En Passant Mutagenesis: A Two Step Marker less Red Recombination System
En Passant Mutagenesis: A Two Step Marker less Red Recombination System
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DOI：
10.1007/978-1-60761-652-8_30
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发表时间：
2010-01-01
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影响因子：
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通讯作者：
Osterrieder, Nikolaus
中科院分区：
文献类型：
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作者：
Tischer, B. Karsten;Smith, Gregory A.;Osterrieder, Nikolaus
细菌人工染色体用于在大肠杆菌中维持和修饰不同来源的大片段序列。除了基于RecA的穿梭诱变外，Red重组也常用于序列修饰。由于在突变的细菌人工染色体（BAC）克隆中，诸如抗生素抗性基因以及frt或loxP位点等外源序列通常是不需要的，我们开发了一种基于Red的技术，该技术能够无痕产生点突变、缺失以及插入大小不同的序列。该方法利用在第一次重组步骤中插入到目标序列中的序列重复，以及通过体内I - SceI切割和第二次Red重组来切除选择标记。为了在不使用额外质粒的情况下实现方便且高效的诱变，构建了具有染色体编码的可诱导Red和I - SceI表达的大肠杆菌菌株GS1783。
Bacterial artificial chromosomes arc used to maintain and modify large sequences of different origins in Escherichia coli. In addition to RecA-based shuttle mutagenesis, Red recombination is commonly used for sequence modification. Since foreign sequences, such as antibiotic resistance genes as well as frt- or loxP-sites are often unwanted in mutant BAC clones, we developed a Red-based technique that allows for the scarless generation of point mutations, deletions, and insertion of smaller and larger sequences. The method employs a sequence duplication that is inserted into the target sequence in the first recombination step and the excision of the selection marker by in vivo I-Scel cleavage and the second Red recombination. To allow for convenient and highly efficient mutagenesis without the use of additional plasmids, the E. coli strain GS1783 with a chromosomal encoded inducible Red- and I-SceI-expression was created.