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. We explored the possibility of supplementing TX-TL with ClpXP, an AAA+ protease pair that selectively degrades tagged proteins [11], to provide finely-tuned degradation. The mechanism of ClpXP degradation has been extensively studied both in vitro and in vivo [12-15]. However, it has not been characterized for use in synthetic circuits – metrics such as toxicity, ATP usage, degradation variation over time, and cellular loading need to be determined. In particular, TX-TL in batch mode is known to be resource limited [16], and ClpXP is known to require significant amounts of ATP to unfold different protein targets [17, 18]. We find that ClpXP’s protein degradation dynamics is dependent on protein identity, but can be determined experimentally. Degradation follows Michaels-Menten kinetics, and can be fine tuned by ClpX or ClpP concentration. Added purified ClpX is also not toxic to TX-TL reactions. Therefore, ClpXP provides a controllable way to introduce protein degradation and dynamics into synthetic circuits in TX-TL.
Wheat germ cell-free expression system as a pathway to improve protein yield and solubility for the SSGCID pipeline
