Scarless Recombineering of Phage in Lysogenic State.
Scarless Recombineering of Phage in Lysogenic State.
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DOI：
10.1007/978-1-0716-2233-9_1
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发表时间：
2022-01-01
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影响因子：
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通讯作者：
Jaramillo, Alfonso
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文献类型：
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作者：
Ababi, Maria;Tridgett, Matthew;Jaramillo, Alfonso
我们提出了一种无瘢痕的重组为基础的方法引入多个点突变到一个温和的噬菌体的基因组。该方法使用λ Red重组工程系统来促进外源ssDNA寡核苷酸在宿主基因组复制期间在前噬菌体滞后链上退火。通过诱导甲基指导的错配修复系统的显性负突变蛋白的表达来抑制DNA修复。筛选没有选择标记的重组细胞是可行的，因为其重组频率高，估计在六个循环后超过40%。该方法能够在4- 5天内对噬菌体的基因组进行无瘢痕编辑。
We present a scarless recombineering-based method for introducing multiple point mutations into the genome of a temperate phage. The method uses the lambda Red recombineering system to promote exogenous ssDNA oligos to anneal on the prophage lagging strand during host genome replication. DNA repair is suppressed by inducing the expression of a dominant-negative mutant protein of the methyl-directed mismatch repair system. Screening for recombinant cells without a selection marker is feasible due to its high recombination frequency, estimated as more than 40% after six cycles. The method enables scarless editing of the genome of a bacteriophage in 4-5days.