Synthetic tetracycline-inducible regulatory networks: computer-aided design of dynamic phenotypes

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p

Multiscale Hy3S: Hybrid stochastic simulation for supercomputers

BACKGROUND: Stochastic simulation has become a useful tool to both study natural biological systems and design new synthetic ones. By capturing the intrinsic molecular fluctuations of "small" systems, these simulations produce a more accurate picture of single cell dynamics, including interesting phenomena missed by deterministic methods, such as noise-induced oscillations and transitions between stable states. However, the computational cost of the original stochastic simulation algorithm can be high, motivating the use of hybrid stochastic methods. Hybrid stochastic methods partition the system into multiple subsets and describe each subset as a different representation, such as a jump Markov, Poisson, continuous Markov, or deterministic process. By applying valid approximations and self-consistently merging disparate descriptions, a method can be considerably faster, while retaining accuracy. In this paper, we describe Hy3S, a collection of multiscale simulation programs. RESULTS: Building on our previous work on developing novel hybrid stochastic algorithms, we have created the Hy3S software package to enable scientists and engineers to both study and design extremely large well-mixed biological systems with many thousands of reactions and chemical species. We have added adaptive stochastic numerical integrators to permit the robust simulation of dynamically stiff biological systems. In addition, Hy3S has many useful features, including embarrassingly parallelized simulations with MPI; special discrete events, such as transcriptional and translation elongation and cell division; mid-simulation perturbations in both the number of molecules of species and reaction kinetic parameters; combinatorial variation of both initial conditions and kinetic parameters to enable sensitivity analysis; use of NetCDF optimized binary format to quickly read and write large datasets; and a simple graphical user interface, written in Matlab, to help users create biological systems and analyze data. We demonstrate the accuracy and efficiency of Hy3S with examples, including a large-scale system benchmark and a complex bistable biochemical network with positive feedback. The software itself is open-sourced under the GPL license and is modular, allowing users to modify it for their own purposes. CONCLUSION: Hy3S is a powerful suite of simulation programs for simulating the stochastic dynamics of networks of biochemical reactions. Its first public version enables computational biologists to more efficiently investigate the dynamics of realistic biological systems

How to Build Kinetic Models of BioBricks

This BioBricks Foundation Request for Comments (BBF RFC) provides instructions on how to automatically generate a model of any BioBrick sequence. These models can be used in computer simulations of the dynamic behavior of all molecular components of the BioBrick

Malignant potential of intrahepatic biliary papillomatosis: a case report and review of the literature

BACKGROUND: Biliary papillomatosis (BP) is a rare disease entity with a strong malignant potential. It is characterized by multiple papillary adenomas involving both the intrahepatic and extrahepatic biliary tree. BP was considered in the past to be a disease with low malignant potential. However, a current review of the English literature revealed a high rate of malignant occurrence of approximately 41% and histological analysis along with the expression pattern of mucin core proteins (MUC) and mucin carbohydrate antigens suggests that BP is a borderline or low grade malignant neoplasm with a high malignant potential. CASE PRESENTATION: A 68 year-old male patient was referred to our hospital due to the presence of sudden right upper quadrant abdominal pain, nausea and dark urine. Imaging workup demonstrated dilatation of the left hepatic duct without the presence of a space-occupying lesion. A left hepatectomy and cholecystectomy were carried out and histological analysis revealed a moderately to poorly differentiated carcinoma of the left hepatic duct in the background of biliary papillomatosis. Postoperative course was uneventful. Unfortunately, two years after initial diagnosis the patient rapidly deteriorated and died from multiple pulmonary secondary deposits. CONCLUSION: BP should not be considered to be a benign disease. The clinical behavior, the high recurrence rate and the even higher malignant transformation occurrence, as well as the presence of carcinogenetic indicators (K-ras mutation, overexpression of p53, MUC and Tn antigens) strongly support that BP is a low-grade neoplasm with high malignant potential

Synthetic tetracycline-inducible regulatory networks: computer-aided design of dynamic phenotypes-2

