Untangling Resource Competition Towards Predictable Mammalian Cell Engineering

Despite the many achievements in synthetic biology, building new engineered systems still poses numerous challenges, such as poor predictability and reliability. This is mainly because heterologous constructs rely on the very same pool of resources used by cells to sustain their physiology. As a consequence, synthetic constructs impose an extra load – also referred to as burden – to engineered cells that have to re-allocate their finite amount of cellular resources to match the transcriptional and translational needs of the engineered systems(Ceroni et al., 2015; Borkowski et al., 2016). This greatly reduces cellular gene expression and metabolic capacities, leading to unexpected behaviours and hampering a straightforward engineering(Tan, Marguet and You, 2009; Gyorgy et al., 2015).

Here, we investigate the parameters to be considered when the design of low-burden synthetic construct for mammalian cell engineering is sought. As reported in previous findings(Huliák et al., 2012; Frei et al., 2020), we confirm that inducible expression of heterologous proteins heavily impacts the behaviour of a co-transfected constitutive reporter. This has already been pointed out as a consequence of resource competition arising between the two co-transfected modules(Huliák et al., 2012; Frei et al., 2020). We have also built a library of constructs with different intracellular localisation, transcriptional strength, and translational efficiency, showing how these three parameters are important when the tuning of such competition is sought.

We next aim to investigate the role played by burden when working with integrated circuits in mammalian cells, with the ultimate aim of individuating new design rules predictive of reduced burden. This will greatly benefit numerous bioproduction and biotherapeutic applications relying on mammalian cell engineering.