Mechanistic modelling of a recombinase-based two-input temporal logic gate

Site-specific recombinases (SSRs) mediate efficient manipulation of DNA sequences in vitro and in vivo. In particular, serine integrases have been identified as highly effective tools for facilitating DNA inversion, enabling the design of genetic switches that are capable of turning the expression of a gene of interest on or off in the presence of a SSR protein. The functional scope of such circuitry can be extended to biological Boolean logic operations by incorporating two or more distinct integrase inputs. To date, mathematical modelling investigations have captured the dynamical properties of integrase logic gate systems in a purely qualitative manner, and thus such models are of limited utility as tools in the design of novel circuitry. Here, the authors develop a detailed mechanistic model of a two-input temporal logic gate circuit that can detect and encode sequences of input events. Their model demonstrates quantitative agreement with time-course data on the dynamics of the temporal logic gate, and is shown to subsequently predict dynamical responses relating to a series of induction separation intervals. The model can also be used to infer functional variations between distinct integrase inputs, and to examine the effect of reversing the roles of each integrase on logic gate output

Mechanistic modeling of a rewritable recombinase addressable data module

Many of the most important applications predicted to arise from Synthetic Biology will require engineered cellular memory with the capability to store data in a rewritable and reversible manner upon induction by transient stimuli. DNA recombination provides an ideal platform for cellular data storage and has allowed the development of a rewritable recombinase addressable data (RAD) module, capable of efficient data storage within a chromosome. Here, we develop the first detailed mechanistic model of DNA recombination, and validate it against a new set of in vitro data on recombination efficiencies across a range of different concentrations of integrase and gp3. Investigation of in vivo recombination dynamics using our model reveals the importance of fully accounting for all mechanistic features of DNA recombination in order to accurately predict the effect of different switching strategies on RAD module performance, and highlights its usefulness as a design tool for building future synthetic circuitry

Modeling the architecture of the regulatory system controlling methylenomycin production in Streptomyces coelicolor

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

The breakthrough listen search for intelligent life: 1.1–1.9 GHz observations of 692 nearby stars

We report on a search for engineered signals from a sample of 692 nearby stars using the Robert C. Byrd Green Bank Telescope, undertaken as part of the Breakthrough Listen Initiative search for extraterrestrial intelligence. Observations were made over 1.1−1.9 GHz (L band), with three sets of five-minute observations of the 692 primary targets, interspersed with five-minute observations of secondary targets. By comparing the “ON” and “OFF” observations we are able to identify terrestrial interference and place limits on the presence of engineered signals from putative extraterrestrial civilizations inhabiting the environs of the target stars. During the analysis, eleven events passed our thresholding algorithm, but a detailed analysis of their properties indicates they are consistent with known examples of anthropogenic radio frequency interference. We conclude that, at the time of our observations, none of the observed systems host high-duty-cycle radio transmitters emitting between 1.1 and 1.9 GHz with an Equivalent Isotropic Radiated Power of ∼ 1013 W, which is readily achievable by our own civilization. Our results suggest that fewer than ∼ 0.1% of the stellar systems within 50 pc possess the type of transmitters searched in this survey

Mechanistic mathematical models for the design of synthetic biological systems : DNA recombination, recombinase-based temporal logic gates and antibiotic production.

Synthetic biology is the design and implementation of novel biological devices via the application of engineering principles to biological systems research. Mathematical modelling is an invaluable tool in developing our understanding of biological system dynamics and characterising small parts and circuits for the assembly of higher-level systems. In this thesis, mathematical modelling approaches are applied to three biological circuits of interest. A novel mechanistic model of the DNA recombination reactions comprising a genetic switch reveals the input criteria and operational specifications required of a digital data storage module. Specific layering of the components comprising recombinase-based genetic switches can provide cellular Boolean logic operations. A novel mechanistic model of a two-input temporal logic gate is able to simulate and predict in vivo dynamical responses captured by a large experimental dataset. Experimental implementation of recombinase-based circuitry is unpredictable and can lead to lengthy development times, providing clear evidence of the advantages of utilising mathematical models in synthetic biology. Antibiotic resistance has become one of the most prominent challenges facing medicine today, placing immense importance on the characterisation of new natural products. The rst detailed mathematical model of the methylenomycin A producing gene cluster in the bacterium Streptomyces coelicolor is developed through the application of model selection to a large set of candidate system architectures. Mathematical models presented in this thesis can be adapted and expanded to suit many different experimental conditions and system responses, facilitating the design of novel synthetic biological circuitr

A mechanistic model of the BLADE platform predicts performance characteristics of 256 different synthetic DNA recombination circuits

AbstractBoolean 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.</jats:p

A mechanistic model of the BLADE platform predicts performance characteristics of 256 different synthetic DNA recombination circuits

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.</jats:p

A mechanistic model of the BLADE platform predicts performance characteristics of 256 different synthetic DNA recombination circuits.

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