<p><b>Copyright information:</b></p><p>Taken from "Synthetic tetracycline-inducible regulatory networks: computer-aided design of dynamic phenotypes"</p><p>BMC Systems Biology 2007;1():7-7.</p><p>Published online 9 Jan 2007</p><p>PMCID:PMC1885862.</p><p></p>cules (WT/w 2000 Tc, green line) or 5000 molecules (WT/w 2000 Tc, red line) of Tc are added into the medium at time 2 × 10sec, using wild-type kinetics. (B) Average number of GFP molecules when 2000 molecules of Tc are added into the medium at time 2 × 10sec, using wild-type kinetics (WT/w 2000 Tc, blue line), a 20 fold (Des 1.1/w 2000 Tc, green line) and a 50 fold (Des 1.2/w 2000 Tc, red line) increase in the dissociation constant of induced Tet-ON from of the gene encoding Tet-ON. (C) Average number of GFP molecules when 2000 molecules of Tc are added into the medium at time 2 × 10sec and 6 × 10sec, using wild-type kinetics (WT/w 2000 Tc (x2), blue line), a 20 fold (Des 1.1/w 2000 Tc (x2), green line) increase in the dissociation constant of induced Tet-ON from of the gene encoding Tet-ON. (D) Average number of GFP molecules when 2000 molecules of Tc are added into the medium at time 2 × 10sec, using wild-type kinetics (WT/w 2000 Tc, blue line), a doubled (Des 1.3/w 2000 Tc, green line) and a quadrupled (Des 1.4/w 2000 Tc, red line) half-life of Tet-ON

Synthetic tetracycline-inducible regulatory networks: computer-aided design of dynamic phenotypes-1

<p><b>Copyright information:</b></p><p>Taken from "Synthetic tetracycline-inducible regulatory networks: computer-aided design of dynamic phenotypes"</p><p>BMC Systems Biology 2007;1():7-7.</p><p>Published online 9 Jan 2007</p><p>PMCID:PMC1885862.</p><p></p>resent genes and are numbered according to Figure 1. Arrows represent repression or activation

Synthetic tetracycline-inducible regulatory networks: computer-aided design of dynamic phenotypes-3

<p><b>Copyright information:</b></p><p>Taken from "Synthetic tetracycline-inducible regulatory networks: computer-aided design of dynamic phenotypes"</p><p>BMC Systems Biology 2007;1():7-7.</p><p>Published online 9 Jan 2007</p><p>PMCID:PMC1885862.</p><p></p>cules (WT/w 2000 Tc, green line) or 5000 molecules (WT/w 5000 Tc, red line) of Tc are added into the medium at time 2 × 10sec, using wild-type kinetics. (B) Average number of GFP molecules when 2000 molecules of Tc are added into the medium at time 2 × 10sec, using wild-type kinetics (WT/w 2000 Tc, blue line), a 10 fold (Des 2.1/w 2000 Tc, green line) and a 50 fold (Des 2.2/w 2000 Tc, red line) increase in the dissociation constant of both TetR and induced Tet-ON from of the gene encoding Tet-ON. (C) Average number of GFP molecules when 2000 molecules of Tc are added into the medium at time 2 × 10sec, using wild-type kinetics (WT/w 2000 Tc, blue line). All other plots have a 10 fold increase in the dissociation constant of both TetR and induced Tet-ON from of the gene encoding Tet-ON, but differ in a 5 fold (Des 2.3/w 2000 Tc, green line) and 20 fold (Des 2.4/w 2000 Tc, red line) increase in the dissociation constant of both TetR and induced Tet-ON from of the gene encoding TetR. (D) Average number of GFP molecules when 2000 molecules of Tc are added into the medium at time 2 × 10sec, using wild-type kinetics (WT/w 2000 Tc, blue line). All other plots have mutated TetR variants that do not bind Tc and show a decreased binding affinity for (20 fold decrease in the dissociation constant), but differ in the half-life of Tet-ON, 40 min (Des 2.5/w 2000 Tc, green line) and 24 hr (Des 2.6/w 2000 Tc, red line)