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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM063525-01
Application #
6359816
Study Section
Special Emphasis Panel (ZGM1-GDB-1 (01))
Program Officer
Anderson, James J
Project Start
2001-06-02
Project End
2004-05-31
Budget Start
2001-06-02
Budget End
2002-05-31
Support Year
1
Fiscal Year
2001
Total Cost
$236,631
Indirect Cost
Name
University of California Berkeley
Department
Biomedical Engineering
Type
Schools of Engineering
DUNS #
094878337
City
Berkeley
State
CA
Country
United States
Zip Code
94704
Ham, Timothy S; Lee, Sung K; Keasling, Jay D et al. (2008) Design and construction of a double inversion recombination switch for heritable sequential genetic memory. PLoS One 3:e2815
Anderson, J Christopher; Clarke, Elizabeth J; Arkin, Adam P et al. (2006) Environmentally controlled invasion of cancer cells by engineered bacteria. J Mol Biol 355:619-27
Ham, Timothy S; Lee, Sung Kuk; Keasling, Jay D et al. (2006) A tightly regulated inducible expression system utilizing the fim inversion recombination switch. Biotechnol Bioeng 94:1-4
Khlebnikov, Artem; Keasling, Jay D (2002) Effect of lacY expression on homogeneity of induction from the P(tac) and P(trc) promoters by natural and synthetic inducers. Biotechnol Prog 18:672-4
Khlebnikov, A; Skaug, T; Keasling, Jay D (2002) Modulation of gene expression from the arabinose-inducible araBAD promoter. J Ind Microbiol Biotechnol 29:34-7
Khlebnikov, A; Datsenko, K A; Skaug, T et al. (2001) Homogeneous expression of the P(BAD) promoter in Escherichia coli by constitutive expression of the low-affinity high-capacity AraE transporter. Microbiology 147:3241-7