Fluorescence-tagged Escherichia coli cells can be made to "blink" in unison by means of a constructed network of genes and proteins that coordinates oscillations within the growing cell population, according to Jeff Hasty and colleagues from the University of California, San Diego (UCSD) in La Jolla. In 2008 the team produced "flashing" microbial cells, but now they endowed those flashing bacteria with the capacity to synchronize their colony-building efforts.

This new form of a "molecular clock" was assembled in E. coli from
borrowed quorum sensing components- luxI from Vibrio fischeri and aiiA from Bacillus thuringiensis. LuxI synthase, encoded by the transplanted luxI genes, produces the quorum sensing signal acyl-homoserine lactone (AHL), a small molecule that diffuses readily across cell membranes of neighboring bacteria and binds to their constitutively produced LuxR. The resulting LuxR-AHL complex activates the luxI promoter (PluxI)- driven transcription of luxI and also stimulates expression of green fluorescent protein (yemGFP). Negative regulation comes from AiiA, which catalyses AHL degradation. "It's an autoinducing circuit that depends on the dynamic interactions of positive and negative feedback loops to produce regular AHL pulses, and these become the molecular clock's timekeepers," says Tal Danino, a leader of the UCSD team.

Local cell density can be modulated with a microfluidic device consisting of amain channel feeding into a chamber in which the E. coli cells are housed. "Once seeded, a monolayer of cells grows in the trapping chamber and are eventually pushed into a main channel and then into the waste port," Hasty says. The design allows researchers to feed the colony nutrients, inducers, inhibitors, and GFP reporter genes to sustain its exponentially dividing cells for 4 days. Importantly, the flow rate is externally controlled, enabling the scientists to tinker with optimal cell and inducer densities. As would be expected, oscillations do not synchronize at low cell densities. Studies of uncoupled E. coli cells confirm that AHL diffusion provides the means for synchronizing activities at higher cellular concentrations. "AHL has a dual role in this system," Hasty says. "It both activates the genes necessary for intracellular oscillations and mediates the coupling between cells."

"Genetically engineering E. coli, microbes in continual frenzied motion, to express a fluorescent protein in synchrony is no small achievement," says synthetic biologist Martin Fussenegger at the Swiss Federal Institute of Technology in Zurich, who likens the feat to "engineering all the world's traffic lights to blink on and off at the same time." While similar to previously reported synthetic oscillators, "the use of the rhythmic synthesis of molecules such as AHL as a pacemaker to coordinate the behavior of individual oscillators in a growing population of cells is a quantum leap in molecular-clock design."

The potential applications of this research are vast. Synchronized waves control sleep-wake cycles and underlie neuronal behavior and the regular release of hormones. Understanding naturally occurring time-keeping processes could provide insights into pathologies behind sleep disorders and epileptic seizures as well as enable the development of cell implants that deliver hormones such as insulin or other therapeutic proteins at specific times and in precise doses. The Hasty group is now designing larger and more complex biological systems, including molecular networks that function as spatially distributed sensors and synthetic machinery "for coupling complex dynamical processes across a multicenter population," he says.

The synchronized bacterial colonies could be considered a nicely timed anniversary gift for the field of synthetic biology, which began in January 2000 with a report of the first synthetic biological oscillator and a bistable gene regulatory network known as a toggle switch.

Details of the recent UCSD work can be found in the 21 January 2010 Nature [doi:10.1038/nature08753]. Also see Nature 403, 335-338 (20 January 2000) doi:10.1038/35002125 and Nature 403, 339-342 (20 January 2000) doi:10.1038/35002131.

Marcia Stone
Marcia Stone is a science writer based in New York City. More of her work can be seen at http://www.mstoneworks.net.