The use of biological components for the construction of artificial genetic circuits has the potential to create novel behaviors in host organisms and to help researchers develop 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 often do not meet the design requirements for a desired function when moved to artificial systems. 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 activities 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 have designed a minimal genetic AND gate based upon interactions between LuxR and the LacI repressor at a hybrid promoter, and have examined how the circuit's logical properties can be improved using our mutants. Application of the mutants to the AND gate was suggested by the use of a simple mathematical model, which predicted improvements to the logical properties of the AND gate for LuxR mutants with increased rates of transcriptional activation. In agreement with the mathematical predictions, incorporation of the LuxR mutants into the AND gates resulted in improvement of the circuit's logic with a maximum improvement of ~1.5-fold.

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