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MccY recombinant integrated engineering bacterium as well as construction method and application thereof

A construction method, a technology for engineering bacteria, applied in applications, genetic engineering, chemical instruments and methods, etc., and can solve problems such as staying

Active Publication Date: 2022-06-17
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the current research on MccY is still at the level of plasmid expression. How to realize microecological preparations, how to safely use and efficiently and continuously scale up the production of MccY still needs further exploration.

Method used

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  • MccY recombinant integrated engineering bacterium as well as construction method and application thereof
  • MccY recombinant integrated engineering bacterium as well as construction method and application thereof
  • MccY recombinant integrated engineering bacterium as well as construction method and application thereof

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Embodiment 1

[0051] 1. Construction of MccY recombinant integration engineered bacteria

[0052] Using Escherichia coli MG1655 (strain number ATCC700926) as the basic strain, using CRISPR-Cas9 gene editing technology, the recombinant integration of MccY was carried out at the hsds of the genome to construct MccY recombinant integration engineering bacteria.

[0053] 1.1 Plasmid construction

[0054] Referring to the full gene sequence of MccY in the NCBI gene bank (NCBI serial number: AAFXLL010000013.1), the expression direction of mcyA was adjusted to be consistent with the expression direction of mcyBCD, and the promoter gene was inserted in front of it. Its sequence is shown in SEQ ID NO.3, mcyA The 50bp~211bp of SEQ ID NO.3 and the 227bp~4163bp of mcyBCD of SEQ ID NO.3 were entrusted to General Bio (Anhui) Co., Ltd. to integrate the MccY sequence into the pet28a(+) vector. The plasmid pet28a-MccY with MccY expression activity was constructed.

[0055] 1.2 Amplification of repair frag...

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Abstract

The invention discloses an MccY recombinant integrated engineering bacterium as well as a construction method and application thereof. The construction method of the MccY recombinant integrated engineering bacterium comprises the following steps: (1) connecting an sgRNA fragment (SEQ ID NO.2) with a T vector to obtain a pHsgRNA plasmid; (2) transforming the pCas9 plasmid into escherichia coli, and preparing competent cells; and then transferring the optimized repair fragment (SEQ ID NO.1) of the MccY and the pH sgRNA plasmid into competent cells, identifying, culturing, and carrying out continuous passage screening to eliminate a strain of double plasmids, so as to obtain the MccY recombinant integrated engineering bacterium. The constructed MccY recombinant integrated engineering bacterium does not have plasmids and resistance genes, does not need an inducer, can continuously and efficiently secrete and express MccY, and has an obvious inhibition effect on salmonella typhimurium, escherichia coli, shigella sonnei, shigella dysenteriae and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, and particularly relates to a MccY recombination and integration engineering bacterium and a construction method and application thereof. Background technique [0002] In recent years, the overuse of antibiotics has caused many problems, and the phenomenon of antibiotic resistance and multi-drug resistance has attracted more and more attention. Among the proposed alternatives, natural antimicrobials have great potential as effective materials for next-generation drugs. [0003] Microcin (Mcc) is a low molecular weight, ribosome-produced, highly stable bacterial inhibitory molecule. It has significant advantages such as certain resistance to proteases. About 15 microcins have been identified, which are divided into two categories. Class I microcins are less than 5 kDa and are encoded by plasmids, and class II microcins are about 5-10 kDa, which can be divided into plasmid encoding and chromosome enc...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N9/22C12N15/113C12N1/21C07K14/255C12N15/31A61K38/16A61K35/74A61P31/04A23K20/147C12R1/19
CPCC12N15/70C12N9/22C12N15/113C12N1/20C07K14/255A61K38/164A61K35/74A61P31/04A23K20/147C12N2310/20Y02A50/30
Inventor 代绘琳秦苗苗曾知为江金飞谢倩梅冯赛祥许斯祺张广文陈爱华
Owner SOUTH CHINA AGRI UNIV
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