We propose the design and implementation of a protocol for the forward design of complex genetic circuitry for precise control of gene expression in a given cell type subject to a set of environmental or other cellular inputs. The ability to efficiently and accurately design and build such circuitry will facilitate a number of central biotechnological goals. For example, circuits might be designed to: 1) """"""""instrument"""""""" cells to read out complex states, 2) maximize protein expression under different metabolic conditions, 3) perform a particular action (synthesis of a protein, initiation of a host-cell process) when particular conditions are met, or 4) provide a controllable mechanism by which a particular cellular system may be perturbed in a designed way to understand cellular function. Applications for such technology span the design of microorganisms for industrial protein production, to precise design of vectors for gene therapy. During the course of the project a theoretical and experimental framework to characterize naturally occurring genetic control circuits and to assemble novel genetic control circuits from the characterized parts to meet a particular control strategy will be developed.
The specific aims may be concisely stated as: 1) to create and implement an experimental protocol designed to rapidly characterize and tune biological parts (promoters, terminators, transcripts, etc.) to sufficient detail that accurate mathematical models may be derived for the kinetics of each, 2) to create very detailed, experimentally validated models of cellular environmental sensing networks in which there are varying degrees of previous knowledge to hone the circuit analysis technology and provide conceptual models for future circuit designs; 3) using these results, to develop a streamlined protocol for computer-aided design of gene expression followed by implementation of a number of """"""""canonical"""""""" circuit designs. As exemplars, we will study mathematically, computationally and experimentally, three """"""""orthogonal"""""""" examples of genetic expression switches in E coli: the chemosensing arabinose promoter system, the type-1C pili phase variation control network, and the OmpR-mediated osmoregulatory system.
