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DOI: http://dx.doi.org/10.7551/978-0-262-31050-5-ch063
Pages 481-488
First published 2 July 2012





Design and construction of a prototype CMY (Cyan-Magenta-Yellow) genetic circuit as a mutational readout device to measure evolutionary stability dynamics and determine design principles for robust synthetic systems

Sean C. Sleight, Herbert M. Sauro

Abstract

Synthetic biology is the engineering discipline for constructing novel organisms with functions that do not exist in nature. We recently engineered a prototype CMY genetic circuit that visually produces cyan, magenta, and yellow colors independently and in combination using different inducer molecules. Since the production of each color can be independently controlled, this allows for the production of a spectrum of colors that can be visualized in normal light conditions, and each color can be quantified using fluorescence measurements. We performed an evolution experiment to measure the evolutionary stability dynamics of this prototype CMY genetic circuit in 88 replicate populations of Escherichia coli, propagated with all colors turned on. Our results using particular inducer concentrations show that all 88 replicate populations change from a dark, green-brown color to a cyan-ish color after only 40 generations. In order to visualize the results of this experiment, we washed and concentrated the cells from each population into a different well of a 384-well plate at different evolutionary timepoints. The color change seen visually is confirmed with quantitative data that demonstrates the loss-of-function of both magenta and yellow colors with variation between replicate populations. We sequenced a single clone from four independently evolved populations and all clones have the same loss-of-function deletion mutation between homologous transcriptional terminators that removes the magenta and yellow expression cassettes. This parallel evolution was somewhat expected from results of previous work, but we expect that randomized and re-engineered versions of this circuit without repeats will produce more divergent results due to more stochastic loss-of-function mutations. This prototype CMY circuit serves as a mutational readout device and allows for a colorimetric and quantitative demonstration of evolution in action using synthetic biology.