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Escherichia coli “Marionette” strains with 12 highly optimized small-molecule sensors


Cellular processes are carried out by many genes, and their study and optimization requires multiple levers by which they can be independently controlled. The most common method is via a genetically encoded sensor that responds to a small molecule. However, these sensors are often suboptimal, exhibiting high background expression and low dynamic range. Further, using multiple sensors in one cell is limited by cross-talk and the taxing of cellular resources. Here, we have developed a directed evolution strategy to simultaneously select for lower background, high dynamic range, increased sensitivity, and low cross-talk. This is applied to generate a set of 12 high-performance sensors that exhibit >100-fold induction with low background and cross-reactivity. These are combined to build a single “sensor array” in the genomes of E. coli MG1655 (wild-type), DH10B (cloning), and BL21 (protein expression). These “Marionette” strains allow for the independent control of gene expression using 12 small-molecule inducers.

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Fig. 1: A dual selection for sensor optimization.
Fig. 2: Improved sensor performance.
Fig. 3: Marionette-Wild performance.
Fig. 4: Marionette-Wild expression level optimization metabolic pathway balancing using Marionette-Wild.

Data availability

Additional data supporting this study are available from the corresponding author upon reasonable request. The sequences of the following plasmids and strains are provided in GenBank: pAJM.711 (PPhlF-YFP) MH101715; pAJM.712 (PCymRC-YFP) MH101716; pAJM.713 (PLuxB-YFP) MH101717; pAJM.714 (PVanCC-YFP) MH101718; pAJM.715 (PTac-YFP) MH101719; pAJM.717 (PTet*-YFP) MH101720; pAJM.716 (PBAD-YFP) MH101721; pAJM.718 (PBetI-YFP) MH101722; pAJM.719 (PTtg-YFP) MH101723; pAJM.1459 (P3B5C-YFP) MH101724; pAJM.721 (PSalTTC-YFP) MH101725; pAJM.944 (PCin-YFP) MH101726; pAJM.847 (PhlFAM + PPhlF-YFP) MH101727; pAJM.657 (CymRAM + PCymRC-YFP) MH101728; pAJM.474 (LuxR + PLuxB-YFP) MH101729; pAJM.773 (VanRAM + PVanCC-YFP) MH101730; pAJM.336 (LacIAM + PTac-YFP) MH101731; pAJM.011 (TetR + PTet*-YFP) MH101732; pAJM.677 (AraCAM + AraE + PBAD-YFP) MH101733; pAJM.683 (BetIAM + PBetI-YFP) MH101734; pAJM.661 (TtgRAM + PTtg-YFP) MH101735; pAJM.690 (PcaUAM + P3B5B-YFP) MH101736; pAJM.771 (NahRAM + PSalTTC-YFP) MH101737; pAJM.1642 (CinRAM + PCin-YFP) MH101738; pAJM.884 (AcuRAM + PAcu-YFP) MH101739; pAJM.969 (MphRAM + EryR + PMph-YFP) MH101740. The following plasmids and strains can be acquired from Addgene: pAJM.711 (PPhlF-YFP) 108512; pAJM.712 (PCymRC-YFP) 108513; pAJM.713 (PLuxB-YFP) 108514; pAJM.714 (PVanCC-YFP) 108515; pAJM.715 (PTac-YFP) 108516; pAJM.717 (PTet*-YFP) 108517; pAJM.716 (PBAD-YFP) 108518; pAJM.718 (PBetI-YFP) 108519; pAJM.719 (PTtg-YFP) 108520; pAJM.1459 (P3B5C-YFP) 108521; pAJM.721 (PSalTTC-YFP) 108522; pAJM.944 (PCin-YFP) 108523; pAJM.847 (PhlFAM + PPhlF-YFP) 108524; pAJM.657 (CymRAM + PCymRC-YFP) 108525; pAJM.474 (LuxR + PLuxB-YFP) 108526; pAJM.773 (VanRAM + PVanCC-YFP) 108527; pAJM.336 (LacIAM + PTac-YFP) 108528; pAJM.011 (TetR + PTet*-YFP) 108529; pAJM.677 (AraCAM + AraE + PBAD-YFP) 108530; pAJM.683 (BetIAM + PBetI-YFP) 108531; pAJM.661 (TtgRAM + PTtg-YFP) 108532; pAJM.690 (PcaUAM + P3B5B-YFP) 108533; pAJM.771 (NahRAM + PSalTTC-YFP) 108534; pAJM.1642 (CinRAM + PCin-YFP) 108535; pAJM.884 (AcuRAM + PAcu-YFP) 108536; pAJM.969 (MphRAM + EryR + PMph-YFP) 108537; sAJM.1504 (Marionette-Clo) 108251; sAJM.1505 (Marionette-Pro) 108253; sAJM.1506 (Marionette-Wild) 108254.


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This work was supported by the US Office of Naval Research Multidisciplinary University Research Initiative grant #N00014-16-1-2388 (A.J.M., T.H.S.-S., E.G., J.Z., and C.A.V.). This work was supported in part by the Koch Institute Support (core) Grant P30-CA14051 from the National Cancer Institute. We would like to thank A.M. Kunjapur and K.L.J. Prather (Department of Chemical Engineering, Massachusetts Institute of Technology) for providing DNA templates for the amplification of PVan, P3B5, vanR, and pcaU and performing the initial characterization of VanR in E. coli. We would also like to thank S. Liu at the MIT-Broad Foundry for assisting in the RNA-seq and ribosome profiling sequencing run.

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A.J.M. and C.A.V. conceived the study and designed the experiments; E.G. performed the mass spectrometry experiments; J.Z. performed the RNA sequencing and ribosome profiling experiments; A.J.M. performed all other experiments. A.J.M., T.H.S.-S., E.G., and J.Z., analyzed the data; A.J.M. and C.A.V. wrote the manuscript with input from all the authors.

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Correspondence to Christopher A. Voigt.

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Supplementary Tables 1–12, Supplementary Figures 1–21, Supplementary Notes 1–4

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Meyer, A.J., Segall-Shapiro, T.H., Glassey, E. et al. Escherichia coli “Marionette” strains with 12 highly optimized small-molecule sensors. Nat Chem Biol 15, 196–204 (2019).

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