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.

Constructing “quantized quorums” to guide emergent phenotypes through quorum quenching capsules

“…To test whether the HE “controller cell” interfere with QS‐dependent actions, we incubated the cells with W3110 pCT6 pET‐EGFP, a reporter strain that both generates and transduces the AI‐2 signal to produce enhanced green fluorescent protein (EGFP) (Tsao et al, ). Previously, we showed that by incubating with an already induced controller cell population, LW12 pACDBFG, at a ratio of 1:1 with the reporter strain, AI‐2 mediated gene expression could be quenched (Zargar et al, ). In Figure B, we co‐cultured W3110 pCT6 pET‐EGFP with SH1c pTrcHisB (control) and SH1c pLsrHE at a range of mixture ratios.…”

Incorporating LsrK AI‐2 quorum quenching capability in a functionalized biopolymer capsule

“…Activity of the modified enzyme, both in solution and attached to chitosan, was verified using three methods: an ATP assay, Kinase‐Glo® Plus (Promega, Madison, WI); the commonly‐used V. fischeri BB170 AI‐2 assay (Bassler et al, ; Zargar, Quan, Emamian, et al, ); and using a previously developed E. coli reporter cell, CT104 (Tsao et al, ). The AI‐2 assays are based on biological activity and the ATP assay is based on fluorescence.…”

“…There have been a variety of methods explored to interrupt autoinducer‐mediated communication networks (Rampioni, Leoni, & Williams, ). These typically involve their chemical rearrangement (Roy, Fernandes, Tsao, & Bentley, ), their sequestration (Zargar, Quan, Emamian, et al, ), or developing molecular analogues that compete for receptor binding sites (Roy et al, ; Sintim et al, ). The various techniques developed often align with specific features of the autoinducers, the targeted bacteria, and/or the signal transduction mechanisms.…”

Communicating artificial cells

“…However, rather than monitoring the cell density in order to, for example, initiate biofilm formation, quorum sensing is combined with different output functions, such as the synthesis of a toxin in order to maintain a desired concentration of cells [9]. Alternatively, bacteria can be engineered to overexpress components of the uptake and degradation pathways for a quorum signal, thereby depleting the concentration to levels below the detection threshold, effectively silencing communication [10]. Engineered communication paths can also be used to increase the robustness of a population.…”