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bioRxiv2024

Engineered bacteria that self-assemble ''bioglass'' polysilicate coatings display enhanced light focusing

Photonic devices are cutting-edge optical materials that produce narrow, intense beams of light, but their synthesis typically requires toxic, complex methodology. Here we employ a synthetic biology approach to produce environmentally-friendly, living microlenses with tunable structural properties. We engineered Escherichia coli bacteria to display the silica biomineralization enzyme silicatein from aquatic sea sponges. Our silicatein-expressing bacteria can self-assemble a shell of polysilicate "bioglass" around themselves. Remarkably, the polysilicate-encapsulated bacteria can focus light into intense nanojets that are nearly an order of magnitude brighter than unmodified bacteria. Polysilicate-encapsulated bacteria are metabolically active for up to four months, potentially allowing them to sense and respond to stimuli over time. Our data demonstrate that engineered bacterial particles have the potential to revolutionize the development of multiple optical and photonic technologies. Significance StatementIn this work, we apply the principles of synthetic biology to create living optical devices. Utilizing the ability of sea sponges to polymerize bioglass from silica precursors in the ocean water using only a single enzyme, silicatein, we have fused this same enzyme to the surface of Escherichia coli bacterial cells. The modified bacteria can polymerize a layer of bioglass at their surface. This bioglass shell allows the bacteria to act as engineered optical devices that are able to scatter high intensity, focused light while also surviving for several months, opening the door to a wide range of sense-and-respond applications. ClassificationBiological Sciences, Applied Biological Sciences

synthetic biology↗

bioRxiv2016

How Cancer Genomics Drives Cancer Biology: Does Synthetic Lethality Explain Mutually Exclusive Oncogenic Mutations?

Large-scale analyses of cancer genomes are revealing patterns of mutations that suggest biologically significant ideas about many aspects of cancer, including carcinogenesis, classification, and preventive and therapeutic strategies. Among those patterns is \"mutual exclusivity\", a phenomenon observed when two or more mutations that are commonly observed in samples of a type of cancer are not found combined in individual tumors. We have been studying a striking example of mutual exclusivity: the absence of co-existing mutations in the KRAS and EGFR proto-oncogenes in human lung adenocarcinomas, despite the high individual frequencies of such mutations in this common type of cancer. Multiple lines of evidence suggest that toxic effects of the joint expression of KRAS and EGFR mutant oncogenes, rather than loss of any selective advantages conferred by a second oncogene that operates through the same signaling pathway, are responsible for the observed mutational pattern. We discuss the potential for understanding the physiological basis of such toxicity, for exploiting it therapeutically, and for extending the studies to other examples of mutual exclusivity.

cancer biology↗

bioRxiv2021

A microfluidic platform for sequential assembly and separation of synthetic cell models

Cell-sized vesicles like giant unilamellar vesicles (GUVs) are established as a promising biomimetic model for studying cellular phenomena in isolation. However, the presence of residual components and by-products, generated during vesicles preparation and manipulation, severely limits the utility of GUVs in applications like synthetic cells. Therefore, with the rapidly growing field of synthetic biology, there is an emergent demand for techniques that can continuously purify cell-like vesicles from diverse residues, while GUVs are being simultaneously synthesized and manipulated. We developed a microfluidic platform capable of purifying GUVs through stream bifurcation, where a stream of vesicles suspension is partitioned into three fractions - purified GUVs, residual components, and a washing solution. Using our purification approach, we showed that giant vesicles can be separated from various residues - that range in size and chemical composition - with a very high efficiency (e = 0.99), based on size and deformability of the filtered objects. In addition, by incorporating the purification module with a microfluidic-based GUV-formation method, octanol-assisted liposome assembly (OLA), we established an integrated production-purification microfluidic unit that sequentially produces, manipulates, and purifies GUVs. We demonstrate the applicability of the integrated device to synthetic biology through sequentially fusing SUVs with freshly prepared GUVs and separating the fused GUVs from extraneous SUVs and oil droplets at the same time.

synthetic biology↗

bioRxiv2023

Engineering of Robust Host Strains: Enhancing Escherichia coli Abiotic Stress Resistance through Ornithine Lipid Formation

