Phage integrase-based circuits are an alternative approach to relying on transcriptional and translational repression for biomolecular circuits. Previous research has shown that circuits based on integrases can perform a variety of functions, including counters, Boolean logic operators, memory modules and temporal event detectors. It is therefore essential to develop a principled theoretical and experimental framework for the design, implementation and study of such circuits. One of the fundamental questions that such a framework should address concerns the functionality limitations and temporal dynamics of the integrases as regulatory elements. Here, we test the functionality of several large serine integrases from a recently published library in a cell-free transcription-translation (TX-TL) platform. Additionally, we use a combination of experimental data and models to investigate integrase dynamics as a function of enzyme concentration and number of binding sites. We report that sequestration of integrase molecules, either in the form of monomers or dimers, by the integrase's own binding sites dominates integrase dynamics, and that the delay in the activation of the reporter is negatively correlated with integrase plasmid concentration. We have validated our sequestration hypothesis by building a model with MATLAB's SimBiology toolbox, and running simulations with various integrase and binding sites concentrations. The simulation results qualitatively match the experimental results, and offer further insights into the system.

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