The plasmid with the SRI+ phenotype (pCC-CDI) was constructed from the plasmid pDAL661 (45) by introducing a constitutive promoter J23119 (http://parts.igem.org/Main_Page) upstream of the contact-dependent inhibition (CDI) operon. The copy-controlled plasmid, pCC1BAC, was purchased from Epicentre and used in the SRI− phenotype. The plasmid with the LRI+ phenotype (pCC-AHL) was constructed by cloning a constitutive Ptrc promoter from pChemoK (46), the luxI gene, and the rnpT1 terminator into the pCC1BAC vector. The plasmid with LRI− phenotype (pCC-CcdB) has a copy of the AHL-inducible ccdB gene. It was constructed by cloning the luxR-PluxR-PluxI cassette from pTD103LuxI_sfGFP (47), a mutant version of ccdB gene (ccdB [9delA], a truncated ccdB gene with a reduced toxicity) and rnpT1 into pCC1BAC. The plasmid pCC-CDI-CcdB was constructed from pCC-CDI by introducing the AHL-inducible ccdB gene from pCC-CcdB. It was used in conjugation with the plasmid pCC-AHL for bidirectional competition.

All plasmid components were used as untagged except for luxI, which has a 3′ ssrA degradation tag (AANDENYALAA) for rapid protein degradation (48). All plasmids used in this study have the same backbone as the plasmid pCC1BAC. The copy number of the plasmids can be switched from single to multiple (~10 to 12) upon induction with either CopyControl Induction Solution provided by the manufacturer or l-(+)-arabinose solution [0.2% (w/v)]. The plasmid maps and their characteristics are listed in fig. S1. GenBank accession numbers for SRI and LRI plasmids are as follows: MG867730, MG867731, MG867732, and MG867733.

Two E. coli EPI300 strains that constitutively express yemGFP or mKate2 (EPI300 lacZ::PJ23119-yemGFP and EPI300 lacZ::PJ23119-mKate2) were constructed with a lambda Red–mediated homologous recombination system by replacing lacZ with PJ23119-driven yemGFP or mKate2 using modified pKD4 plasmids, pKD4-PJ23119-yemGFP and pKD4-PJ23119-mKate2, as previously described (49). The resulting strains, used in range expansion experiments, were named as EPI300-yemGFP and EPI300-mKate2. Another two strains (EPI300-P21-yemGFP and EPI300-P21-mKate2) that have stronger fluorescence expression were constructed for single-cell experiments using the clonetegration method through the integration of PJ23119-yemGFP or PJ23119-mKate2 into the attB site of phage P21 on the chromosome of EPI300 (50). All strains used in this study are described in table S1.

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