Synthetic tetracycline-inducible regulatory networks: computer-aided design of dynamic phenotypes.
BACKGROUND
Tightly regulated gene networks, precisely controlling the expression of protein molecules, have received considerable interest by the biomedical community due to their promising applications. Among the most well studied inducible transcription systems are the tetracycline regulatory expression systems based on the tetracycline resistance operon of Escherichia coli, Tet-Off (tTA) and Tet-On (rtTA). Despite their initial success and improved designs, limitations still persist, such as low inducer sensitivity. Instead of looking at these networks statically, and simply changing or mutating the promoter and operator regions with trial and error, a systematic investigation of the dynamic behavior of the network can result in rational design of regulatory gene expression systems. Sophisticated algorithms can accurately capture the dynamical behavior of gene networks. With computer aided design, we aim to improve the synthesis of regulatory networks and propose new designs that enable tighter control of expression.


RESULTS
In this paper we engineer novel networks by recombining existing genes or part of genes. We synthesize four novel regulatory networks based on the Tet-Off and Tet-On systems. We model all the known individual biomolecular interactions involved in transcription, translation, regulation and induction. With multiple time-scale stochastic-discrete and stochastic-continuous models we accurately capture the transient and steady state dynamics of these networks. Important biomolecular interactions are identified and the strength of the interactions engineered to satisfy design criteria. A set of clear design rules is developed and appropriate mutants of regulatory proteins and operator sites are proposed.


CONCLUSIONS
The complexity of biomolecular interactions is accurately captured through computer simulations. Computer simulations allow us to look into the molecular level, portray the dynamic behavior of gene regulatory networks and rationally engineer novel ones with useful applications. We are able to propose, test and accept or reject design principles for each network. Guided by simulations, we develop a set of design principles for novel tetracycline-inducible networks.
Sotiropoulos
Vassilios
V
Department of Chemical Engineering and Materials Science, and Digital Technology Center, University of Minnesota, 421 Washington Ave SE, Minneapolis, MN 55455 USA. sotiropo@cems.umn.edu
Kaznessis
Yiannis N
YN
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D003198:Computer Simulation; D017076:Computer-Aided Design; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D053263:Gene Regulatory Networks / Q000187:drug effects*; D005818:Genetic Engineering; D008957:Models, Genetic; D009154:Mutation; D009876:Operon / Q000187:drug effects; D010641:Phenotype; D013752:Tetracycline / Q000494:pharmacology*; D013753:Tetracycline Resistance / Q000235:genetics
D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C083139:Tet O resistance protein, Bacteria; C470187:TetR(B) protein, E coli; D013752:Tetracycline