ABSTRACT
Boolean logic and arithmetic through DNA excision (BLADE) is a recently developed platform for implementing inducible and logical control over gene expression in mammalian cells, which has the potential to revolutionise cell engineering for therapeutic applications. This 2-input 2-output platform can implement 256 different logical circuits that exploit the specificity and stability of DNA recombination. Here, we develop the first mechanistic mathematical model of the 2-input BLADE platform based on Cre- and Flp-mediated DNA excision. After calibrating the model on experimental data from two circuits, we demonstrate close agreement between model outputs and data on the other 111 circuits that have so far been experimentally constructed using the 2-input BLADE platform. Model simulations of the remaining 143 circuits that have yet to be tested experimentally predict excellent performance of the 2-input BLADE platform across the range of possible circuits. Circuits from both the tested and untested subsets that perform less well consist of a disproportionally high number of STOP sequences. Model predictions suggested that circuit performance declines with a decrease in recombinase expression and new experimental data was generated that confirms this relationship.
Subject(s)
Computer Simulation , DNA/genetics , Recombination, Genetic , Algorithms , Calibration , HEK293 Cells , Humans , Stochastic Processes , Synthetic BiologyABSTRACT
Inducible genetic switches based on tyrosine recombinase-based DNA excision are a promising platform for the regulation and control of chimeric antigen receptor (CAR) T cell activity in cancer immunotherapy. These switches exploit the increased stability of DNA excision in tyrosine recombinases through an inversion-excision circuit design. Here, the authors develop the first mechanistic mathematical model of switching dynamics in tyrosine recombinases and validate it against experimental data through both global optimisation and statistical approximation approaches. Analysis of this model provides guidelines regarding which system parameters are best suited to experimental tuning in order to establish optimal switch performance in vivo. In particular, they find that the switching response can be made significantly faster by increasing the concentration of the inducer drug 4-OHT and/or by using promoters generating higher expression levels of the FlpO recombinase.
ABSTRACT
BACKGROUND: The antibiotic methylenomycin A is produced naturally by Streptomyces coelicolor A3(2), a model organism for streptomycetes. This compound is of particular interest to synthetic biologists because all of the associated biosynthetic, regulatory and resistance genes are located on a single cluster on the SCP1 plasmid, making the entire module easily transferable between different bacterial strains. Understanding further the regulation and biosynthesis of the methylenomycin producing gene cluster could assist in the identification of motifs that can be exploited in synthetic regulatory systems for the rational engineering of novel natural products and antibiotics. RESULTS: We identify and validate a plausible architecture for the regulatory system controlling methylenomycin production in S. coelicolor using mathematical modeling approaches. Model selection via an approximate Bayesian computation (ABC) approach identifies three candidate model architectures that are most likely to produce the available experimental data, from a set of 48 possible candidates. Subsequent global optimization of the parameters of these model architectures identifies a single model that most accurately reproduces the dynamical response of the system, as captured by time series data on methylenomycin production. Further analyses of variants of this model architecture that capture the effects of gene knockouts also reproduce qualitative experimental results observed in mutant S. coelicolor strains. CONCLUSIONS: The mechanistic mathematical model developed in this study recapitulates current biological knowledge of the regulation and biosynthesis of the methylenomycin producing gene cluster, and can be used in future studies to make testable predictions and formulate experiments to further improve our understanding of this complex regulatory system.
