Abstract
A large number of genetic studies in yeast rely on the use of expression vectors. To facilitate the experimental approach of these studies, several collections of expression vectors have been generated (YXplac, pRS series, etc.). Subsequently, these collections have been expanded by adding more diversity to many of the plasmid features, including new selection markers and new promoter sequences. However, the ever growing number of plasmid features makes it unrealistic for research labs to maintain an up-to-date collection of plasmids. Here, we developed the COSPLAY toolbox: a Golden Gate approach based on the scheme of a simple modular plasmid that recapitulates and completes all the properties of the pRS plasmids. The COSPLAY toolbox contains a basal collection of individual functional modules. Moreover, we standardized a simple and rapid, software-assisted protocol which facilitates the addition of new personalized modules. Finally, our toolbox includes the possibility to select a genomic target location and to perform a single copy integration of the expression vector.
Highlights
Over the last decade, the explosion of genome-wide analyses, as well as the rise of synthetic/ systems biology has profoundly transformed the methodology used to study model organisms [1]
The organization of a yeast expression vector generated with the COSPLAY toolbox is based on six functional modules assembled in a specific order into a destination vector
Our toolbox is based on a ready-to-use collection of individual functional modules and a one-step protocol to rapidly assemble a combination of modules into a functional expression vector
Summary
The explosion of genome-wide analyses, as well as the rise of synthetic/ systems biology has profoundly transformed the methodology used to study model organisms [1]. 10 μl of this reaction are transformed into DH-5 α or TOP10 bacterial strains and plated on LB plates supplemented with Ampicillin and X-Gal for Blue/White screening. For primer design it is mandatory to check whether the target sequence of interest contains BsaI sites. If so, these have to be mutated by PCR
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