Department of Medicine, Division of Allergy and Infectious Diseases, University of Washington, Harborview Medical Center, Seattle, Washington.
Department of Global Health, University of Washington, Harborview Medical Center, Seattle, Washington.
Curr Protoc. 2022 Aug;2(8):e507. doi: 10.1002/cpz1.507.
Although the isolation of Treponema pallidum subsp. pallidum (T. pallidum) from a syphilis patient dates to 1912, for the duration of the 20 century, this pathogen has remained an exceedingly difficult organism to study due to the lack of a system to support its viability in vitro. This limitation, in turn, has precluded the application of genetic engineering techniques via transformation and subsequent selection of T. pallidum transformants. A recently described method for in vitro cultivation of T. pallidum, however, has made it possible for us to experiment with transformation and selection methods. Here we describe the approach that we adopted to successfully transform T. pallidum with foreign DNA and select the resulting recombinant strain using kanamycin. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Transformation of T. pallidum Support Protocol 1: Quantification of T. pallidum in suspensions using dark-field microscopy Support Protocol 2: Counting cells using a hemacytometer Basic Protocol 2: Selection, initial passaging, and expansion of transformed cultures Basic Protocol 3: Isolation of a clonal strain through limiting dilution.
虽然梅毒螺旋体亚种苍白密螺旋体（T. pallidum）于 1912 年从梅毒患者中分离出来，但在整个 20 世纪，由于缺乏体外支持其存活的系统，这种病原体仍然难以研究。这种局限性又使得无法通过转化和随后选择梅毒转化体来应用遗传工程技术。然而，最近描述的一种梅毒螺旋体体外培养方法使我们能够尝试转化和选择方法。在这里，我们描述了我们成功地用外源 DNA 转化梅毒螺旋体并使用卡那霉素选择重组菌株的方法。© 2022 Wiley Periodicals LLC. 基本方案 1：梅毒螺旋体的转化 支持方案 1：使用暗场显微镜对悬液中的梅毒螺旋体进行定量 支持方案 2：用血球计数器计数细胞 基本方案 2：转化培养物的选择、初步传代和扩增 基本方案 3：通过有限稀释分离克隆株。
