Pattern Formation in Synthetic Bacterial Colonies
Our synthetic biology project explores the engineering and control of cell differentiation in multi-cellular systems. In particular, we will analyse the phenotype differentiation of microbial colonies guided by the interchange of molecular signals. We have included in the design of our circuit transcriptional and post-transcriptional levels of regulation. The transcriptional control is exerted by the regulators LuxR and RhlR, which bind to their cognate molecular signals 3-oxo-C6-HSL (3OC6) and C4-HSL (C4), respectively. These regulators are part of two orthogonal quorum sensing systems, the rhl system from Pseudomonas aeruginosa and the lux system from Vibrio fisheri. Moreover, we used four well characterised repressors LacI and TetR (from Escherichia coli), CI and CI434 from phage lambda and bacteriophage 434, respectively. The system is also regulated at the post-transcriptional level. This control consists of a theophylline responsive riboswitch regulating translational initiation. Furthermore all the regulators and the enzymes present in the circuit were tagged with degradation tags (a fast- degradation tag and a slow-degradation tag), which conferred different half- lives to the corresponding proteins. The resulting phenotypes are associated to the expression of two fluorescent proteins (GFP and RED-Mcherry) and two signal molecules 3OC6 and C4 synthesised by the LuxI and RhlI.
All these components are wired in a double negative feedback loop genetic circuit, the dynamics of which exhibits a bi-stable behaviour. The system modulates the expression of two possible phenotypes depending on the signal that is first sensed. If the system receives first C4 it becomes insensitive to 3OC6 and starts producing 3OC6 and RED-Mcherry (red phenotype). On the other hand, if the system receives first 3OC6 it becomes insensitive to C4 and produces C4 and GFP (green phenotype).
Our synthetic double negative feedback loop has been designed using our software tool, Infobiotics workbench, that unites computational and molecular biology protocols by providing a multi-compartmental and rule-based modelling language, and related analysis tools, with a library of modular components modelling the different parts of our system. The instantiation and combination of these modules corresponds to different lab protocols such as ligations and transformations. Computer simulations of an initially homogeneous culture of bacteria carrying our design predict the emergence of spatial patterns consisting of alternating green/red stripes.