An in vitro recombination method to convert restriction- and ligation-independent expression vectors.
An in vitro recombination method to convert restriction- and ligation-independent expression vectors.
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DOI：
10.1002/biot.200700170
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发表时间：
2008-03-01
影响因子：
4.7
通讯作者：
Zhang, Yu-Zhu
中科院分区：
文献类型：
--
作者：
Guo, Feng;Chiang, Ming-Yi;Zhang, Yu-Zhu
近年来，基于位点特异性重组酶的高效无限制重组克隆系统已被证明是非常成功的。因此，希望将现有的常规载体转化为重组载体。在这份报告中，我们描述了一组广泛使用的传统载体的网关重组表达载体的转换。将侧翼有几个限制性酶位点的attB盒插入克隆载体中，然后亚克隆到现有载体中以转化为含有attB盒的中间载体，然后通过体外重组将其转化为重组表达载体。本研究中产生的中间载体可用于释放attB盒，以使用本文所述的相同方案转化其他载体。随着用该方案构建的重组载体数量的增加，从现有载体释放attB盒而不是用PCR合成它的可能性将增加。最终的表达载体也可用于释放attR盒以构建新载体。
In recent years, restriction-less recombination cloning systems based on site-specific recombinase with high efficiency have been proven to be very successful. Thus, it is desirable to convert existing conventional vectors to recombination vectors. In this report, we describe the conversion of a set of widely used conventional vectors to Gateway recombination expression vectors. An attB cassette flanked by several restriction enzyme sites was inserted in a cloning vector, and then subcloned into existing vectors to be converted to construct intermediate vectors containing the attB cassette, which were then converted to recombination expression vectors by in vitro recombination. The intermediate vectors generated in this study can be used for releasing the attB cassette to convert other vectors using the same protocol described here. With the increasing number of recombination vectors constructed with this protocol, the likeliness of releasing the attB cassette from an existing vector, rather than synthesizing it with PCR, will increase. The final expression vectors can also be used for releasing the attR cassette for constructing new vectors.