Resource-Aware Construct Design in Mammalian Cells

Despite recent advances in mammalian synthetic biology, the design of predictable and robust synthetic constructs still proves challenging. The competition for intracellular resources has been reported as one relevant cause of circuit failure in both bacterial and mammalian cells, and mitigation strategies to address this problem are starting to emerge (Ceroni et al., 2015; Frei et al., 2020; Jones et al., 2020; Di Blasi et al., 2021).

While in bacteria the comparison among different construct design parameters (e.g. promoter strength, RBS) has led to more robust expression over time, in mammalian cells such an analysis has not been performed yet, precluding potential improvement of mammalian synbio applications.

Here, we investigate the rules for efficient circuits assembly by testing the composition of different design parameters in a panel of mammalian cell lines. In particular, we show how tuning promoter strength, Kozak and polyadenylation efficiency results in designs that place a different demand on cellular resources when transiently expressed. We also test the secretion system as further bottleneck in the context of resource shortage.

This study lays initial foundations to identify new design rules predictive of reduced resource competition in mammalian cells, leading the way towards a more robust cell engineering that can benefit both biotherapy and bioproduction.