An in vitro recombination method to convert restriction- and ligation-independent expression vectors.
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.
Guo
Feng
F
Department of Biology, Illinois Institute of Technology, Chicago, IL 60616, USA.
Chiang
Ming-Yi
MY
Wang
Yingtong
Y
Zhang
Yu-Zhu
YZ
D011088:DNA Ligases; D005822:Genetic Vectors / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics*; D011995:Recombination, Genetic / Q000235:genetics*
D011994:Recombinant Proteins; D011088:DNA Ligases