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CRISPR-Cas9 dual vector system suitable for genetic modification of serratia

A Serratia and genetic modification technology, applied in the field of genetic engineering, can solve the problem of unreported gene editing

Active Publication Date: 2020-07-07
SHAANXI NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, only Serratia sp.ATCC 39006 has been reported to contain type I CRISPR-Cas system, while gene editing using CRISPR-Cas9 system has not been reported in other Serratia sp.

Method used

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  • CRISPR-Cas9 dual vector system suitable for genetic modification of serratia
  • CRISPR-Cas9 dual vector system suitable for genetic modification of serratia
  • CRISPR-Cas9 dual vector system suitable for genetic modification of serratia

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1: Construction of vector pCAS-p15A-sacBΔλ-Red

[0066] 1. Test method:

[0067] 1.1 Obtaining of fragments pCAS, p15A and sacB:

[0068] Using the plasmid pCAS as a template, primers pCAS-overlap-F, pCAS-overlap-R, and TaKaRa high-fidelity enzyme PrimeSTAR Max DNA Polymerase R045A were used to amplify the pCAS fragment by PCR, which was designated as pCAS. The PCR reaction system (50 μL) of pCAS fragments includes: 2×Prime STAR Max Premix 25 μL, Template(pCAS) (100ng / uL) 1 μL, pCAS-overlap-F (10 μM) 1 μL, pCAS-overlap-R (10 μM) 1 μL ,ddH 2 O 22 μL. The PCR reaction program of the pCAS fragment is: 98°C for 10s, 55°C for 15s, 72°C for 60s, a total of 35 cycles.

[0069] Using the plasmid pSTV29 as a template, the p15A fragment was amplified by PCR using primers p15A-overlap-F and p15A-overlap-R, which was designated as p15A. The p15A fragment PCR reaction system (50 μL) includes: 2×Prime STAR Max Premix 25 μL, Template (pSTV29) (100ng / uL) 1 μL, p15A-overlap...

Embodiment 2

[0101] Embodiment 2: Construction of vector pTarget F-Gm

[0102] 1. Test method:

[0103] 1.1 Obtaining fragment Gm:

[0104] Using the plasmid pBBR1MCS-5 as a template, the Gm fragment was amplified by PCR using primers Gm-F and Gm-F, which was denoted as Gm. PCR reaction system (50 μL) includes: 2×Prime STAR Max Premix 25 μL, Template (pBBR1MCS-5) (100ng / uL) 1 μL, Gm-F (10 μM) 1 μL, Gm-F (10 μM) 1 μL, ddH 2 O 22 μL. The PCR reaction program was: 98°C for 10s, 55°C for 15s, 72°C for 10s, a total of 35 cycles.

[0105] 1.2 Gel recovery of fragment Gm: the method steps are the same as 1.2 in Example 1.

[0106] 1.3 Digestion and purification of fragment Gm:

[0107] According to the concentration of the recovered fragment Gm, the fragment was double-digested with restriction endonucleases Mlu I and Xho I. After the digestion, the PCR product was purified strictly according to the operation steps of the Tiangen DNA Purification and Recovery Kit. The purified DNA was quant...

Embodiment 3

[0119] Example 3: Investigation of transformation of Serratia YD25 by vector pCAS-p15A-sacBΔλ-Red

[0120] 1. Test method:

[0121] 1.1 Preparation of Serratia YD25 Competent Cells:

[0122] Take the Serratia YD25 glycerol bacteria stored in the -80°C refrigerator and put them on ice to thaw, dip a small amount of bacterial liquid and streak on the LB solid plate, and place it in a 30°C constant temperature incubator for upside-down cultivation. After 16 hours, a single clone was picked and inoculated in 3 mL of LB liquid medium, and placed in a constant temperature shaker at 30°C at 220 rpm for overnight culture.

[0123] The next day, 1% transfer, take 1mL of the cultured YD25 bacteria solution and add it to the Erlenmeyer flask containing 100mL LB liquid medium, 30°C, 220rpm, shaking culture to OD 6000.4-0.6. Transfer the bacterial solution to two 50mL centrifuge tubes, place on ice for 5min, and then centrifuge in a refrigerated centrifuge at 4°C, 2000g, 15min, 4°C. Di...

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Abstract

The present invention discloses a CRISPR-Cas9 dual vector system suitable for genetic modification of serratia. The CRISPR-Cas9 dual vector system comprises a vector pCAS-p15A-sacB delta lambda-Red and a vector pTarget F-Gm. The CRISPR-Cas9 dual vector system makes up for limitations of a serratia gene editing system and provides a platform for verification of serratia gene functions and a research on a mechanism of production of secondary metabolites of the serratia strain. The constructed CRISPR-Cas9 dual vector system is used to conduct gene luxS knock out and replenishment experiments anda preliminary study is made on a co-regulatory mechanism of serratia YD25 secondary metabolite prodigiosin and Serrawettin W2. A positive regulation of a gene luxS on synthesis of the prodigiosin andSerrawettin W2 is determined.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a CRISPR-Cas9 dual vector system suitable for genetic modification of Serratia. Background technique [0002] Serratia is an opportunistic pathogen with a very wide range of hosts, which can secrete a series of virulence factors to damage human cells and tissues. Swimming ability and hemolytic performance of bacteria are the main virulence factors of pathogenic bacteria. The surfactants in the secondary metabolites of Serratia can reduce the surface tension, thereby promoting the swimming of the bacteria; the surfactants Serrawettin W1 and Serrawettin W2 produced by it can act as hemolysins to lyse the cells. Therefore, the biosynthesis of surfactants in Serratia can directly affect the pathogenicity of the pathogen. At the same time, the surfactants produced by Serratia have a new mechanism of action and better biological activity. The screening and transformation ...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12N15/65C12N15/66C12N15/90
CPCC12N15/74C12N15/65C12N15/66C12N15/902
Inventor 苏春焦乙雪赵武帅江鸿标温浩宇郑汪东
Owner SHAANXI NORMAL UNIV
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