Crawford I T, Greis K D, Parks L, Streips U N
Department of Microbiology and Immunology, School of Medicine, University of Louisville, Kentucky 40292.
J Bacteriol. 1987 Dec;169(12):5423-8. doi: 10.1128/jb.169.12.5423-5428.1987.
We describe a method for maximizing the rate of conversion of Bacillus thuringiensis subsp. kurstaki vegetative cells to osmotically fragile forms in the absence of exogenously added enzymes. Optimal generation of autoplasts occurred in 50 mM sodium acetate buffer (pH 7.0) at 37 degrees C with 10% (wt/vol) polyethylene glycol as an osmotic stabilizer. The maximum autolytic rate resulted in a conversion of greater than 90% of bacilli to spherical autoplasts in 6 min. Autoplasts regained bacillary morphology upon plating on DM3-G regeneration medium, with reversion frequencies ranging from 1.2 x 10(-1) to 5.3 x 10(-3). The autoplasts could efficiently take up exogenously added plasmid DNA. The presence of plasmids was verified by Southern hybridization analysis.
我们描述了一种在不添加外源酶的情况下，使苏云金芽孢杆菌库尔斯塔克亚种营养细胞转化为渗透敏感型细胞的转化率最大化的方法。在37℃下，以10%（重量/体积）聚乙二醇作为渗透稳定剂，于50 mM醋酸钠缓冲液（pH 7.0）中可实现原生质体的最佳生成。最大自溶速率导致在6分钟内超过90%的杆菌转化为球形原生质体。将原生质体接种在DM3-G再生培养基上后可恢复杆菌形态，回复频率范围为1.2×10⁻¹至5.3×10⁻³。原生质体能够有效地摄取外源添加的质粒DNA。通过Southern杂交分析验证了质粒的存在。
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