体内一般亲本克隆的消除和定点诱变。

In vivo elimination of parental clones in general and site-directed mutagenesis.

作者信息

Holland Erika G, Acca Felicity E, Belanger Kristina M, Bylo Mary E, Kay Brian K, Weiner Michael P, Kiss Margaret M

机构信息

AxioMx, Inc., 688 E. Main St., Branford, CT 06405, United States.

University of Illinois at Chicago, 845 West Taylor Street, Chicago, IL 60607, United States.

出版信息

J Immunol Methods. 2015 Feb;417:67-75. doi: 10.1016/j.jim.2014.12.008. Epub 2014 Dec 15.

DOI:10.1016/j.jim.2014.12.008

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC4394739/

Abstract

The Eco29k I restriction endonuclease is a Sac II isoschizomer that recognizes the sequence 5'-CCGCGG-3' and is encoded, along with the Eco29k I methylase, in the Escherichia coli strain 29k. We have expressed the Eco29k I restriction-methylation system (RM2) in E. coli strain TG1 to produce the strain AXE688. We have developed a directed molecular evolution (DME) mutagenesis method that uses Eco29k I to restrict incoming parental DNA in transformed cells. Using our DME method, we have demonstrated that our AXE688 strain results in mutated directed molecular evolution libraries with diversity greater than 10(7) from a single transformation and with greater than 90% recombinant clones.

摘要

Eco29k I 限制性内切酶是一种Sac II 同裂酶，它识别序列5'-CCGCGG-3'，并与Eco29k I 甲基化酶一起在大肠杆菌29k菌株中编码。我们已在大肠杆菌TG1菌株中表达了Eco29k I 限制-甲基化系统（RM2），以产生AXE688菌株。我们开发了一种定向分子进化（DME）诱变方法，该方法使用Eco29k I 限制转化细胞中进入的亲本DNA。使用我们的DME方法，我们已经证明，我们的AXE688菌株能够产生多样性大于10（7）的突变定向分子进化文库，单次转化的重组克隆率大于90%。

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