In Vitro Transformation and Selection of Treponema pallidum subsp. pallidum.
In Vitro Transformation and Selection of Treponema pallidum subsp. pallidum.
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DOI：
10.1002/cpz1.507
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发表时间：
2022-08
期刊：
影响因子：
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通讯作者：
Giacani, Lorenzo
中科院分区：
文献类型：
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作者：
Phan, Amber;Romeis, Emily;Tantalo, Lauren;Giacani, Lorenzo
虽然分离到的梅毒螺旋体亚种为梅毒螺旋体。梅毒患者的梅毒(梅毒螺旋体)可追溯到1912年，由于在20世纪缺乏支持其体外生存能力的系统，这种病原体一直是一个极其困难的有机体。这种限制反过来又阻碍了通过转化和随后选择梅毒螺旋体转化体来应用基因工程技术。然而，最近描述的一种体外培养梅毒螺旋体的方法使我们有可能进行转化和选择方法的实验。在这里，我们描述了我们采用的方法，成功地将外源DNA转化为梅毒螺旋体，并用卡那霉素筛选得到了重组菌株。基本议定书1：梅毒螺旋体的转化。支持方案1：使用暗视野显微镜定量检测悬浮液中的梅毒螺旋体。支持方案2：使用血细胞分析仪进行细胞计数。基本议定书2：转化培养物的选择、初始传代和扩展。基本方案3：通过有限稀释法分离克隆菌株。
Although the isolation of Treponema pallidum subsp. pallidum (T. pallidum) from a syphilis patient dates to 1912, this pathogen has remained an exceedingly difficult organism to study due to the lack of a system to support its viability in vitro for the duration of the 20th century. Such limitation, in turn, 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 we adopted to successfully transform T. pallidum with foreign DNA and select the resulting recombinant strain using kanamycin. 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.