Self-Organization in a Synthetic Consortium Figure 1. Design and initial characterization of the biofilm-forming consortium. (A) The synthetic symbiotic consortium. The blue population cannot synthesize diaminopimelate or lysine; when cultured without lysine or diaminopimelate, this population forms only a scant biofilm. The yellow population cannot form biofilms alone but is otherwise healthy. It synthesizes C4HSL, which diffuses freely and activates production of diaminopimelate in the blue population. Yellow cells become bound within the biofilm formed by the blue population and rescue growth. Together, the two populations form viable biofilms that persist. (B) The symbiotic consortium functions as designed. The blue population control forms a biofilm which eventually dies (blue bars), and the yellow population accumulates very little biomass (insignificant, not shown). When the yellow and blue populations are inoculated into a flow chamber in a 50/50 mixture, more biomass accumulates than in either control (solid yellow areas, total yellow biomass in the biofilm; solid blue areas, total blue biomass in the biofilm; the sum of blue and yellow areas is the overall total biomass in the biofilm at each time point; all errors are s.d.). All biofilm measurements were derived from images quantified with COMSTAT [22]. doi:10.1371/journal.pone.0016791.g001 population, which is identified by constitutive expression of cyan fluorescent protein (CFP, the ‘‘blue population’’), can form initially healthy biofilms. However, it cannot synthesize a set of metabolites critical for cell growth and division and therefore quickly dies. To engineer this strain we interrupted a critical pathway responsible for synthesis of diaminopimelate and lysine by deleting a key gene, DapD (E. coli MG1655DDapD) [27]. We then restored DapD under the control of a RhlR-dependent promoter. The transcription factor RhlR is activated by a small-molecule autoinducer, butanoyl-homoserine lactone (C4HSL) [28], which is provided by a second population. This ‘‘yellow population,’’ identified by constitutive expression of yellow fluorescent protein (YFP), cannot form biofilms alone but is otherwise healthy. To engineer this strain we identified genetic loci implicated in E. coli biofilm formation and, by trial-and-error, found a subset of these loci which when deleted significantly reduce biofilm formation without compromising growth rate. This set of deletions, removing genes involved in expression of type I pili, curli, colanic acid, and capsular polysaccharides, was concurrently identified elsewhere (E. coli MG1655Dfim, DwcaL–wza, DcsgC-csgG) [29]. Finally, strong PLoS ONE | www.plosone.org 2 February 2011 | Volume 6 | Issue 2 | e16791