Transformation method of two sorangium cellulosum strains

A technology of Cystobacter fibroblastus and donor bacteria, which is applied in the field of genetic engineering and can solve the problem of time-consuming recombinant plasmids and the like

Inactive Publication Date: 2014-05-28
GUANGDONG INST OF MICROORGANISM
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  • Abstract
  • Description
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Problems solved by technology

Xia Zhijie et al. (Xia Z-J, Wang J, Hu W, Liu H, Gao X-Z, Wu Z-H, Zhang P-Y, Li Y-Z. Improving conjugation efficacy of Sorangium cellulosum by the addition of dual selection antibiotics. Journal of industrial microbiology & biotechnology, 2008, 35( 10): 1157-1163) used combined transfer and natural transformation methods to transfer foreign genes into S. cellulosus, but the recombinant plasmid construction and transformation process took a long time, although Jaoua et al. in 1992 (Jaoua, S., S .Neff,T.Schupp.Transfer of mobilizable plasmids to Sorangium cellulosum and evidence for the integration into the chromosome.Plasmid,1992,(28):157-165) established the use of IncPCR plasmid pME305 to mediate PSJB55 binding transfer to strain So ce26 medium, but due to the strain-specificity of S. cellulosus, this method is not suitable for other strains

Method used

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  • Transformation method of two sorangium cellulosum strains
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  • Transformation method of two sorangium cellulosum strains

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Experimental program
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Embodiment 1

[0022] Embodiment 1: the protoplast transformation of S. cellulosus So ce M1 comprises the following steps:

[0023] (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 EDTA iron sodium, 1g / L MgSO 4 .7H 2 O, 1g / L CaCl 2 , 0.5g / LTris, the solvent is water, adjust the pH to 7.4 with hydrochloric acid. Sterilize at 115°C for 25min. Inoculate S. cellulosus Soce M1 into 30ml of liquid medium with an inoculation amount of 2.5%, cultivate at 30°C and 180rpm until the OD595nm is about 0.8, centrifuge at 8000rpm for 10min to obtain the bacteria, wash twice with PBS solution, and then wash with 14% Fully suspend Soce M1 cells with mannitol solution, then treat the cells with 15% EDTA solution for 30 minutes, wash with PBS, then suspend the cells with 1ml PBS, add 100μl 3mg / ml lysozyme, act at 30℃ for 2 hours, centrifuge at 8000rpm for 10min, wash with 1...

Embodiment 2

[0032] Embodiment 2: the three parents of S. cellulosus So ce M6 are combined and transformed, comprising the following steps:

[0033] (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.

[0034] (2) Transformation of exogenous plasmids by three-parent combination method: inoculate donor bacteria containing PLA2917-vgb, helper bacteria containing plasmid PRK2013 (in the DH5α strain) and So ce M6 recipient bacteria, and the donor bacteria and helper bacteria were inoculated respectively. The OD of the recipient bacteria is about 0.6, and the OD of the recipient bacteria is about 0.8. The three kinds of bacteria are mixed wi...

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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

Technical field: [0001] 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: [0002] 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. (Xia Z-J, Wang J, Hu W, Liu H, Gao X-Z, Wu Z-H, Zhang P-Y, Li Y-Z. Improving conjugation efficacy of Sorangium cellulosum by the addition of dual selection antibiotics. Journal of industrial microbiology & biotechnology, 2008, 35( 10): 1157-1163) used combine...

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 MICROORGANISM
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