Conversion method for two fiber sorangium cellulosum strains

A technology of S. cellulosus and donor bacteria, which is applied in the field of genetic engineering and can solve the problems of time-consuming construction of recombinant plasmids and transformation processes.

Inactive Publication Date: 2015-11-18
GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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Problems solved by technology

Xia Zhijie et al. (XiaZ-J, WangJ, HuW, LiuH, GaoX-Z, WuZ-H, ZhangP-Y, LiY-Z. Improving conjugation efficacy of Sorangium cellulosum by the addition of dual selection antibiotics. Journal of industrial microbiology & biotechnology, 2008, 35(10): 16157-1 and other methods are used to transfer exogenous genes into Sorangium cellulosum, but the construction of recombinant plasmids and the conversion process take a long time, although in 1992 Jaoua et al. (Jaoua, S., S.Neff, T.Schupp. (28):157-165) established the use of IncPCR plasmid pME305 to mediate the transfer of PSJB55 binding to the strain Soce26, but due to the strain specificity of S. cellulosus, this method is not applicable to other strains

Method used

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  • Conversion method for two fiber sorangium cellulosum strains
  • Conversion method for two fiber sorangium cellulosum strains
  • Conversion method for two fiber sorangium cellulosum strains

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Experimental program
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Embodiment 1

[0023] Embodiment 1: the protoplast transformation of S. cellulosus SoceM1 comprises the following steps:

[0024] (1) Preparation of protoplasts: Prepare liquid medium according to the following formula: 8g / L potato starch, 2g / L glucose, 2g / L soybean protein hydrolyzate, 2g / L yeast extract, 8mg / L LEDTA iron sodium, 1g / L MgSO 4 .7H 2 O, 1g / LCaCl 2 , 0.5g / LTris, the solvent is water, adjust the pH to 7.4 with hydrochloric acid. Sterilize at 115°C for 25min. Inoculate Socesia cellulosus SoceM1 into 30ml liquid medium with 2.5% inoculation amount, culture at 30°C and 180rpm until OD595nm is about 0.8, centrifuge at 8000rpm for 10min to obtain the bacteria, wash twice with PBS solution, and then use 14% mannitol Suspend the SoceM1 cells in the solution, then treat the cells with 15% EDTA solution for 30min, wash with PBS, then suspend the cells with 1ml of PBS, add 100μl of 3mg / ml lysozyme at 30°C for 2h, centrifuge at 8000rpm for 10min, and fully wash with 14% mannitol solutio...

Embodiment 2

[0033] Embodiment 2: the three parents of S. cellulosus SoceM6 are combined and transformed, comprising the following steps:

[0034] (1) Construction of the recombinant plasmid: add HindIIII and PstI restriction sites at both ends of the hemoglobin gene vgb gene of Vitella hyaline, use HindIIII and PstI to perform double enzyme digestion on the vgb gene and the broad host vector PLA2917, and connect overnight at 16°C. Transformed into DH5α competent cells, picked a single clone to expand culture, identified by PCR and sent for sequencing.

[0035] (2) Transformation of exogenous plasmids by three-parent combination method: inoculate donor bacteria containing PLA2917-vgb, helper bacteria (in DH5α strain) and SoceM6 recipient bacteria containing plasmid PRK2013 respectively, and the OD of donor bacteria and helper bacteria were inoculated. 0.6, and the OD of the recipient bacteria is about 0.8. The three kinds of bacteria are mixed in the ratio of the total number of cells as d...

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Abstract

The invention discloses a transformation method of two sorangium cellulosum strains. In consideration of the condition that the available plasmid vectors in the conventional sorangium cellulosum transformation method are relatively less, exogenous genes (vgb) are respectively led into the two sorangium cellulosum strains by anti-double-stranded shuttle plasmid vectors and anti-double-stranded wide host vectors. A foundation is laid for the establishment of a genetic operation system of the sorangium cellulosum and the promotion of the gene engineering modification of the sorangium cellulosum, so that the abundance and the yield of active secondary metabolites in the sorangium cellulosum are improved.

Description

[0001] This divisional application is a divisional application of the patent application with application number: 201410049382.7, title of invention: transformation method of two S. cellulosus strains, and application date: February 12, 2014. Technical field: [0002] The invention belongs to the field of genetic engineering, and in particular relates to a transformation method of two strains of S. cellulosus. Background technique: [0003] Sotrophys cellulosus is a kind of myxobacteria that can degrade and utilize cellulose. It has attracted extensive attention because of its rich active metabolites. 48.4% of the total. For example, epothilone produced by S. cellulosus has strong antitumor activity. Therefore, by establishing an appropriate genetic manipulation system, it is of great significance to use genetic engineering to increase the yield of its secondary metabolites. The genetic manipulation of S. cellulosus has progressed slowly. Xia Zhijie et al. (XiaZ-J, WangJ,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/74
Inventor 叶伟章卫民李浩华李赛妮王磊谭国慧
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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