AbstractOverexpression of synthetic genes depletes cellular resources, particularly ribosomes, which leads to lower expression of other synthetic genes and decreased growth rate. These burden effects can be detrimental to genetic circuit performance and hinders the process of modularly composing genetic circuits to create complex biomolecular systems with novel functions. No solution exists that allows the expression of any gene to a desired level without hindering the expression level of all other genes and growth rate. Here, we engineer an actuator that upregulates ribosome production. The key component of the actuator is a genetic cassette that expresses the hydrolysis domain of the SpoT enzyme (SpoTH) in a cell strain with elevated basal levels of ppGpp. We demonstrate that our actuator is capable of increasing protein production rates (proxy for free ribosomes) by over 150% and growth rate by over 80%. We use the actuator to engineer a feedforward controller, in which SpoTH is co-expressed with a target gene. Expressing the target gene without SpoTH purges the expression of a constitutive gene by more than 80% and cellular growth rate by 40%. By contrast, with SPOTH, the feedforward controller can be tuned to guarantee less than 10% change in the expression of a constitutive gene while keeping the expression of a the target gene at any desired level without any decrease in growth rate (however growth can increase by ≈40%). Alternatively, the feedforward controller can be tuned to guarantee less than 10% deviations in growth rate while also providing 30% higher expression of a constitutive gene relative to the case of expressing the target gene without SpoTH. Therefore, this solution allows desired target gene overexpression without burden, which is instrumental for predictable composition of genetic circuits.
A burden-free gene overexpression system
