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Gene editing method based on streptomyces virginiae IBL14 gene cas7-5-3

A technology of gene editing and Streptomyces, applied in biochemical equipment and methods, viruses/bacteriophages, using vectors to introduce foreign genetic materials, etc., can solve problems that have not been discovered, and have not seen CRISPR-CasI type systems, etc., to achieve effective gene The effect of editing

Pending Publication Date: 2017-06-13
ANHUI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although the CRISPR-Cas type I system _ENREF_1 has been found in Escherichia coli (Semenova, E.; Savitskaya, E.; Musharova, O.; Strotskaya, A.; Vorontsova, D.; Datsenko, K.A.; Logacheva, M. D.; Severinov, K., Highly efficient primed spaceacquisition from targets destroyed by the Escherichia coli type I-E CRISPR-Cas interfering complex. Proceedings of the National Academy of Sciences of the United States of America 2016, 113 (27), 7626-7631), but so far There has not been any report on gene editing by CRISPR-Cas type I system in Escherichia coli; in particular, no CRISPR-Cas system has been found in Bacillus subtilis_ENREF_1(http: / / crispr.u-psud.fr / )

Method used

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  • Gene editing method based on streptomyces virginiae IBL14 gene cas7-5-3
  • Gene editing method based on streptomyces virginiae IBL14 gene cas7-5-3
  • Gene editing method based on streptomyces virginiae IBL14 gene cas7-5-3

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 (Three-plasmid one-step co-transformation method to knock out EC JM109 lacZ Gene)

[0034] (1) Protein expression plasmid pHT304- cas 7-5-3 Construction

[0035] according to SV IBL14 whole-genome sequencing information and plasmid pHT-304 sequence information, designed with the complementary sequence of plasmid pHT-304 cas 7-5-3 Gene-specific primers cas -F and cas -R (Table 2). extract SV IBL14 genomic DNA was processed using TransStart FastPfu DNA Polymerase produced by Quanshijin Biotechnology Co., Ltd. cas Gene PCR amplification, reaction conditions: 95°C for 5 min, 94°C for 30 s, 55°C for 30 s, 72°C for 2 min, 2.5 U Pfu DNA Polymerase produced by Sangon Bioengineering (Shanghai) Co., Ltd. (50 μl reaction system ), 30 cycles at 72°C for 10 min. The PCR product was detected by 1% agarose electrophoresis, recovered by the kit, and purified cas full-length gene. in one step cas The full-length gene sequence was connected with the plasmid p...

Embodiment 2

[0052] Embodiment 2 (two-step co-transformation knockout of three plasmids EC JM109 lacZ Gene)

[0053] (1) Protein expression plasmid pHT304- cas 7-5-3 Construction

[0054] Same as embodiment 1 step (1)

[0055] (2) Gene editing plasmid pKC1139- lacZ - Construction of t-DNA

[0056] Same as embodiment 1 step (2)

[0057] (3) Acquisition and inspection of recombinants

[0058] (A) Plasmid pHT304- cas 7-5-3 Transformation and EC JM109-pHT304- cas 7-5-3 Competent preparation

[0059] The plasmid pHT304- cas 7-5-3 Transforms to EC In the JM109 competent state, the transformant was screened by erythromycin resistance, and then the transformant was used to prepare a competent state to obtain a plasmid containing pHT304- cas 7-5-3 EC JM109-pHT304- cas Competence of 7-5-3 strain. The competent preparation method is the same as step (3A) in Example 1.

[0060] (B) Plasmids pKD46 and pKC1139- lacZ - Co-transformation of t / g-DNA

[0061] Plasmids pKD46 and...

Embodiment 3

[0064] Embodiment 3 (two plasmid one-step co-transformation method knockout EC JM109 lacZ Gene)

[0065] (1) Protein expression plasmid pHT304- cas 7-5-3 Construction

[0066] Same as embodiment 1 step (1)

[0067] (2) Gene editing plasmid pKD46- lacZ - Construction of t / g-DNA

[0068] In addition to the homology arm and guide DNA- lacZ The target gene fragments are sequentially connected to the plasmid pKD46 to form the gene editing plasmid pKD46- lacZ Except for -t / g-DNA, other steps are the same as step (2) in Example 1.

[0069] (3) Acquisition and inspection of recombinants

[0070] Same as embodiment 1 step (3)

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PUM

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Abstract

The invention discloses a gene editing method based on a streptomyces virginiae IBL14 gene cas7-5-3. The gene editing method has the advantages that the gene editing on prokaryotic genomes is realized for the first time by CRISPR-Cas-I system; a new supplementing and selection type is provided for the gene editing on the CRISR-Cas II type CAS; by using a system, the prokaryotic genomes, such as escherichia coli and bacillus subtilis, can be conveniently, quickly and effectively subject to gene editing; the optimized gene editing system may be applied to the gene editing of other organisms.

Description

technical field [0001] The invention relates to gene editing technology in the field of biotechnology, specifically a gene editing method based on Streptomyces virginia IBL14 gene cas 7-5-3 approach to gene editing. Background technique [0002] Streptomyces virginiae IBL14 (Streptomyces virginiae IBL14) is an actinomycete isolated and screened from the sludge near the steroid drug manufacturer. It can effectively transform and degrade progesterone, flavonoids, cortisol and cholesterol and other steroidal compounds_ENREF_1(Wang, F.-Q.; Zhang, C.-G.; Li, B.; Wei,D.-Z.; Tong, W.-Y., New microbiological transformations of steroids by Streptomyces virginiae IBL-14. Environmental science & technology 2009, 43(15), 5967-5974). CRISPR (clustered regμlarly interspaced short palindromic repeat sequences) is called clustered regularly interspaced short palindromic repeat sequences, which were first discovered in Escherichia coli_ENREF_1 in the 1980s (Ishino, Y.; Shinagawa, H.; Makin...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/74C12N15/75
CPCC12N15/70C12N15/74C12N15/75C12N2800/101C12N2800/80C12N2810/10
Inventor 童望宇邱彩花杨兴旺王安静
Owner ANHUI UNIVERSITY
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