The removal of unwanted genetic material is a key aspect in many synthetic biology efforts, and often requires preliminary knowledge of which genomic regions are dispensable. Typically, these efforts are guided by transposon mutagenesis studies, coupled to deep-sequencing (TnSeq) to identify insertion points and gene essentiality. However, epistatic interactions can cause unforeseen changes in essentiality after the deletion of a gene, leading to the redundancy of these essentiality maps. Here, we present LoxTnSeq, a new methodology to generate and catalogue libraries of genome reduction mutants. LoxTnSeq combines random integration of lox sites by transposon mutagenesis, and the generation of mutants via cre recombinase, catalogued via deep-sequencing. When LoxTnSeq was applied to the naturally genome reduced bacterium Mycoplasma pneumoniae, we obtained a mutant pool containing 285 unique deletions. These deletions spanned from >50 bp to 28 Kb, which represent 21% of the total genome. LoxTnSeq also highlighted large regions of non-essential genes that could be removed simultaneously, and other non-essential regions that could not, providing a guide for future genome reductions.

中文翻译：

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LoxTnSeq：随机转座子插入与cre / lox重组和反选择相结合，产生大的随机基因组减少

不需要的遗传物质的去除是许多合成生物学工作中的关键方面，并且通常需要对哪些基因组区域是可有可无的进行初步了解。通常，这些工作是在转座子诱变研究的指导下进行的，再加上深度测序（TnSeq）来识别插入点和基因必需性。但是，上位性相互作用可能会导致基因缺失后本质上不可预见的变化，从而导致这些本质图谱的重复。在这里，我们介绍LoxTnSeq，这是一种生成和分类基因组还原突变体文库的新方法。LoxTnSeq结合了通过转座子诱变对lox位点的随机整合，以及通过cre重组酶（通过深度测序进行分类）生成突变体。当将LoxTnSeq应用于天然基因组减少的细菌性肺炎支原体时，我们获得了一个包含285个独特缺失的突变池。这些缺失范围从> 50 bp到28 Kb，占总基因组的21％。LoxTnSeq还强调了可以同时删除的大部分非必需基因区域，以及不能删除的其他非必需区域，为将来的基因组减少提供了指导。