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Construction method of penicillin-producing recombined strain of streptomyces virginiae IBL14

A technology for recombinant strains and construction methods, which is applied in the field of biomedicine and can solve problems such as no reports of penicillin recombinant strains

Inactive Publication Date: 2016-04-20
ANHUI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Using the CasI-SV14B system of Streptomyces virginiae IBL-14 to edit its penicillin-producing genes to obtain recombinant penicillin-producing strains has not been reported

Method used

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  • Construction method of penicillin-producing recombined strain of streptomyces virginiae IBL14
  • Construction method of penicillin-producing recombined strain of streptomyces virginiae IBL14
  • Construction method of penicillin-producing recombined strain of streptomyces virginiae IBL14

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 (Isopenicillin-N isomerase gene sviIPI / GVGL005789 knockout in bacterial strain IBL-14)

[0042] (1) Primer design and DNA amplification of gene sviIPI01

[0043] According to the whole genome sequencing information, gene sviIPI-specific primers sviIPI-F and sviIPI-R were designed (Table 2). Genomic DNA of Streptomyces virginia IBL-14 was extracted, and PCR amplification of sviIPI gene was performed using PfuDNAPolymerase produced by Shanghai Sangon Bioengineering Co., Ltd., reaction conditions: 95°C for 5min, 94°C for 30s, 52°C for 30s, 72°C for 2min , 2.5U PfuDNAPolymerase (50μl reaction system) produced by Sangon, 30 cycles, 72°C for 10min. The PCR product was detected by 1% agarose electrophoresis, the kit was recovered, and the purified sviIPI full-length gene fragment was obtained for future use.

[0044] (2) Preparation of upstream and downstream homology arms

[0045] According to the complete sequence of the sviIPI gene (Table 1), the upstream homol...

Embodiment 2

[0059] Example 2 (Knockout of β-lactamase gene sviLT / GVGL000792 in bacterial strain IBL-14)

[0060] (1) Gene sviLT primer design and DNA amplification

[0061] The designed gene primers were sviLT-F and sviLT-R (Table 2). Using IBL-14 genomic DNA as a template, the sviLT gene fragment was amplified. Reaction conditions: 95°C for 5 min, 94°C for 30 s, 52°C for 30 s, 72°C for 3 min, 2.5 U of PfuDNA Polymerase produced by Sangon (50 μl reaction system), 30 cycles, 72°C for 10 min. The PCR product was detected by 1% agarose electrophoresis, the kit was recovered, and the purified sviLT full-length gene fragment was obtained for future use. Specifically with embodiment 1 step (1).

[0062] (2) Preparation of upstream and downstream homology arms

[0063] The amplification of the upstream and downstream homology arms is specifically the same as step (2) of Example 1. The designed upper and lower homology arm primers were sviLT-UF, sviLT-UR, sviLT-DF, sviLT-DR (Table 2). Using...

Embodiment 3

[0076] Example 3 (knockout of penicillin amidase gene sviPA / GVGL002963 in bacterial strain IBL-14)

[0077] (1) Gene sviPA primer design and DNA amplification

[0078] The designed gene primers were sviPA-F and sviPA-R (Table 2). Using IBL-14 genomic DNA as a template, the sviPA gene fragment was amplified. Reaction conditions: 95°C for 5 min, 94°C for 30 s, 55°C for 30 s, 72°C for 3.5 min, 2.5 U of PfuDNA Polymerase produced by Sangon (50 μl reaction system), 30 cycles, 72°C for 10 min. The PCR product was detected by 1% agarose electrophoresis, the kit was recovered, and the purified sviPA full-length gene fragment was obtained for future use. Specifically with embodiment 1 step (1).

[0079] (2) Preparation of upstream and downstream homology arms

[0080] The amplification of the upstream and downstream homology arms is specifically the same as step (2) of Example 1. The designed upper and lower homology arm primers are sviPA-UF, sviPA-UR, sviPA-DF, sviPA-DR (Table 2)...

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Abstract

The invention discloses a construction method of a penicillin-producing recombined strain of streptomyces virginiae IBL14. The method involves sequence properties of a beta-lactamase gene containing hydrolysis penicillin and gene clusters producing penicillin, and the whole process of a construction method of the penicillin-producing recombined strain constructed on this basis. Related genes are compiled through the penicillin-producing gene and the CRISPR-Cas I-B gene of streptomyces virginiae IBL14, and therefore the aim of increasing the penicillin yield is achieved. A new route and method are provided for increasing the types of biological medicine, improving the production level and improving the product quality.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a method for constructing a penicillin-producing recombinant strain of Streptomyces virginia IBL14. Background technique [0002] Penicillin is an antibiotic with low toxicity and high efficacy accidentally discovered by the British Alexander Fleming in 1928 (Xu Zhongwei. (2005) found penicillin. Health Expo, 29). In 1939, Fleming provided the strains cultivated for 10 years to Australian pathologist Howard Florey and British biochemist Earnest Chain at Oxford University. In 1940, they completed the preparation of penicillin crystals and animal experiments (Lin Yin. (2007) The glorious past of penicillin. Science 24 hours, 40-42). In 1941, when the Second World War broke out, the U.S. government ordered the arduous task of mass-producing penicillin for wartime needs. At that time, Pfizer used its unique deep tank fermentation technology to complete the task and became the world's larg...

Claims

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Application Information

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IPC IPC(8): C12N15/76
CPCC12N15/76C12N2800/101C12N2800/80
Inventor 童望宇李雪邱彩花雍德祥
Owner ANHUI UNIVERSITY
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