Rudenko Oleksandra, Barnes Andrew C
The University of Queensland, School of Biological Sciences and Centre for Marine Science, St Lucia Campus, Brisbane, Queensland 4072, Australia.
The University of Queensland, School of Biological Sciences and Centre for Marine Science, St Lucia Campus, Brisbane, Queensland 4072, Australia.
J Microbiol Methods. 2018 Jan;144:157-163. doi: 10.1016/j.mimet.2017.11.023. Epub 2017 Nov 28.
Allelic exchange mutagenesis that relies on RecA-mediated homologous recombination up- and downstream from the targeted gene is a generalizable method of site-specific bacterial gene knock-out and knock-in. However, generation of a mutagenic DNA construct (alternative allele flanked by regions surrounding the gene target) and subsequent mutant selection are laborious procedures. Here we demonstrate allelic exchange knock-out facilitated by Gibson Assembly in Streptococcus iniae. Gibson Assembly allows rapid construction of a large allelic exchange cassette simultaneous with cloning, as well as rapid reconstruction of complete recombinant vector sequence when required. Additionally, we show that during two-step mutant selection, absence of recombination at one of the homologous regions (single cross-over) might be rapidly detected by colony PCR of meroploid clones and resolved by extension/shifting of corresponding sequence in DNA construct. The combination of Gibson Assembly for mutagenic DNA construction/redesign with colony PCR screening of meroploids to detect recombination at both sides of the exchange target may significantly accelerate generation of chromosomal mutants in a wide range of bacterial taxa.
依赖RecA介导的靶向基因上下游同源重组的等位基因交换诱变是一种通用的位点特异性细菌基因敲除和敲入方法。然而，诱变DNA构建体（基因靶点周围区域侧翼的替代等位基因）的生成以及随后的突变体选择是繁琐的程序。在这里，我们展示了在海豚链球菌中由吉布森组装促进的等位基因交换敲除。吉布森组装允许在克隆的同时快速构建大的等位基因交换盒，并且在需要时快速重建完整的重组载体序列。此外，我们表明，在两步突变体选择过程中，通过部分二倍体克隆的菌落PCR可以快速检测到同源区域之一（单交换）不存在重组，并通过在DNA构建体中延伸/移位相应序列来解决。将用于诱变DNA构建/重新设计的吉布森组装与部分二倍体的菌落PCR筛选相结合，以检测交换靶点两侧的重组，可能会显著加速在广泛的细菌分类群中产生染色体突变体。
