Department of Biology, Texas A&M University, College Station, Texas, USA.
Department of Biology, Texas A&M University, College Station, Texas, USA
mSphere. 2020 Mar 4;5(2):e00941-19. doi: 10.1128/mSphere.00941-19.
An important risk factor for acquiring infection is antibiotic use. Therefore, a detailed knowledge of the physiology and the virulence factors can help drive the development of new diagnostic tools and nonantibiotic therapeutic agents to combat these organisms. Several genetic systems are available to study in the laboratory environment, and all rely on stably replicating or segregationally unstable plasmids. Currently, the transfer of plasmids into can only be performed by conjugation using or as conjugal donors. Here we report a method to introduce plasmid DNA into using electroporation and test factors that might contribute to higher transformation efficiencies: osmolyte used to stabilize weakened cells, DNA concentration, and recovery time postelectroporation. Depending on the strain and plasmid used, this transformation protocol achieves between 20 and 200 colonies per microgram of DNA and is mostly influenced by the recovery time postelectroporation. Based on our findings, we recommend that each strain be tested for the optimum recovery time in each lab. Understanding the underlying biology of pathogens is essential to develop novel treatment options. To drive this understanding, genetic tools are essential. In recent years, the genetic toolbox available to researchers has expanded significantly but still requires the conjugal transfer of DNA from a donor strain into Here we describe an electroporation-based transformation protocol that was effective at introducing existing genetic tools into different strains.
获得感染的一个重要危险因素是抗生素的使用。因此，详细了解生理学和毒力因子可以帮助开发新的诊断工具和非抗生素治疗剂来对抗这些生物体。有几种遗传系统可用于实验室环境中的研究，并且都依赖于稳定复制或分离不稳定的质粒。目前，只能通过使用或作为共轭供体的共轭将质粒转入。在这里，我们报告了一种使用电穿孔将质粒 DNA 导入的方法，并测试了可能有助于提高转化效率的因素：用于稳定弱化细胞的渗透剂、DNA 浓度和电穿孔后的恢复时间。根据使用的菌株和质粒，该转化方案每微克 DNA可实现 20 至 200 个菌落，并且主要受电穿孔后的恢复时间影响。基于我们的发现，我们建议每个实验室针对每种菌株测试最佳的恢复时间。了解病原体的基础生物学对于开发新的治疗方法至关重要。为了推动这种理解，遗传工具是必不可少的。近年来，可供研究人员使用的遗传工具包有了显著扩展，但仍需要从供体菌株中将 DNA 共轭转移到。在这里，我们描述了一种基于电穿孔的转化方案，该方案可有效地将现有的遗传工具引入不同的菌株。
