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Plasmid pMMB1 for enterobacter gene editing as well as construction method and application of plasmid pMMB1

A construction method and plasmid technology, applied in the biological field, can solve the problem of inability to host cell gene editing

Pending Publication Date: 2021-02-12
XUZHOU NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since most genetically engineered plasmids are derived from Enterobacteriaceae Escherichia coli, their origin of replication can be identified in a variety of Enterobacteriaceae biological cells, making the plasmids do not need to be integrated into the Enterobacteriaceae host cell chromosome. Replicate, making it impossible to gene edit the host cell

Method used

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  • Plasmid pMMB1 for enterobacter gene editing as well as construction method and application of plasmid pMMB1
  • Plasmid pMMB1 for enterobacter gene editing as well as construction method and application of plasmid pMMB1
  • Plasmid pMMB1 for enterobacter gene editing as well as construction method and application of plasmid pMMB1

Examples

Experimental program
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Effect test

Embodiment 1

[0041] Example 1: Construction of the plasmid pMMB1 used for gene editing of Enterobacteriaceae according to the present invention

[0042] Using the pKNG101 plasmid as a template, the primer pair R6KF / R6KR was used for PCR amplification to obtain the amplified product λpir protein-dependent replicon oriR6K. The amplified product is recovered with a universal nucleic acid recovery kit.

[0043] Wherein, the amplification primers R6KF and R6KR in the PCR amplification system are respectively:

[0044] R6KF: 5-CCGCTCGAGTCTGAAGATCAGCAGTTCAACCTG-3

[0045] R6KR: 5-CCGCTCGAGAGCTTTGTCATCACCAAATTTCCTT-3

[0046]The PCR amplification system is 25 μL, including:

[0047] 10×DNA polymerase buffer 2.5μL; magnesium ion 1.5mmol / L; 0.2mmol / L dATP; 0.2mmol / L dTTP; 0.2mmol / L dCTP; R6KR 0.3μmol / L; template DNA 0.1μg; KOD high-fidelity DNA polymerase 0.5U; supplement the remaining volume with sterilized double distilled water.

[0048] The reaction conditions for PCR amplification are: ...

Embodiment 2

[0061] Embodiment 2: Utilize the method for constructing a gene knockout plasmid using the pMMB1 plasmid of the present invention

[0062] This example introduces a method for using the pMMB1 plasmid in Example 1 to construct a gene knockout vector pMMB1-DpigA for knocking out the pigA gene (Ser39006_020695) in Serratia sp.ATCC 39006. The method includes:

[0063] Use restriction endonuclease PstI and restriction endonuclease BamHI to carry out enzyme digestion to described pMMB1 plasmid, and use general type nucleic acid purification and recovery kit to reclaim the pMMB1 plasmid after digestion;

[0064] Using the Serratia sp.ATCC 39006 genomic DNA as a template, PCR amplifies the upstream homology arm and downstream homology arm of the pigA gene to obtain fragments of the upstream homology arm and downstream homology arm, using general-purpose nucleic acid purification and recovery reagents Cassette purification and recovery of the upstream homology arm and downstream hom...

Embodiment 3

[0090] Embodiment 3: The method that utilizes pMMB1-DpigA plasmid to carry out gene knockout

[0091] This example introduces a method for knocking out the pigA gene in Serratia sp.ATCC 39006 using the pMMB1-DpigA plasmid in Example 2. The pigA gene encodes the key enzyme in the process of prodigiosin synthesis, so Serratia sp.ATCC 39006 no longer produces prodigiosin after knocking out the gene. The method includes:

[0092] The Serratia sp.ATCC 39006 strain was inoculated in LB liquid medium and cultured overnight in a constant temperature shaker at 30°C and 200rpm;

[0093] Escherichia coli S17-1 (λpir) containing the above pMMB1-DpigA plasmid was inoculated in LB liquid medium containing 50 μg / mL kanamycin. Cultivate the above-mentioned liquid culture medium with strains in a constant temperature shaker at 37°C and 220rpm overnight;

[0094] Centrifuge the above-mentioned Escherichia coli S17-1 (λpir) culture solution containing the pMMB1-DpigA plasmid in a high-speed...

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Abstract

The invention provides a plasmid pMMB1. The nucleotide sequence of the plasmid pMMB1 is as shown in SEQ ID NO. 1. The invention also provides a construction method of the plasmid pMMB1. The inventionalso provides an application of the plasmid pMMB1 in gene editing. The plasmid provided by the invention has a conjugational transfer site oriT (RP4), and can be transferred into a receptor strain through conjugational transfer, transformation and the like; according to the present invention, the plasmid has multiple cloning sites (MCS), such that the exogenous fragment can be conveniently and rapidly inserted; meanwhile, the plasmid can be transferred into an enterobacteriaceae receptor strain in various modes and is used for carrying out gene editing on enterobacteriaceae bacteria.

Description

technical field [0001] The invention relates to a plasmid pMMB1 for gene editing of Enterobacteriaceae and its construction method and application, belonging to the field of biotechnology. Background technique [0002] In genetic engineering, plasmids are commonly used gene carriers, which are used to carry foreign genes and transfer them into host cells to play a role. Most common genetic engineering plasmids are derived from Escherichia coli. In order to realize the gene editing of host cells, it is necessary to provide screening conditions in the outside world so that the plasmid can be integrated into a specific site on the host chromosome. This requires that the plasmid has the following characteristics: 1. It has a suitable selection marker, which is convenient for screening to obtain recombinant transformants transferred into the plasmid; 2. The plasmid cannot replicate in the host cell, so that external selection pressure forces the plasmid to integrate into the hos...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N1/21C12N15/64C12R1/425
CPCC12N15/74C12N15/64
Inventor 孙地刘聪刘伟杰刘佳文朱静榕蒋虹
Owner XUZHOU NORMAL UNIVERSITY