Once the BP and/or LR reactions are performed, the next step is to transform competent E. As in the BP reaction, a DNA fragment containing the ccd B gene is excised from the destination vector. As a result, an expression clone with the DNA of interest flanked by att B sites is generated. This reaction is catalyzed by the LR Clonase enzyme mix. The LR Reaction takes place between the att L sites of the generated entry clone and the att R sites of the destination vector. The LR reaction creates an expression clone with all of the components necessary for gene expression. The BP reaction creates an attL-flanked entry clone. Under certain conditions, the att L and att R sites can recombine, leading to the excision of the phage from the bacterial chromosome and the regeneration of att P and att B sites.įigure 2: The Gateway system adopts phage integration into the BP and LR reactions. As a result of recombination between the attP and attB sites, the phage integrates into the bacterial genome flanked by two new recombination sites ( att L-left- and att R-right-, Figure 1). I n vivo, t hese recombination reactions are facilitated by the recombination of attachment sites from the phage ( att P) and the bacteria ( att B). The Gateway cloning method, developed by Invitrogen, is an in vitro version of the integration and excision recombination reactions that take place when lambda phage infects bacteria. Keep reading to learn more about the Gateway cloning method and its advantages. With the appropriate entry and destination vectors, one can use Gateway to clone a gene of interest into a variety of expression systems. Since its invention in the late 1990s, Gateway cloning technology has become very popular as a rapid and highly efficient way to move DNA sequences into multiple vector systems. Instead, you can choose a molecular cloning technique that will work well with a given set of resources, time, and experimental needs. When facing a cloning project, scientists are no longer limited to traditional restriction enzyme cloning.
0 kommentar(er)
