Biology subjects

Kubaczka, E.

Publications and source records attributed to Kubaczka, E..

6 recordsLinked to original sources

bioRxiv2026

A Yeast Logic Toolkit for Fast and Efficient Assembly of Genetic Circuits

Synthetic biologists aim for standardization and simplicity in order to ensure reproducibility and reliability. Toolkits offer both, making it easier to share DNA sequences between labs while minimizing context dependent effects from sequences surrounding the construct of interest. The Yeast Toolkit (YTK) for the model organism Saccharomyces cerevisiae has found wide adoption within the scientific community. While the Toolkit and its existing extensions offer a large variety of different parts, there is a lack of characterized logic gates and insulator containing backbones for the assembly of genetic circuits with the yeast toolkit syntax. In this work, we present the Yeast Logic Toolkit (YLT). YLT contains 40 pre-assembled backbones with dropouts, 109 parts for NOT/NOR gate assembly, and a subdivided promoter Type 2 part that allows more flexible 5'UTR design. For insulation, the backbones contain spacer sequences and self-cleaving ribozymes which buffer against transcriptional read-through from upstream sequences. Due to our subdivision of Type 2 parts, we are able to manipulate the 5' untranslated region without a cloning scar upstream of the start codon. We also characterized the NOT and NOR gates from the Cello library for yeast in the sequence context of the yeast toolkit cloning scheme. This toolkit extension will make it easier and faster for scientists to assemble genetic circuits in yeast while providing a minimum of context dependent effects such as genetic read-trough due to the standardization of up- and downstream sequences surrounding the transcriptional units.

synthetic biology↗

bioRxiv2025

The Coli Toolkit (CTK): An extension of the modular Yeast Toolkit for use in E. coli

Genetic circuits are a cornerstone of synthetic biology, enabling programmable control of cellular behavior for applications in health, sustainability, and biotechnology. While Genetic Design Automation (GDA) tools have optimized and streamlined the design of such circuits, rapid and efficient assembly of DNA remains a bottleneck in the DBTL cycle. Here we present the Coli Toolkit (CTK), a modular Golden Gate-based cloning system, adapted from the Yeast Toolkit (YTK) for use in Escherichia coli. The CTK expands on the original YTK architecture by introducing a more flexible control of transcription and translation through subdividing the former promoter part into subparts; promoter, insulating ribozyme, and ribosome binding site (RBS). We provide a range of basic parts that enable the assembly of a wide range of constructs, as well as characterization data for all constitutive and inducible promoters provided. Additionally, we provide characterization data, as well as calibrated models, for all 20 NOT gates from the Cello library, and we provide the NOT gates as preassembled basic parts, which enables rapid cloning of larger genetic circuits. With this toolkit, we leverage the strengths of the YTK architecture to enable rapid and high-efficiency assembly of genetic circuits in E. coli, filling a key gap in the infrastructure of bacterial synthetic biology. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/663901v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@189a5c2org.highwire.dtl.DTLVardef@1e18f6dorg.highwire.dtl.DTLVardef@18f5ddeorg.highwire.dtl.DTLVardef@15bd410_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

NAND Hybrid Riboswitch Design by Deep Batch Bayesian Optimization

The design of large genetic circuits requires genetic regulatory devices capable of performing complex logic operations. Hybrid riboswitches, synthetically enhanced compact RNA elements (<100 nucleotides) that form a tertiary structure with the ability to specifically bind two different target molecules, can be used to design genetic regulators that emulate Boolean logic. When inserted into the 5 UTR of an mRNA, these devices can regulate translation initiation upon specific binding of one or both ligands. The goal of this study is to design hybrid riboswitches that emulate Boolean NAND logic in yeast. We propose a novel machine learning-based design framework combining high-throughput in vivo screening and deep Bayesian optimization. Through an initial screening, we discovered a hybrid riboswitch with NAND behavior. Using batch Bayesian optimization with an ensemble neural network as surrogate, we further improve the NAND functionality of our hybrid riboswitch with respect to a performance score, thereby achieving near digital NAND behavior. With its focus on model-based and score-driven design, our proposed method can complement experiment driven approaches by allowing fine grained adaptation of functionality, including constructs sensitive to single nucleotide changes.

