Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.
Genes (Basel). 2020 Aug 20;11(9):965. doi: 10.3390/genes11090965.
is the causative agent of a multitude of diseases, and further study into its pathogenies is vital. The pneumococcus is genetically malleable, and several tools are available to manipulate this pathogen. In this study, we attempted to utilize one such tool, the Sweet Janus cassette, to replace the capsule locus with other capsule loci in our strain background and found that the efficiency of allelic replacement was low and the number of revertant false-positive colonies was high. We determined that the capacity to recombine capsule varied by the initial isolated colony, suggesting that frequency of reversion is dependent on the bacterial clone. Alternative selection markers may further expand the application of Sweet Janus. We created novel cassettes that utilized chlorinated phenylalanine as an alternative counter-selection agent in conjunction with the Janus or Sweet Janus cassette, providing a new dual or triple selection marker. Moreover, we created cassettes that do not require engineered resistance in the background strain, including both single and dual selection markers. We were able to utilize all constructs in allelic replacement of the capsule loci. These novel constructs provide a new means for generating gene deletions in that expand experimental applications.
是许多疾病的病原体，进一步研究其发病机制至关重要。肺炎球菌具有遗传可变性，并且有几种工具可用于操纵这种病原体。在这项研究中，我们试图利用这样一种工具，即甜蜜的双面盒，用其他荚膜基因座取代我们菌株背景中的荚膜基因座，但发现等位基因替换的效率较低，回复突变的假阳性菌落数量较多。我们发现，荚膜的重组能力因初始分离的菌落而异，这表明回复的频率取决于细菌克隆。替代选择标记可能会进一步扩展甜蜜双面盒的应用。我们创建了新的盒，利用氯苯丙氨酸作为替代的反向选择剂，与双面盒或甜蜜双面盒一起使用，提供了新的双或三重选择标记。此外，我们创建了不需要在背景菌株中进行工程抗性的盒，包括单选择和双选择标记。我们能够在荚膜基因座的等位基因替换中使用所有构建体。这些新的构建体为在 中生成基因缺失提供了一种新的手段，从而扩展了实验应用。
