Prediction of Host-Circuit Interactions Using Cell-Free Protein System

The design of synthetic circuits rarely accounts for the impact production of orthologous proteins have on the host cell. However, in bacteria the burden triggered by the production of synthetic parts can lead to significant decreases in protein production efficiency, to growth rate decreases and even cell death. Moreover, alteration of cell physiology considerably impacts the behaviour of synthetic circuits complicating our abilities to produce reliable predictions and efficient designs. To address this, we combined Cell-Free Protein Systems (CFPS), measurements in E. coli and modelling to enable prediction of the impact synthetic circuits have on cells.

 CFPS has been used by others to rapidly obtain protein expression measurements and predict protein production in engineered E. coli. Here, we extend this further and use CFPS as a platform to measure competition for resources between different synthetic circuits, allowing us predict in vivo burden. We measured the expression and resource-use of different genes of interest, combined with different parts in both CFPS and in vivo with E. coli. The competition for resources between our previously described capacity monitor and the different genes of interest revealed a strong correlation between CFPS and in vivo measurements thus reinforces the utility of CFPS measurements for predicting complex behaviours in cells.

 In parallel, we also developed a whole cell model to extend predictions of both in vivo circuit performance and burden in different growth conditions and also in cells producing multiple genes of interest. By combining these efforts, we will provide a novel method for rapid in vitro screening of synthetic parts that then allows robust prediction of synthetic circuit behaviours, efficiencies and their impacts on the host cell.