A split methyl halide transferase and gate that reports by synthesizing an indicator gas

Emily M. Fulk, Dongkuk Huh, Joshua T. Atkinson, Margaret Lie, Caroline A. Masiello, Jonathan J. Silberg

Research output: Contribution to journalArticlepeer-review

10 Scopus citations

Abstract

Monitoring microbial reactions in highly opaque or autofluorescent environments like soils, seawater, and wastewater remains challenging. To develop a simple approach for observing post-translational reactions within microbes situated in environmental matrices, we designed a methyl halide transferase (MHT) fragment complementation assay that reports by synthesizing an indicator gas. We show that backbone fission within regions of high sequence variability in the Rossmann domain yields split MHT (sMHT) AND gates whose fragments cooperatively associate to synthesize CH3Br. Additionally, we identify a sMHT whose fragments require fusion to pairs of interacting partner proteins for maximal activity. We also show that sMHT fragments fused to FKBP12 and the FKBP-rapamycin binding domain of mTOR display significantly enhanced CH3Br production in the presence of rapamycin. This gas production is reversed in the presence of the competitive inhibitor of FKBP12/FKPB dimerization, indicating that sMHT is a reversible reporter of post-translational reactions. This sMHT represents the first genetic AND gate that reports on protein-protein interactions via an indicator gas. Because indicator gases can be measured in the headspaces of complex environmental samples, this assay should be useful for monitoring the dynamics of diverse molecular interactions within microbes situated in hard-to-image marine and terrestrial matrices.

Original languageEnglish (US)
Pages (from-to)3104-3113
Number of pages10
JournalACS Synthetic Biology
Volume9
Issue number11
DOIs
StatePublished - Nov 20 2020
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biomedical Engineering
  • Biochemistry, Genetics and Molecular Biology (miscellaneous)

Keywords

  • AND gate
  • Biosensor
  • Indicator gas
  • Methyl halide transferase
  • Protein engineering
  • Protein-fragment complementation assay
  • Reporter

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