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Robustness and reproducibility of simple and complex synthetic logic circuit designs using a DBTL loop.

ABSTRACT: Computational tools addressing various components of design-build-test-learn (DBTL) loops for the construction of synthetic genetic networks exist but do not generally cover the entire DBTL loop. This manuscript introduces an end-to-end sequence of tools that together form a DBTL loop called Design Assemble Round Trip (DART). DART provides rational selection and refinement of genetic parts to construct and test a circuit. Computational support for experimental process, metadata management, standardized data collection and reproducible data analysis is provided via the previously published Round Trip (RT) test-learn loop. The primary focus of this work is on the Design Assemble (DA) part of the tool chain, which improves on previous techniques by screening up to thousands of network topologies for robust performance using a novel robustness score derived from dynamical behavior based on circuit topology only. In addition, novel experimental support software is introduced for the assembly of genetic circuits. A complete design-through-analysis sequence is presented using several OR and NOR circuit designs, with and without structural redundancy, that are implemented in budding yeast. The execution of DART tested the predictions of the design tools, specifically with regard to robust and reproducible performance under different experimental conditions. The data analysis depended on a novel application of machine learning techniques to segment bimodal flow cytometry distributions. Evidence is presented that, in some cases, a more complex build may impart more robustness and reproducibility across experimental conditions. Graphical Abstract.

SUBMITTER: Cummins B

PROVIDER: S-EPMC10105856 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

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Robustness and reproducibility of simple and complex synthetic logic circuit designs using a DBTL loop.

Cummins Breschine B Vrana Justin J Moseley Robert C RC Eramian Hamed H Deckard Anastasia A Fontanarrosa Pedro P Bryce Daniel D Weston Mark M Zheng George G Nowak Joshua J Motta Francis C FC Eslami Mohammed M Johnson Kara Layne KL Goldman Robert P RP Myers Chris J CJ Johnson Tessa T Vaughn Matthew W MW Gaffney Niall N Urrutia Joshua J Gopaulakrishnan Shweta S Biggers Vanessa V Higa Trissha R TR Mosqueda Lorraine A LA Gameiro Marcio M Gedeon Tomáš T Mischaikow Konstantin K Beal Jacob J Bartley Bryan B Mitchell Tom T Nguyen Tramy T TT Roehner Nicholas N Haase Steven B SB

Synthetic biology (Oxford, England) 20230328 1

Computational tools addressing various components of design-build-test-learn (DBTL) loops for the construction of synthetic genetic networks exist but do not generally cover the entire DBTL loop. This manuscript introduces an end-to-end sequence of tools that together form a DBTL loop called Design Assemble Round Trip (DART). DART provides rational selection and refinement of genetic parts to construct and test a circuit. Computational support for experimental process, metadata management, stand ...[more]

PMID: 37073283

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Project description:Variation in the concentration of biological components is inescapable for any cell. Robustness in any biological circuit acts as a cushion against such variation and enables the cells to produce homogeneous output despite the fluctuation. The two-component system (TCS) with a bifunctional sensor kinase (that possesses both kinase and phosphatase activities) is proposed to be a robust circuit. Few theoretical models explain the robustness of a TCS, although the criteria and extent of robustness by these models differ. Here, we provide experimental evidence to validate the extent of the robustness of a TCS signaling pathway. We have designed a synthetic circuit in Escherichia coli using a representative TCS of Mycobacterium tuberculosis, MprAB, and monitored the in vivo output signal by systematically varying the concentration of either of the components or both. We observed that the output of the TCS is robust if the concentration of MprA is above a threshold value. This observation is further substantiated by two in vitro assays, in which we estimated the phosphorylated MprA pool or MprA-dependent transcription yield by varying either of the components of the TCS. This synthetic circuit could be used as a model system to analyze the relationship among different components of gene regulatory networks.IMPORTANCE Robustness in essential biological circuits is an important feature of the living organism. A few pieces of evidence support the existence of robustness in vivo in the two-component system (TCS) with a bifunctional sensor kinase (SK). The assays were done under physiological conditions in which the SK was much lower than the response regulator (RR). Here, using a synthetic circuit, we varied the concentrations of the SK and RR of a representative TCS to monitor output robustness in vivo. In vitro assays were also performed under conditions where the concentration of the SK was greater than that of the RR. Our results demonstrate the extent of output robustness in the TCS signaling pathway with respect to the concentrations of the two components.

Dataset Information

Robustness and reproducibility of simple and complex synthetic logic circuit designs using a DBTL loop.

Publications

Robustness and reproducibility of simple and complex synthetic logic circuit designs using a DBTL loop.

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