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Double-plasmid system and application thereof

A plasmid and gene technology, applied in the field of dual-plasmid system for genome editing of Acinetobacter baumannii, can solve the genome editing problems of Acinetobacter baumannii

Active Publication Date: 2019-11-08
SHANGHAI TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current CRISPR-Cas9 system still has great limitations and cannot be directly used for genome editing of Acinetobacter baumannii. Further exploration and development are needed to achieve high-efficiency genome editing of Acinetobacter baumannii

Method used

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  • Double-plasmid system and application thereof
  • Double-plasmid system and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: pCasAb plasmid construction

[0046] The composition of the pCasAb plasmid is attached figure 1 As shown in A, its sequence is SEQ ID NO: 1, and its specific construction method is as follows:

[0047] (1) The abramycin resistance gene fragment of aprR was amplified from the pCasKP-apr plasmid by polymerase chain reaction (PCR). The primer sequences used for PCR amplification are:

[0048] aprR-F: 5'-GCGTCAATTCACGGATCCGGTTCATGTGCAGCTCCATCAGC-3' (SEQ ID NO: 3)

[0049] aprR-R: 5'-AAACTTGGTCTGACAGTCAGCCAATCGACTGGCGA-3' (SEQ ID NO: 4)

[0050] Use Takara's PrimerSTAR HS DNA Polymerase to amplify the above DNA fragments, the reaction system is: 32 μL ddH 2 O, 4 μL dNTP Mixture (2.5mM each), 10 μL 5×Primestar Buffer, 1.5 μL aprR-F (10 μM), 1.5 μL aprR-R (10 μM), 0.5 μL pCasKP-apr plasmid template (1ng / μL), 0.5 μL PrimerSTAR HS DNA Polymerase.

[0051] After the system configuration is completed, carry out PCR amplification. Amplification parameters: pre-dena...

Embodiment 2

[0075] Embodiment two: pSGAb plasmid construction

[0076] The composition of the pSGAb plasmid is attached figure 1 As shown in B, its sequence is SEQ ID NO: 2, and its specific construction method is as follows:

[0077] (1) The WH1266 plasmid replicon DNA fragment was amplified from the pWH1266 plasmid by polymerase chain reaction (PCR). The primer sequences used for PCR amplification are:

[0078] WH1266-ori-F: 5'-TGAGAGTGCACCATAGGATTTTAACATTTTGCGTTGTTC-3' (SEQ ID NO: 9)

[0079] WH1266-ori-R: 5'-ATTTCACACCGCATAGCCAAGATCGTAGAAATATCTATGA-3' (SEQ ID NO: 10)

[0080] Use Takara's PrimerSTAR HS DNA Polymerase to amplify the above DNA fragments, the PCR reaction system is: 32 μL ddH 2 O, 4μL dNTP Mixture (2.5mM each), 10μL 5×Primestar Buffer, 1.5μL WH1266-ori-F (10μM), 1.5μL WH1266-ori-R (10μM), 0.5μL pWH1266 plasmid template (1ng / μL), 0.5 μL PrimerSTAR HS DNA Polymerase.

[0081] After the system configuration was completed, PCR amplification was carried out. The amplifi...

Embodiment 3

[0090] Example three: inserting the spacer fragment into the pSGAb plasmid

[0091] In order to enable the sgRNA and Cas9 nuclease complex to locate at a specific target site of Acinetobacter baumannii genomic DNA for cutting, the spacer sequence of the target site needs to be inserted into the sgRNA expression gene, and the insertion method is as follows:

[0092] (1) First, select the 20 base sequences before a certain NGG (N stands for any base of A / T / G / C) sequence on the target gene (these 20 base sequences are called spacers, and NGG is not included in Among them, the GC ratio of the spacer sequence is controlled at 20-80%). The feature of this step is that in order to insert the spacer fragment into the pSGAb plasmid, it is necessary to add a tagt adapter at the 5' end of the sense strand of the spacer sequence, and at the same time add an aaac adapter at the 5' end of the antisense strand of the spacer sequence. The specific primer design template as follows:

[0093]...

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Abstract

The invention provides a double-plasmid system. The double-plasmid system is characterized in that the system comprises first plasmid and second plasmid, the first plasmid is named pCasAb with a sequence being SEQ ID NO:1, and the second plasmid is named pSGAb with a sequence being SEQ ID NO:2. Genome DNA of an acinetobacter baumanii strain can be efficiently, rapidly and tracelessly edited, including gene insertion, gene knockout and single base mutation. The technology has broad application prospects in the aspects such as gene function research, resistance mechanism analysis, new drug target screening and new treatment method development of acinetobacter baumanii.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a dual-plasmid system for genome editing of Acinetobacter baumannii and its application. Background technique [0002] Acinetobacter baumannii is a highly harmful Gram-negative human pathogenic bacterium that can cause a series of serious hospital-acquired infections such as sepsis, pneumonia, endocarditis and secondary meningitis. Disease, and it is very easy to spread among patients and medical staff. The strong pathogenicity and transmission ability of Acinetobacter baumannii are mainly attributed to two aspects. On the one hand, Acinetobacter baumannii has the ability to tolerate drying and various disinfectants, so that it can survive on the surface of clinical medical materials for a long time, and it is difficult to be completely killed; on the other hand, Acinetobacter baumannii can absorb foreign substances. The source of drug-resistant genes acquires resistance to a varie...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N15/70C12N1/21C12R1/01C12R1/19
CPCC12N15/74C12N15/70C12N15/113C12N9/22C12N2310/10
Inventor 季泉江王宇
Owner SHANGHAI TECH UNIV
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