Study on Efficient Transformation Technology of Food Microorganism Staphylococcus carnosus
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(College of Food Science and Biotechnology, Zhejiang Gongshang University, Key Laboratory for Food Microbial Technology of Zhejiang Province, Hangzhou 310018)

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    Abstract:

    Staphylococcus carnosus is a food microorganism widely used in fermented meat products. However, the growth and metabolism mechanism of staphylococcus carnosus in fermented meat products is still unknown at present, which is closely bound up to the difficulty in plasmid transformation and the inability to establish a mature molecular biological system in genetic engineering operations. Staphylococcus carnosus has a thick and dense rigid cell wall and a restrictive modification (RM) system, causing poor reproducibility and inefficient of electrical conversion. In this study, the effects of shock buffer, electric field intensity, plasmid concentration, recovery medium, plasmid modification and heat treatment of competent cells on the electrical conversion efficiency of Staphylococcus carnosus pCA44 were studied, and the electrical conversion scheme was optimized. The results showed that: The competent cells were washed with 10% glycerol and 0.5 mol/L mannitol, 500 ng/100 μL plasmid modified by Escherichia coli DC10B was added, the competent cells were treated at 56 ℃ for 2 min, electric field intensity was 10 kV/cm, the mixture of plasmid and cell was shocked, and RGM2 containing 0.5 mol/L mannitol was added immediately after electric shock, which obtained the highest transformation efficiency, reaching 6.26×105 CFU/μg, and all the randomly selected transformants were positive for PCR verification. The electrical transfer scheme obtained in this study significantly improved the electric shock conversion efficiency of Staphylococcus carnosus pCA44, which could provide technical support for the analysis of its fermentation and metabolism mechanism, and provide reference for other staphylococcus strains with RM system barrier in the future.

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History
  • Received:August 25,2022
  • Revised:
  • Adopted:
  • Online: September 01,2023
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