Department of Microbiology, Heersink School of Medicine, The University of Alabama at Birmingham, Birmingham, Alabama, USA.
Department of Microbiology and Immunology, Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, Maryland, USA.
Appl Environ Microbiol. 2024 Sep 18;90(9):e0101024. doi: 10.1128/aem.01010-24. Epub 2024 Aug 14.
The ability to genetically manipulate bacteria is a staple of modern molecular microbiology. Since the 2000s, marker-less mutants of () have been made by allelic exchange predominantly using the cassette known as "Janus." The conventional Janus protocol involves two transformation steps using multiple PCR-assembled products containing the Janus cassette and the target gene's flanking DNA. We present an innovative strategy to achieve marker-less allelic replacement through a single transformation step. Our strategy involves integrating an additional copy of the target's downstream region before the Janus cassette, leading to a modified genetic arrangement. This single modification reduced the number of required PCR fragments from five to four, lowered the number of assembly reactions from two to one, and simplified the transformation process to a single step. To validate the efficacy of our approach, we implemented this strategy to delete in serotype 4 strain TIGR4 the virulence gene the entire capsular polysaccharide synthesis locus , and to introduce a single-nucleotide replacement into the chromosome. Notably, beyond streamlining the procedure, our method markedly reduced false positives typically encountered during negative selection with streptomycin when employing the traditional Janus protocol. Furthermore, and as consequence of reducing the amount of exogenous DNA required for construct synthesis, we show that our new method is amendable to the use of commercially available synthetic DNA for construct creation, further reducing the work needed to obtain a mutant. Our streamlined strategy, termed easyJanus, substantially expedites the genetic manipulation of facilitating future research endeavors.
We introduce a new strategy aimed at streamlining the process for marker-less allelic replacement in , a Gram-positive bacterium and leading cause of pneumonia, meningitis, and ear infections. Our approach involves a modified genetic arrangement of the Janus cassette to facilitate self-excision during the segregation step. Since this new method reduces the amount of exogenous DNA required, it is highly amendable to the use of synthetic DNA for construction of the mutagenic construct. Our streamlined strategy, called easyJanus, offers significant time and cost savings while concurrently enhancing the efficiency of obtaining marker-less allelic replacement in .
基因操纵细菌的能力是现代分子微生物学的基础。自 21 世纪初以来，主要通过使用称为“杰纳斯”的盒式元件进行等位基因交换，已经制造出了（）的无标记突变体。传统的杰纳斯方案涉及两步转化，使用包含杰纳斯盒和目标基因侧翼 DNA 的多个 PCR 组装产物。我们提出了一种通过单步转化实现无标记等位基因替换的创新策略。我们的策略涉及在杰纳斯盒之前整合目标下游区域的额外副本，导致遗传排列发生改变。这一单一改变将所需的 PCR 片段数量从五个减少到四个，将组装反应数量从两个减少到一个，并简化了转化过程为一个步骤。为了验证我们方法的有效性，我们在 4 型血清型 TIGR4 菌株中实施了这种策略，以删除毒力基因和整个荚膜多糖合成基因座，并在染色体上引入单个核苷酸替换。值得注意的是，除了简化程序外，我们的方法还显著减少了传统杰纳斯方案中使用链霉素进行负选择时通常遇到的假阳性。此外，由于减少了构建体合成所需的外源性 DNA 量，我们表明我们的新方法适用于使用商业上可获得的合成 DNA 进行构建体创建，进一步减少了获得突变体所需的工作量。我们简化的策略，称为 easyJanus，极大地加速了在导致肺炎、脑膜炎和耳部感染的革兰氏阳性菌中的无标记等位基因替换的遗传操作，为未来的研究工作提供了便利。
我们引入了一种新策略，旨在简化在（一种革兰氏阳性菌，也是肺炎、脑膜炎和耳部感染的主要病原体）中进行无标记等位基因替换的过程。我们的方法涉及对杰纳斯盒进行改良的遗传排列，以促进在分离步骤中的自我切除。由于这种新方法减少了所需的外源性 DNA 量，因此非常适合使用合成 DNA 构建诱变构建体。我们简化的策略称为 easyJanus，在提高获得无标记等位基因替换的效率的同时，还显著节省了时间和成本。
