Fig. 1. The broad-host-range pS4 plasmid harboring the inverter circuit was introduced into 6 distinct hosts from the Gammaproteobacteria class. (IMAGE)

Nanjing Agricultural University The Academy of Science

Fig. 1. The broad-host-range pS4 plasmid harboring the inverter circuit was introduced into 6 distinct hosts from the Gammaproteobacteria class.

Caption

 (A) Schematic of Ara- and aTc-inducible inverter circuit. In the presence of aTc, the upstream cassette is up-regulated, leading to production of AraC transcription factor and mKate reporter protein. In the absence of Ara (Ara−), AraC acts as a repressor and binds to its cognate PBAD promoter to down-regulate the downstream cassette. When bound to Ara (Ara+), AraC instead acts as an activator and up-regulates expression of sfGFP and TetR, creating a negative feedback loop of AraC expression. RiboA and RiboB are autocatalytic ribozyme insulators. Un-RBSn are BASIC linkers with a RBS in their adapter region. Number in Un indicates the BASIC linker family (1, 2, or 3). Number in RBSn indicates relative translational strength of the RBS, from 1 (weakest) to 3 (strongest). (B) Plasmid pS4 was transformed by electroporation into six bacterial Gammaproteobacteria hosts. Hosts are color-coded. E. co, E. coli; H. ae, H. aestusnigri; H. oc, H. oceani; P. de, P. deceptionensis; P. fl, P. fluorescens; P. pu, P. putida. (C) Specific growth rate (μ) and (D) carrying capacity (A) of WT strains in the presence of aTc (10 ng/ml) and Ara (20 mM) and no inducer (NI). Error bars show standard error of the mean (n = 3 biological replicates, with 4 technical replicates each). sfGFP (E) and mKate (F) autofluorescence normalized by OD600 over time by WT strains in the absence of inducer. Blank is autofluorescence of wells containing only LB medium. (G) The 172 single-copy genes in the Gammaproteobacteria hidden Markov model set from GToTree used for phylogeny inference grouped by functional annotation. Numbers of genes in the largest grouped are indicated. (H) Phylogenomic tree inferred from multi-locus sequence alignment of concatenated gene hits. Xanthomonas fragariae PD855 was chosen as outgroup. Scale bar indicates the number of amino acid substitutions per site between 2 sequences. Tree was inferred using the neighbor-joining method in MEGAX. The percentage of replicate trees in which the associated taxa clustered together in bootstrap test (1,000 replicates) are shown next to the branches.

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BioDesign Research

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