Conditional plasmid conjugation as wiring for multicellular biocircuitsindividual-based modeling and simulation

Resumo

Synthetic biology is a young science focused on the control of the way programmed living cells interpret, compute and react to different inner and environmental signals. This is achieved through the engineering of the DNA. Synthetic biology still has to overcome the challenge of programming multicellular distributed behaviors. For this, proper regulation over the intercellular communication mechanisms is key. Conjugation is a physical contact mediated DNA transfer mechanism used by cells for the exchange of plasmid DNA. PLASWIRES European project, under which this thesis is developed, proposes the engineering of conjugation to control the transmission of synthetic plasmids for the distributed computation of logic functions in a bacterial multicellular colony. Here the control of the regulatory proteins needed for conjugation is suggested as a way to conditionally regulate conjugation. The aim of this thesis is to evaluate the feasibility of conditional conjugation as a scalable and programmable intercellular communication mechanism for distributed gene regulatory networks by means of an individual-based model: gro, a two dimensional bacterial colony simulator. In the first part of this thesis, the gro simulator is re-designed and improved for the simulation of the proposed scenarios. Main redesigns include the approximation of protein concentration values as binary variables and a probabilistic timed automata for the calculation of gene network dynamics. The presented features enable the re-purposed gro to simulate larger colonies for longer simulation times unreachable by the original version. In addition, a new architecture is presented for the description and computation of cellular behavior. Results show how the new abstraction is still able to reproduce the complex dynamics while achieving a higher performance. In the second part of this thesis, feasibility analysis of conditional conjugation is assessed by studying the horizontal spread of conditional conjugative plasmids in scenarios where its transmission is expected. Additionally, different studies analyze the capacity of conditionally conjugated plasmids to be sequentially regulated, forming more complex network motifs. To do this, four different distributed gene regulatory network designs are examined: a 3 input multiAND, a SOP (Sum Of Products), a POS (Product Of Sums) and a PLA (Programmable Logic Array) design. Studies of these designs focus on the exploration of the conditions under which the High/Low readouts statistically differ the most. Results show the feasibility of many of the presented multicellular distributed circuits. While low conjugation frequency remains the biggest limitation, conditional conjugation is shown to be a feasible mechanisms to be implemented for the distribution of synthetic gene regulatory circuits in bacterial populations. Results also expose a large dynamical range between the Low and High readouts of a PLA circuit where two logic functions are evaluated in parallel. Simulations of the PLA in heterogeneous patchy colonies evidence local accuracy for the computing and reporting stages, ensuring a faithful performance with spatial discrimination. This thesis remarks conditional conjugation as a highly interesting mechanism for the communication and dynamic distribution of gene regulatory networks worth being further researched. Results show how efforts should be done in order to increase the experimental conjugation frequency. The study of more complex distributed motifs apart from the cascades motifs presented here is another future relevant research line to be continued.