Heterologous gene activation causes non-physiological burden on cellular resources that cells are unable to adjust to. Here, we introduce a feedforward controller that actuates growth rate upon activation of a gene of interest (GOI) to compensate for such a burden. The controller achieves this by activating a modified SpoT enzyme (SpoTH) with sole hydrolysis activity, which lowers ppGpp level and thus increases growth rate. An inducible RelA+ expression cassette further allows to precisely set the basal level of ppGpp, and thus nominal growth rate, in any bacterial strain. Without the controller, activation of the GOI decreased growth rate by more than 50%. With the controller, we could activate the GOI to the same level without growth rate defect. A cell strain armed with the controller in co-culture enabled persistent population-level activation of a GOI, which could not be achieved by a strain devoid of the controller. The feedforward controller is a tunable, modular, and portable tool that allows dynamic gene activation without growth rate defects for bacterial synthetic biology applications.
Overexpression 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
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.