571ad Improvement of Artificial Genetic Circuits Using Mutant Libraries of the Luxr Transcriptional Activator

Daniel J. Sayut and Lianhong Sun. Chemical Engineering, University of Massachusetts, 159 Goessmann Laboratory, 686 North Pleasant St, Amherst, MA 01003

The use of biological components for the construction of artificial genetic circuits has the potential for creating novel behaviors in host organisms, and developing a deeper understanding of the design principles underlying natural systems. Natural components, however, have been evolutionarily optimized to function in their specific cellular contexts and commonly do not meet the design requirements for a desired function when moved to artificial systems. This problem is made worse by uncertainties in the design properties that govern natural systems, and in the properties of the components, both resulting from the inability to accurately measure biological parameters. The directed evolution of biological components has been suggested as a way to overcome this problem, as it can be used to generate mutant libraries that have diverse properties while retaining the original function of the mutated protein. Following this approach we have generated a library of mutant LuxR transcriptional activators that have a ten-fold range of increased sensitivities to the 3-oxo-C6 homoserine lactone signaling molecule. Previously, we used this library to improve the properties of artificial positive feedback loops. As an extension of this work, we will show how the LuxR mutant library can also be used to improve the logical outputs of a genetic AND gate where circuit responses are dependent on interactions between LuxR and the LacI repressor. The increased logic of the AND gate was related to the increased sensitivities of the LuxR mutants and a maximum logical improvement of ~1.5-fold was obtained. Finally, we will discuss recent progress on the use of the mutant library to alter the dynamic outputs of a minimal LuxR-LacI based relaxation oscillator.