Akaby strain
Cell-free protein expression system
Akaby is a RecB nuclease knockout E.coli strain. Cell-free translation system made from Akaby strain allows for expression of linear DNA templates without the need to use nuclease inhibitors.
The name was inspired by Arcade game Pac-Man, where Akabei is Japanese name for the Blinky ghost nemesis of Pac-Man. The ghost incapacitates the Pac-Man, analogous to Akaby strain with incapacitated major nuclease.
Full characterization of Akaby cell-free protein expression system:
https://doi.org/10.1371/journal.pone.0266272
Cell-free protein expression is increasingly becoming popular for biotechnology, biomedical and research applications. Among cell-free systems, the most popular one is based on Escherichia coli. Endogenous nucleases in E. coli cell-free transcription-translation (TXTL) degrade the free ends of DNA, resulting in inefficient protein expression from linear DNA templates. RecBCD is a nuclease complex that plays a major role in nuclease activity in E. coli, with the RecB subunit possessing the actual nuclease activity.
We created a RecB knockout of an E. coli strain optimized for cell-free expression. We named this new strain Akaby.
We demonstrated that Akaby TXTL successfully reduced linear DNA degradations, rescuing the protein expression efficiency from the linear DNA templates.
The practicality of Akaby for TXTL is an efficient, simple alternative for linear template expression in cell-free reactions.
We also used this work as a model protocol for modifying the TXTL source E. coli strain, enabling the creation of TXTL systems with other custom modifications.