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Application for suppressing expression of gene rsaL in pseudomonas aeruginosa and pseudomonas aeruginosa with suppressed expression of gene rsaL

A Pseudomonas aeruginosa and gene technology, applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve the problems of unstable production performance of strains, easy recovery of physical and chemical mutagenesis, etc., to enhance exercise ability and solve recovery mutations , high yield effect

Inactive Publication Date: 2018-03-23
DONGGUAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the production strain of rhamnolipid is mainly Pseudomonas aeruginosa, and the improvement of its production performance is generally through various physical and chemical mutagenesis techniques to improve the production capacity of strain rhamnolipid, and physical and chemical mutagenesis is easy to reverse the mutation, Cause the instability of strain production performance

Method used

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  • Application for suppressing expression of gene rsaL in pseudomonas aeruginosa and pseudomonas aeruginosa with suppressed expression of gene rsaL
  • Application for suppressing expression of gene rsaL in pseudomonas aeruginosa and pseudomonas aeruginosa with suppressed expression of gene rsaL
  • Application for suppressing expression of gene rsaL in pseudomonas aeruginosa and pseudomonas aeruginosa with suppressed expression of gene rsaL

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Experimental program
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Effect test

Embodiment approach

[0021] According to a preferred embodiment of the present invention, the rhamnolipid is total rhamnolipid synthesized by Pseudomonas aeruginosa.

[0022] According to another preferred embodiment of the present invention, the rhamnolipid is double rhamnolipid Rha-Rha-C synthesized by Pseudomonas aeruginosa 10 -C 10 and the monorhamnolipid for Rha-C 10 -C 10 .

[0023] In the present invention, the Pseudomonas aeruginosa can be existing Pseudomonas aeruginosa that can synthesize rhamnolipids, but the present invention is particularly suitable for Pseudomonas aeruginosa PAO1. Therefore, according to a preferred embodiment of the present invention, the Pseudomonas aeruginosa is Pseudomonas aeruginosa PAO1. Wherein, the Pseudomonas aeruginosa PAO1 can be obtained through conventional commercial channels, or obtained through other available channels. The Pseudomonas aeruginosa PAO1 is described in detail in the literature Geneticrecombination in Pseudomonas aeruginosa. J Gen M...

Embodiment 1

[0039] This example is used to illustrate the knockout of the rsaL gene

[0040]The gene deleted in the RsaL deletion mutant strain ΔrsaL is located at PA1431 on the Pseudomonas aeruginosa PAO1 genome (www.pseudomonas.com). The construction reference of ΔrsaL is Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange. Nature Protocol, 2015 , 10(11):1820-41. The knockout plasmid is pEX18AP, and the reference is A broad-host-range Flp-FRTrecombination system for site-specific excision of chromosomely-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosamutants.Gene,1998,212(1):77-86 . Primers were designed according to the rsaL gene promoter to amplify its upstream and downstream homologous fragments. The forward primer of the upstream homologous fragment is shown in SEQ ID No: 4 (5'-gccagtgccaagcttgagcattacgaccgggc); the reverse primer is shown in SEQ ID No: 5 (3'-gatcagagcaggcaagatcagagagtaataag); the forward prime...

Embodiment 2

[0043] This example is used to illustrate the comparison of the engineering strain PAO1ΔrsaL provided by the present invention with the starting strain PAO1 swarming exercise ability.

[0044] The activated strains were inoculated into LBNS liquid medium, and cultured with shaking at 37°C overnight. Take 1 μl of the bacterial solution and inoculate it on the surface of the swarming plate (Nutrientbroth 8g / L, D-glucose 5g / L, BactoAgar 5g / L, sterilized at 115°C for 15min), and culture overnight at 37°C. Measure the diameter of the bacterial movement area on each plate, the results are shown in figure 2 .

[0045] From figure 2 It can be seen that the swarming ability of PAO1ΔrsaL (6.9±0.3cm) is significantly greater than that of PAO1 (3.1±0.2cm).

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Abstract

The invention relates to the field of biology and particularly relates to an application for suppressing expression of a gene rsaL in pseudomonas aeruginosa and the pseudomonas aeruginosa with suppressed expression of the gene rsaL. The application is improvement of the transcriptional levels of rhamnolipid-synthesizing genes rhlA and rhlB, the rhamnolipid-synthesizing capability and the swarmingmoving capability of the pseudomonas aeruginosa. According to the transcriptional levels of the rhamnolipid-synthesizing genes rhlA and rhlB and synthesis of total rhamnolipid, dirhamnolipid Rha-Rha-C10-C10 and monorhamnolipid Rha-C10-C10 of the strain provided by the invention, the obtained yield is higher, the property is more stable and the problem of mutation restoration in the process of physicochemical mutagenesis is solved. In addition, the swarming moving capability of the rhamnolipid strain provided by the invention is enhanced. Simultaneously, the strain constructed by the inventionhas the beneficial effects that antibiotics do not need to be added in the production process of rhamnolipid, so that the benefit for the following separation purification is achieved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to inhibiting the expression of the gene rsaL in Pseudomonas aeruginosa to improve the transcription level of the rhamnolipid synthesis gene rhlAB of Pseudomonas aeruginosa, the ability to synthesize rhamnolipids and the ability to swarming in Pseudomonas aeruginosa with suppressed expression of the gene rsaL. Background technique [0002] Rhamnolipid is a glycolipid anionic surfactant, because of its good biocompatibility and efficient emulsification, solubilization and surface tension reduction capabilities, it is widely used in petroleum exploration, biomedicine, environmental protection and It is widely used in food and other fields. [0003] At present, the production strain of rhamnolipid is mainly Pseudomonas aeruginosa, and the improvement of its production performance is generally through various physical and chemical mutagenesis techniques to improve the production capacity o...

Claims

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Application Information

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IPC IPC(8): C12N15/31C12N1/21C12N15/78C12R1/385
Inventor 孙艳梅王世伟李萌陈桂华陈慧海
Owner DONGGUAN UNIV OF TECH
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