Periodic Reporting for period 1 - PLATE (Biophysical Genetic Design Automation Technology)
Periodo di rendicontazione: 2022-07-01 al 2023-12-31
Advances in our capabilities to program, synthesize and modify DNA has led to a surge in the field of synthetic biology. Various gene circuits have been proposed and designed in various organisms for application domains ranging from biomedicine over biotechnology to biomaterials. Our current bottleneck is not anymore, our ability to program and synthesize custom DNA, but rather our ability to design regulatory circuit that realize the desired functionality and operate reliably in a specific target host cell or in vitro systems. The largest hurdle that we identify is the context-dependency of synthetic circuits, i.e. their perturbation by other molecular factors belonging to the host cell. Given the complexity of these molecular systems, current design approaches that rely on trial-and error will not be able to produce meaningful designs in a reliable, fast and systematic manner at the scale required for industrial applications. Although genetic design automation tools are available to overcome this unsatisfactory state of affairs their practical impact have been limited. This is due to the fact that the used models are not accurate enough, in particular, they cannot predict reliably the performance of a circuit design when operating within a host cell. The main reason for this limited predictive power is that models do not take into account the named context-dependency of circuits.
The current project PLATE takes on this challenge and leverages methods developed within the ERC Project CONSYN that allow for accurate modeling of context-effects through the use of detailed biophysical models. The aim of PLATE is to integrate all those computational methods into a coherent design environment for the synthetic biology researcher in academia and in industry. The resulting PLATE software suite follows a modular approach where different analysis types and different design methods can be selected according to the specific need of a given academic or industrial project.
The current project PLATE takes on this challenge and leverages methods developed within the ERC Project CONSYN that allow for accurate modeling of context-effects through the use of detailed biophysical models. The aim of PLATE is to integrate all those computational methods into a coherent design environment for the synthetic biology researcher in academia and in industry. The resulting PLATE software suite follows a modular approach where different analysis types and different design methods can be selected according to the specific need of a given academic or industrial project.
Our project aimed to enhance the integration of novel methodologies and algorithms within comprehensive workflows for genetic design automation. By employing sophisticated thermodynamic models of promoter behavior, we investigated the impact of cellular context effects, specifically addressing crosstalk resulting from regulator specificity limitations and the titration of circuit regulators binding to off-target sites on the host genome. To manage the resultant computational complexity, we implemented dedicated branch-and-bound techniques during technology mapping. Through a case study involving the synthesis of combinational logic circuits utilizing a previously published device library (Cello), we analyzed the ramifications of varying degrees and distributions of crosstalk on circuit performance and library usability. Additionally, we proposed a model for non-equilibrium steady-state gene expression, capable of predicting protein count distributions and associated energy consumption rates in response to transcription factor inputs. This model's adaptability to diverse scenarios, including multiple transcription factor binding sites, distinct transcription factor species, and variable transcription rates, facilitates its application across different organisms and levels of abstraction. Leveraging our model, we enable energy-conscious technology mapping for genetic logic circuits, integrating function and energy expenditure optimization through constrained or multi-objective optimization approaches. Moreover, we devised strategies to enhance search space exploration efficiency concerning structural variants.
The scientific results of PLATE provide more accurate and realistic tools for the automation of the design of genetic circuits and the possibility to go beyond bacterial systems and use mammalian cells as targets. This will make it possible to design genetic circuits with a clear application goal (e.g. as therapeutic circuits in the fight against cancer) and to test them effectively in (pre)clinical research.