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Genetic circuit characterization by inferring RNA polymerase movement and ribosome usage

ABSTRACT: When performing a computation, genetic circuits change states through a symphony of genetic parts that turn regulator expression on and off. Debugging is frustrated by an inability to measure part function and identify the origins of failures. Here, we take “snapshots” of a large genetic circuit in different states. RNA-seq is used to visualize circuit function as a changing pattern of RNA polymerase (RNAP) flux along the DNA. Together with ribosome profiling, all 54 genetic parts are parameterized. The circuit behaves as designed; however, it is riddled with errors, including cryptic sense/antisense promoters and translation, attenuation, incorrect start codons and a failed gate. While serendipitously not impacting function, they reduce prediction accuracy and could lead to failures when used in other designs. Finally, the cellular power (RNAPs and ribosomes) required to maintain a circuit state is calculated. This work demonstrates the use of a small number of measurements to fully parameterize a regulatory circuit and quantify its impact on host.

ORGANISM(S): Escherichia coli

PROVIDER: GSE152664 | GEO | 2020/06/18

REPOSITORIES: GEO

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Project description:Mandlik2012 - Synthetic circuit of IPC synthase in Leishmania A genetic circuit for the targeted enzyme inositol phosphorylceramide (IPC) synthase belonging to the protozoan parasite Leishmania is constructed by Mandlik et al. (2013). This model is described in the article: Synthetic circuit of inositol phosphorylceramide synthase in Leishmania: a chemical biology approach Mandlik V., Limbachiya D., Shinde S., Mol M, S., Singh, S. J Chem Biol. January 2013 Abstract: Building circuits and studying their behavior in cells is a major goal of systems and synthetic biology. Synthetic biology enables the precise control of cellular states for systems studies, the discovery of novel parts, control strategies, and interactions for the design of robust synthetic systems. To the best of our knowledge, there are no literature reports for the synthetic circuit construction for protozoan parasites. This paper describes the construction of genetic circuit for the targeted enzyme inositol phosphorylceramide synthase belonging to the protozoan parasite Leishmania. To explore the dynamic nature of the circuit designed, simulation was done followed by circuit validation by qualitative and quantitative approaches. The genetic circuit designed for inositol phosphorylceramide synthase (Biomodels Database—MODEL1208030000) shows responsiveness, oscillatory and bistable behavior, together with intrinsic robustness. This model is hosted on BioModels Database and identified by: MODEL1208030000 . To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models . To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

Project description:Boada2016 - Incoherent type 1 feed-forward loop (I1-FFL) A synthetic-biology mathematical modelling framework that was constructed to provide guidelines for experimental implementation and parameter optimisation resulted in a biological device demonstrating desired behaviour. This model is described in the article: Multi-objective optimization framework to obtain model-based guidelines for tuning biological synthetic devices: an adaptive network case. Boada Y, Reynoso-Meza G, Picó J, Vignoni A. BMC Syst Biol 2016 Mar; 10: 27 Abstract: Model based design plays a fundamental role in synthetic biology. Exploiting modularity, i.e. using biological parts and interconnecting them to build new and more complex biological circuits is one of the key issues. In this context, mathematical models have been used to generate predictions of the behavior of the designed device. Designers not only want the ability to predict the circuit behavior once all its components have been determined, but also to help on the design and selection of its biological parts, i.e. to provide guidelines for the experimental implementation. This is tantamount to obtaining proper values of the model parameters, for the circuit behavior results from the interplay between model structure and parameters tuning. However, determining crisp values for parameters of the involved parts is not a realistic approach. Uncertainty is ubiquitous to biology, and the characterization of biological parts is not exempt from it. Moreover, the desired dynamical behavior for the designed circuit usually results from a trade-off among several goals to be optimized.We propose the use of a multi-objective optimization tuning framework to get a model-based set of guidelines for the selection of the kinetic parameters required to build a biological device with desired behavior. The design criteria are encoded in the formulation of the objectives and optimization problem itself. As a result, on the one hand the designer obtains qualitative regions/intervals of values of the circuit parameters giving rise to the predefined circuit behavior; on the other hand, he obtains useful information for its guidance in the implementation process. These parameters are chosen so that they can effectively be tuned at the wet-lab, i.e. they are effective biological tuning knobs. To show the proposed approach, the methodology is applied to the design of a well known biological circuit: a genetic incoherent feed-forward circuit showing adaptive behavior.The proposed multi-objective optimization design framework is able to provide effective guidelines to tune biological parameters so as to achieve a desired circuit behavior. Moreover, it is easy to analyze the impact of the context on the synthetic device to be designed. That is, one can analyze how the presence of a downstream load influences the performance of the designed circuit, and take it into account. This model is hosted on BioModels Database and identified by: BIOMD0000000696. To cite BioModels Database, please use: Chelliah V et al. BioModels: ten-year anniversary. Nucl. Acids Res. 2015, 43(Database issue):D542-8. To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

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Genetic circuit characterization by inferring RNA polymerase movement and ribosome usage

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