Bacillus subtilis genetically engineered bacterium with group quenching activity as well as construction method and application of bacillus subtilis genetically engineered bacterium

A technology of Bacillus subtilis and genetically engineered bacteria, applied in the fields of biotechnology and genetic engineering, can solve problems such as environmental pollution, endangering human health, and increasing human consumption

Inactive Publication Date: 2021-09-21
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the prevention and control methods of plant soft rot include reasonable crop rotation, avoiding mechanical damage, spraying pesticides or plant water extracts, etc. These methods not only increase manpower consumption, but also cause environmental pollution and endanger human health.

Method used

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  • Bacillus subtilis genetically engineered bacterium with group quenching activity as well as construction method and application of bacillus subtilis genetically engineered bacterium
  • Bacillus subtilis genetically engineered bacterium with group quenching activity as well as construction method and application of bacillus subtilis genetically engineered bacterium
  • Bacillus subtilis genetically engineered bacterium with group quenching activity as well as construction method and application of bacillus subtilis genetically engineered bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: Construction of recombinant Bacillus subtilis BSAHLX01 strain.

[0054] Select the inducible promoter Pgrac carried by the plasmid pHT01 itself to express the population quencher AhlX, and use the software Primer 5 to design the upstream and downstream primers F-A1, F-A1, R-A2, use I-5 TM The DNA polymerase of 2×High-Fidelity Master Mix amplifies the target fragment ahlX. The amplified product ahlX and the expression vector pHT01 were simultaneously digested with BamH I and Xba I restriction enzymes, the digested product was purified with the expression vector pHT01 after double digestion, and then ligated with T4 ligase at 16°C overnight to construct Inducible expression vector pHT01(Pgrac)-ahlX, (its plasmid map is in this patent attached figure 1 ), the successfully constructed vector pHT01(Pgrac)-ahlX was transformed into Bacillus subtilis B. subtilis 168 by electric shock to obtain the BSAHLX01 strain.

Embodiment 2

[0055] Example 2: Construction of recombinant Bacillus subtilis expressed from promoters from different sources.

[0056] The promoters adopted are respectively PgsiB, PaprE, PamyE, PamyQ, PsacB, PsrfA, PxylA, Phoiln, Phpall, Pshuttle-09, P43, (the numbering of the promoter sequence in this patent is respectively SEQ No.4, SEQ No. 5. SEQNo.6, SEQ No.7, SEQ No.8, SEQ No.9, SEQ No.10, SEQ No.11, SEQ No.12, SEQ No.13, SEQNo.14), according to the specific sequence, Synthetic promoter fragments (Nanjing GenScript Co., Ltd.), cloned into pHT01(Pgrac)-ahlX with Spe I and BamH I, replaced the original Pgrac promoter with a new promoter, and constructed ahlX expression vectors with promoters from different sources , (its plasmid map is attached to this patent figure 2 ). The successfully constructed ahlX expression plasmids with promoters from different sources were respectively electrotransformed into Bacillus subtilis 168 strains to obtain recombinant strains such as BSAHLX02, BSA...

Embodiment 3

[0057] Example 3: Construction of recombinant Bacillus subtilis engineering strain B.subtilis 168 / pHT01(Pgrac-ΔlacI)-ahlX.

[0058] Considering that the recombinant strain BSAHLX01 needs IPTG induction, additional inducers cannot be added during plant disease control. Therefore, the lacI region on the original vector was removed, and the repression of the LacI protein on the promoter was released, so that the expression plasmid could be used without adding inducers. Under certain circumstances, while continuously and efficiently expressing the population quenching enzyme AhlX, reducing the cost of industrial production, an expression vector pHT01(Pgrac-△lacI)-ahlX that can constitutively express the AhlX protein was constructed.

[0059] First, using the vector pHT01(Pgrac)-ahlX as a template, using F-△L1 and R-△L2 as upstream and downstream primers, using I-5 TM 2×High-Fidelity Master Mix DNA polymerase inverse PCR amplification, delete the regulatory region gene lacI on pHT0...

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Abstract

The invention relates to the technical field of biotechnology and genetic engineering, in particular to bacillus subtilis genetically engineered bacterium with group quenching activity and application of the bacillus subtilis genetically engineered bacterium. A construction method comprises the steps that acyl homoserine lactonase coding genes are cloned to a bacillus subtilis expression vector, regulatory protein coding genes lacI on the vector are removed, and transferring into bacillus subtilis is carried out to obtain the bacillus subtilis genetically engineered bacterium with group quenching activity. The recombinant bacillus subtilis for expressing AHL lactonase constructed by the invention can continuously express high-activity quorum sensing quenching enzyme AhlX without additionally adding an inducer, the used host is the environment-friendly bacillus subtilis, and the bacillus subtilis has natural advantages in the aspect of preventing and treating plant bacterial diseases, is pollution-free and non-toxic compared with chemical pesticides, has the advantages of no residue and no harm to the environment, is a green prevention and control measure, and has great potential in the aspect of preventing and controlling bacterial diseases.

Description

technical field [0001] The invention relates to the technical fields of biotechnology and genetic engineering, in particular to a Bacillus subtilis genetically engineered bacterium with quorum quenching activity and its application. Background technique [0002] Plant soft rot is a plant disease caused by Erwinia carotovora. Erwinia can infect plant tissues or organs, causing large-scale rot of plants and causing great harm to agricultural production. At present, the control methods for plant soft rot include reasonable crop rotation, avoiding mechanical damage, spraying pesticides or plant water extracts, etc. These methods not only increase manpower consumption, but also cause environmental pollution and endanger human health. Therefore, new environment-friendly disease control strategies urgently need to be researched and developed. [0003] Quorum Sensing (QS) refers to the phenomenon that bacteria regulate the expression of specific genes and coordinate a series of bio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/75C12N15/55C12N9/18A01N63/22A01P1/00C12R1/125
CPCC12N9/18C12N15/75C12Y301/01081A01N63/22
Inventor 韩笑笑柳鹏福储消和陈艳谢赛雪陈天德
Owner ZHEJIANG UNIV OF TECH
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