Rational design of ‘controller cells’ to manipulate protein and phenotype expression

Abstract

Coordination between cell populations via prevailing metabolic cues has been noted as a promising approach to connect synthetic devices and drive phenotypic or product outcomes. However, there has been little progress in developing ‘controller cells’ to modulate metabolic cues and guide these systems. In this work, we developed ‘controller cells’ that manipulate the molecular connection between cells by modulating the bacterial signal molecule, autoinducer-2, that is secreted as a quorum sensing (QS) signal by many bacterial species. Specifically, we have engineered Escherichia coli to overexpress components responsible for autoinducer uptake (lsrACDB), phosphorylation (lsrK), and degradation (lsrFG), thereby attenuating cell–cell communication among populations. Further, we developed a simple mathematical model that recapitulates experimental data and characterizes the dynamic balance among the various uptake mechanisms. This study revealed two controller ‘knobs’ that serve to increase AI-2 uptake: overexpression of the AI-2 transporter, LsrACDB, which controls removal of extracellular AI-2, and overexpression of the AI-2 kinase, LsrK, which increases the net uptake rate by limiting secretion of AI-2 back into the extracellular environment. We find that the overexpression of lsrACDBFG results in an extraordinarily high AI-2 uptake rate that is capable of completely silencing QS-mediated gene expression among wild-type cells. We demonstrate utility by modulating naturally occurring processes of chemotaxis and biofilm formation. We envision that ‘controller cells’ that modulate bacterial behavior by manipulating molecular communication, will find use in a variety of applications, particularly those employing natural or synthetic bacterial consortia.