食品微生物肉葡萄球菌的高效转化技术研究

Study on Efficient Transformation Technology of Food Microorganism Staphylococcus carnosus

肉葡萄球菌是广泛应用于发酵肉制品的食品微生物,然而,该菌在发酵制品中的生长代谢机理目前尚未可知,究其原因与基因工程操作中质粒转化难、无法建立成熟的分子生物学体系密切相关。肉葡萄球菌具有厚且致密的刚性细胞壁和限制性修饰(RM)系统,导致电转化的再现性差、效率低。本试验研究了电击缓冲液、电场强度、质粒质量浓度、复苏培养基以及质粒修饰和感受态细胞的高温热处理对肉葡萄球菌pCA44电转化效率的影响,并进行电转方案优化。结果表明:用10%甘油与0.5 mol/L甘露醇洗涤制备感受态细胞,加入500 ng/100μL经大肠杆菌DC10B修饰的质粒,56℃高温热处理肉葡萄球菌p CA44感受态细胞2 min后,在电场强度10 k V/cm的条件下电击质粒与细胞混合物,且电击结束后立即加入含有0.5 mol/L甘露醇的复苏培养基RGM2,得到的肉葡萄球菌p CA44电转化效率最高,达到6.26×10^(5)CFU/μg,且随机挑取转化子进行PCR验证均是阳性。本研究获得的电转方案显著提高了肉葡萄球菌p CA44电击转化效率,可为其发酵代谢机制解析提供技术支持,并为后续其它含有RM系统屏障的葡萄球菌系菌株提供参考。

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/100μL 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 6.26×10^(5) 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.