New tools for self-organised pattern formation

Position-dependent gene expression is a critical aspect of the development and behaviour of multicellular organisms. It requires a complex series of interactions to occur between different cell types in addition to intracellular signalling cascades. We used Escherichia coli to study the properties of an artificial signalling system at the interface between two expanding cell populations. We genetically engineered one population to produce a diffusible acyl-homoserine lactone (AHL) signal, and another population to respond to it. Our experiments demonstrate how such a signal can be used to reproducibly generate simple visible patterns with high accuracy in swimming agar. The producing and responding cassettes of two such signalling systems can be linked to produce a symmetric interface for bidirectional communication that can be used to visualise molecular logic. Intracellular feedback between these two cassettes would then create a framework for self-organised patterning of higher complexity. Adapting the experiments of Basu et al. (Basu et al., 2005) using cell motility, rather than a differential response to AHL concentrations as a way to define zones of response, we noted how the interaction of sender and receiver cell populations on a swimming plate could lead to complex pattern formation. Equipping highly motile strains such as E. coli MC1000 with AHL-mediated auto-inducing systems based on Vibrio fischeri luxI/luxR and Pseudomonas aeruginosa lasI/lasR cassettes would allow the amplification of a response to an AHL signal and its propagation. We designed and synthesised codon-optimised auto-inducing luxI/R and lasI/R cassettes as optimal gene expression is crucial for the generation of robust patterns. We still have to complete and test the entire genetic circuitry, although by modelling the system we were able to demonstrate its feasibility. © 2007 The Institution of Engineering and Technology.

An in vitro transposon system for highly regulated gene expression: construction of Escherichia coli strains with arabinose-dependent growth at low temperatures.

Placing a gene of interest under the control of an inducible promoter greatly aids the purification, localization and functional analysis of proteins but usually requires the sub-cloning of the gene of interest into an appropriate expression vector. Here, we describe an alternative approach employing in vitro transposition of Tn Omega P(BAD) to place the highly regulable, arabinose inducible P(BAD) promoter upstream of the gene to be expressed. The method is rapid, simple and facilitates the optimization of expression by producing constructs with variable distances between the P(BAD) promoter and the gene. To illustrate the use of this approach, we describe the construction of a strain of Escherichia coli in which growth at low temperatures on solid media is dependent on threshold levels of arabinose. Other uses of the transposable promoter are also discussed.