting the lacZ gene, the constitutively expressed strong promoter J23119 promoter, the replicon and the ampicillin resistance gene are connected in sequence.

[0095] The high-throughput screening tool for obtaining an effective NHEJ system for Escherichia coli is constructed by the following method:

[0096] (1) Gene synthesis of pDual-...

Embodiment 2

[0108] Example 2 Construction of Ku+ligD plasmid library using pDual-Cas9-Parental as the backbone vector

[0109] In this example, pDual-Cas9-Parental is used as the backbone vector to construct a Ku+ligD plasmid library, including the following steps:

[0110] (1) Obtained five CDS coding sequences of Ku protein and ligD protein derived from microorganisms from NCBI, GenBank accession numbers are WP_010886496 (Bsu-Ku), GAS86454 (Mbr-Ku), YP_889815 (Msm-Ku), NP_215452 ( Mtb-Ku), ACV76561 (Nmu-Ku), NP_389223 (Bsu-ligD), ATD76462 (Bve-ligD), ALI25184 (Mfo-ligD), WP_011730625 (Msm-ligD) and NP_215453 (Mtb-ligD).

[0111] (2) Codon optimization was carried out on the CDS coding sequences of Ku protein and ligD protein for the E. coli host. The CDS coding sequences of the optimized Ku protein did not contain BbsI and BsaI recognition sequences, and the 5' end of the sequence was added with SEQ ID NO.22 The nucleotide sequence shown in the 3' end is added with the nucleotide seque...

Embodiment 3

[0165] Example 3 Screening of an effective NHEJ system in Escherichia coli from the Ku+ligD plasmid library

[0166] This embodiment screens the effective NHEJ system in Escherichia coli, including the following steps:

[0167] (1) The Ku+ligD plasmid library constructed in Example 2 was electrotransformed into MG1655 Escherichia coli competent cells, coated with kanamycin-resistant LB plates, and cultured overnight at 30°C.

[0168] (2) Scrape all the clones on the plate and inoculate the bacterial liquid into LB medium, cultivate it at 30°C and 220rpm, when the bacterial liquid OD 600 When the value reached 0.6, the competent cells were prepared according to the standard electroporation competent cell preparation method.

[0169] (3) Electrotransfer the pDual-sgRNA-lacZ plasmid to the electrotransfer competent cells prepared in step (2), spread the LB plate containing IPTG, X-gal, kanamycin and ampicillin, and culture overnight at 30°C.

[0170] (4) All the plates are whit...

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Abstract

The invention provides a high-throughput screening tool for enabling escherichia coli to obtain an effective NHEJ system and an application of the high-throughput screening tool. The high-throughput screening tool comprises a pDual-Cas9-Parental plasmid vector, a plasmid vector and a screening vector, wherein the pDual-Cas9-Parental plasmid vector contains a DNA helicase gene, a replicon, an antibiotic resistance gene, a nuclease gene, an araC gene, an arabinose promoter and an IIs type restriction enzyme recognition site; and the pDual-sgRNA-lacZ plasmid vector contains an sgRNA sequence of a targeted lacZ gene, a strong promoter for constitutive expression, a replicon and an antibiotic resistance gene. The high-throughput screening tool can perform rapid and high-throughput screening on effective Ku + ligD combinations in escherichia coli, multiple Ku + ligD combination results can be obtained through one screening experiment, the time is short, the screening efficiency is high, and conditions are created for gene editing research of escherichia coli.

Description

technical field [0001] The invention belongs to the technical field of gene editing, and in particular relates to a high-throughput screening tool for obtaining an effective NHEJ system for Escherichia coli and its application. Background technique [0002] Until the early 21st century, biologists still generally believed that the immune system of prokaryotes consisted only of the innate immune system, including a restriction-modification system and a variety of exonucleases, without an adaptive immune system. Therefore, the discovery of the adaptive immune system of prokaryotes is a huge milestone in the history of biological development. Prokaryotes are single-celled organisms, so the adaptive immune system of prokaryotes must store all immune information in a single cell. In contrast, each lymphocyte of the adaptive immune system of jawed vertebrates stores only An immunization message. [0003] CRISPR / Cas is an adaptive immune defense system of prokaryotes, which recor...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/65C12N15/113C12N15/55C12N15/52C12N15/31C12N15/66C12N15/90
CPCC12N15/70C12N15/65C12N15/113C12N9/22C12N9/93C12N15/66C12N15/902C07K14/245C12N2800/22C12N2800/101C12N2310/20
Inventor 薛高旭夏立军方其张艳
Owner GENEWIZ INC SZ
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