Genetic Manipulation of Haloferax Species.
Genetic Manipulation of Haloferax Species.
复制标题
DOI：
10.1007/978-1-0716-2445-6_3
复制
发表时间：
2022-01-01
期刊：
影响因子：
--
通讯作者：
Allers, Thorsten
中科院分区：
文献类型：
--
作者：
Dattani, Ambika;Harrison, Catherine;Allers, Thorsten
在这一章中，我们描述了在以易于操作而闻名的盐藻属古生菌物种中产生靶向基因缺失或替换的反向遗传学方法学。该方法中的各个步骤包括设计基因靶向载体，将其用于转化Haloferax以产生“弹入”和“弹出”克隆，以及验证基因操作菌株的技术。该载体携带500-1000bp的DNA片段，位于目标基因(或突变等位基因)两侧，此外还有用于尿嘧啶生物合成的pyrE2基因(Bitan-Banin等人)。J细菌185：772-778,2003)。后者被用作转化Haloferax的可选择标记，其中载体通过在基因组座位上的同源重组整合而产生“弹入”菌株；这也被称为等位基因偶联交换。在非选择性肉汤中培养这些转化子，然后在含有5-氟甲酸(5-FOA)的培养基上电镀，选择切除载体，产生野生型或突变型“突出型”克隆。使用表型分析、菌落杂交和Southern杂交相结合的方法，对这些5-FOA抗性克隆进行筛选，以确认所需的突变。这种弹入/弹出方法允许重复使用pyrE2标记，从而能够在单个菌株中进行多个基因缺失，从而提供对多个蛋白质的功能以及它们在各自的细胞路径中如何相互作用的见解。
In this chapter, we describe the reverse genetics methodology behind generating a targeted gene deletion or replacement in archaeal species of the genus Haloferax, which are renowned for their ease of manipulation. Individual steps in the method include the design of a gene-targeting vector, its use in transforming Haloferax to yield "pop-in" and "pop-out" clones, and techniques for validating the genetically manipulated strain. The vector carries DNA fragments of 500-1000bp that flank the gene of interest (or a mutant allele), in addition to the pyrE2 gene for uracil biosynthesis (Bitan-Banin et al. J Bacteriol 185:772-778, 2003). The latter is used as a selectable marker for the transformation of Haloferax, wherein the vector integrates by homologous recombination at the genomic locus to generate the "pop-in" strain; this is also known as allele-coupled exchange. Culturing of these transformants in nonselective broth and subsequent plating on 5-fluoroorotic acid (5-FOA)-containing media selects for excision of the vector, yielding either wild type or mutant "pop-out" clones. These 5-FOA-resistant clones are screened to confirm the desired mutation, using a combination of phenotypic assays, colony hybridization and Southern blotting. The pop-in/pop-out method allows for the recycling of the pyrE2 marker to enable multiple gene deletions to be carried out in a single strain, thereby providing insights into the function of multiple proteins and how they interact in their respective cellular pathways.