synthetic biology↗

bioRxiv2024

Energy Aware Technology Mapping of Genetic Logic Circuits

Energy and its dissipation are fundamental to all living systems, including cells. Insufficient abundance of energy carriers -as caused by the additional burden of artificial genetic circuits-shifts a cells priority to survival, also impairing the functionality of the genetic circuit. Moreover, recent works have shown the importance of energy expenditure in information transmission. Despite living organisms being non-equilibrium systems, non-equilibrium models capable of accounting for energy dissipation and non-equilibrium response curves are not yet employed in genetic design automation (GDA) software. To this end, we introduce Energy Aware Technology Mapping, the automated design of genetic logic circuits with respect to energy efficiency and functionality. The basis for this is an energy aware non-equilibrium steady state (NESS) model of gene expression, capturing characteristics like energy dissipation -which we link to the entropy production rate- and transcriptional bursting, relevant to eukaryotes as well as prokaryotes. Our evaluation shows that a genetic logic circuits functional performance and energy efficiency are disjoint optimization goals. For our benchmark, energy efficiency improves by 37.2% on average when comparing to functionally optimized variants. We discover a linear increase in energy expenditure and overall protein expression with the circuit size, where Energy Aware Technology Mapping allows for designing genetic logic circuits with the energy efficiency of circuits that are one to two gates smaller. Structural variants improve this further, while results show the Pareto dominance among structures of a single Boolean function. By incorporating energy demand into the design, Energy Aware Technology Mapping enables energy efficiency by design. This extends current GDA tools and complements approaches coping with burden in vivo. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/601038v2_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@77317borg.highwire.dtl.DTLVardef@151a140org.highwire.dtl.DTLVardef@318c19org.highwire.dtl.DTLVardef@e4be6f_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2022

Context-Aware Technology Mapping in Genetic Design Automation

Genetic design automation (GDA) tools hold promise to speed-up circuit design in synthetic biology. Their wide-spread adoption is hampered by their limited predictive power, resulting in frequent deviations between in-silico and in-vivo performance of a genetic circuit. Context-effects, i.e., the change in overall circuit functioning, due to the intracellular environment of the host and due to cross-talk among circuits components are believed to be a major source for the aforementioned deviations. Incorporating these effects in computational models of GDA tools is challenging but is expected to boost their predictive power, and hence, their deployment. Using fine-grained thermodynamic models of promoter activity we show in this work, how to account for two major components of cellular context effects: (i) Crosstalk due to limited specificity of used regulators and (ii) titration of circuit regulators to off-target binding sites on the host genome. We show, how we can compensate the incurred increase in computational complexity through dedicated branch-and-bound techniques during the technology mapping process. Using the synthesis of several combinational logic circuits based on Cellos device library as a case study, we analyze the effect of different intensities and distributions of crosstalk on circuit performance and on the usability of a given device library.

synthetic biology↗

bioRxiv2021

Automated Design of Robust Genetic Circuits: Structural Variants and Parameter Uncertainty

Genetic design automation methods for combinational circuits often rely on standard algorithms from electronic design automation in their circuit synthesis and technology mapping. However, those algorithms are domain-specific and are hence often not directly suitable for the biological context. In this work we identify aspects of those algorithms that require domain-adaptation. We first demonstrate that enumerating structural variants for a given Boolean specification allows us to find better performing circuits and that stochastic gate assignment methods need to be properly adjusted in order to find the best assignment. Second, we present a general circuit scoring scheme that accounts for the limited accuracy of biological device models including the variability across cells and show that circuits selected according to this score exhibit higher robustness with respect to parametric variations. If gate characteristics in a library are just given in terms of intervals, we provide means to efficiently propagate signals through such a circuit and compute corresponding scores. We demonstrate the novel design approach using the Cello gate library and 33 logic functions that were synthesized and implemented in vivo recently (1). We show that an average 1.3-fold and a peak 6.5-fold performance increase can be achieved by simply considering structural variants and that an average 1.8-fold and a peak 30-fold gain in the novel robustness score can be obtained when selecting circuits according to it. Graphical TOC Entry O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/456094v1_ufig1.gif" ALT="Figure 2"> View larger version (20K): org.highwire.dtl.DTLVardef@3ef8aorg.highwire.dtl.DTLVardef@635957org.highwire.dtl.DTLVardef@7e747eorg.highwire.dtl.DTLVardef@122db0_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