standardized-modular-assembly-polycistronic-operons-with-modular

Standardized Modular Assembly of Polycistronic Operons with Modular Cloning (MoClo) using the <em>In-Cloning</em> toolkit

Modular Cloning (MoClo) toolkits enable the rapid assembly of multigene constructs. They are based on Golden Gate cloning, which uses Type IIS restriction ...

Wageningen University and Research, Agrotechnology and Food Sciences

YeastFab is a Golden Gate-based cloning standard and parts repository. It is designed for modular, hierarchical assembly of transcription units and multi-gene assemblies for expression in Saccharomyces cerevisiae. This makes it a suitable toolbox to optimize the expression strength of heterologous genes in yeast. When cloning heterologous coding sequences into YeastFab vectors, in several cases we have observed toxicity to the cloning host Escherichia coli. The provided protocol details how to clone such toxic genes from multiple synthetic DNA fragments while adhering to the YeastFab standard. The presented cloning strategy includes a C-terminal FLAG tag that allows screening for constructs with a desired protein expression in yeast by western blot. The design allows scarlessly removing the tag through a Golden Gate reaction to facilitate cloning of expression constructs with the native, untagged transgene.

Stefan A. Hoffmann

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