Protein Degradation in a TX-TL Cell-free Expression System Using ClpXP Protease.

摘要

An in vitro S30 - based Escherichia coli expression system (Transcription-Translation, or TX - TL) has been developed as an alternative prototyping environment to the cell for synthetic circuits [1 - 5] . Basic circuit elements, such as switches and cascades, have been shown to function in TX - TL, as well as bacteriophage assembly [2, 6] . Circuits can also be prototyped from basic parts within 8 hours, avoiding cloning and transformation steps [7] . However, most published results have been obtained in a batch mode reaction, where factors that play an important role for in vivo circuit dynamics namely protein degradation and protein dilution are severely hindered or are not present. This limits the complexity of circuits built in TX - TL without steady - state or continuous - flow solutions [8 - 10] . However, alternate methods that enable dilution either require extra equipment and expertise or demand lower reaction throughput.