(116g) Construction of Genetic Programs by Layering Logic Gates in Single Cells

Many applications require cells to integrate multiple environmental signals and to implement synthetic control over biological processes.  Genetic circuits enable cells to perform computational operations, interfacing biosensors and actuators.  Despite advances in the rational construction of genetic circuits, synthetic biologists are still limited by the lack of well-characterized orthogonal parts and predictive computational models.  Here, we harness a toolbox of genetic parts from bacteria to construct three 2-input AND gates.  To this end, genetic parts have been recruited from Type III Secretion System (T3SS) where activator-chaperone pairs interact to regulate virulence gene expression.  Such pairs have been assembled in E. coli and their expression has been tuned by saturation mutagenesis of promoters and RBSs.  These engineered 2-input AND gates have been fully characterized, modified (via directed evolution) to make them orthogonal, and rationally layered to make complex genetic programs, the largest of which is a 4-input AND gate that consists of 11 regulatory proteins.  In addition, transfer function models have been developed to computationally analyze the AND gates.  This work represents a step toward constructing programmed cells that are able to process multiple input signals and to produce desirable outputs for real-world applications.