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bioRxiv · 10.1101/2021.02.11.430724

A burden-free gene overexpression system

Barajas, C.·Gibson, J.·Sandoval, L.·del vecchio, d.
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Abstract

Heterologous gene activation causes non-physiological burden on cellular resources that cells are unable to adjust to. Here, we introduce a feedforward controller that increases ribosome level upon activation of a gene of interest (GOI) to compensate for such a burden. The controller achieves this by activating a modified SpoT enzyme with sole hydrolysis activity, which lowers ppGpp level and thus de-represses ribosomes. 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 a growth rate decrease. 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 for the first time allows dynamic gene activation without growth rate defects for bacterial synthetic biology applications.

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Barajas, C., Gibson, J., Sandoval, L., del vecchio, d.. 2021-02-11. A burden-free gene overexpression system. https://doi.org/10.1101/2021.02.11.430724

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