Escherichia coli is a common host for biotechnology and synthetic biology applications. During growth and fermentation, the microbes are often exposed to stress conditions, such as variations in pH or solvent concentrations. Bacterial membranes play a key role in response to abiotic stresses. Ornithine lipids (OLs) are a group of membrane lipids whose presence and synthesis have been related to stress resistance in bacteria. We wondered if this stress resistance could be transferred to bacteria not encoding the capacity to form OLs in their genome, such as E. coli. In this study, we engineered different E. coli strains to produce unmodified OLs and hydroxylated OLs by expressing the synthetic operon olsFC. Our results showed that OL formation improved pH resistance and increased biomass under phosphate limitation. Transcriptome analysis revealed that OL-forming strains differentially expressed stress- and membrane-related genes. OL-producing strains also showed better growth in the presence of the ionophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP), suggesting reduced proton leakiness in OL-producing strains. Furthermore, our engineered strains showed improved heterologous violacein production at phosphate limitation and also at low pH. Overall, this study demonstrates the potential of engineering the E. coli membrane composition for constructing robust hosts with an increased abiotic stress resistance for biotechnology and synthetic biology applications. KeypointsO_LIThe E. coli membrane composition was engineered by producing ornithine lipids C_LIO_LIOrnithine lipid production increase biomass yield under phosphate limitation C_LIO_LIEngineered strains show enhanced production phenotype under low pH stress C_LIO_LITranscriptome analysis and CCCP experiments revealed reduced proton leakage C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=145 SRC="FIGDIR/small/544863v2_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1e261ecorg.highwire.dtl.DTLVardef@18ae062org.highwire.dtl.DTLVardef@915b57org.highwire.dtl.DTLVardef@103b74d_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2021

An artificial self-assembling nanocompartment for organising metabolic pathways in yeast

Metabolic pathways are commonly organised by sequestration into discrete cellular compartments. Compartments prevent unfavourable interactions with other pathways and provide local environments conducive to the activity of encapsulated enzymes. Such compartments are also useful synthetic biology tools for examining enzyme/pathway behaviour and for metabolic engineering. Here, we expand the intracellular compartmentalisation toolbox for budding yeast (Saccharomyces cerevisiae) with engineered Murine polyomavirus virus-like particles (MPyV VLPs). The MPyV system has two components: VP1 which self-assembles into the compartment shell; and a short anchor, VP2C, which mediates cargo protein encapsulation via binding to the inner surface of the VP1 shell. Destabilised GFP fused to VP2C was specifically sorted into VLPs and thereby protected from host-mediated degradation. In order to access metabolites of native and engineered yeast metabolism, VLP-based nanocompartments were directed to assemble in the cytosol by removal of the VP1 nuclear localisation signal. To demonstrate their ability to function as a metabolic compartment, MPyV VLPs were used to encapsulate myo-inositol oxygenase (MIOX), an unstable and rate-limiting enzyme in D-glucaric acid biosynthesis. Strains with encapsulated MIOX produced ~20% more D-glucaric acid compared to controls expressing free MIOX - despite accumulating dramatically less expressed protein - and also grew to higher cell densities. These effects were linked to enzyme stabilisation and mitigation of cellular toxicity by the engineered compartment. This is the first demonstration in yeast of an artificial biocatalytic compartment that can participate in a metabolic pathway and establishes the MPyV platform as a promising synthetic biology tool for yeast engineering.

synthetic biology↗

bioRxiv2025

An experimentally verified mechanistic model for predicting quorum sensing-based switches

Quorum sensing-based genetic circuits are gaining traction in synthetic biology as they link population-level behaviour to individual cell responses. However, tuning these circuits remains challenging due to complex dynamics, particularly during the Learn phase of the Design-Build-Test-Learn (DBTL) cycle. To accelerate this process, we developed a mathematical model to predict how varying expression levels of the transcription factor and synthase affect the response of the EsaI/EsaR quorum sensing system. A strain library was constructed, and experimental data were used to optimize the model. The final model could successfully differentiate between the effects of these expression levels on the response of the bidirectional promoter. It allowed visualization of all potential system outcomes and emphasized the transcription factors critical role in tuning the circuit. This model offers a valuable tool for fine-tuning EsaI/EsaR-based systems for synthetic biology applications. Moreover, given the homology within the LuxR-family quorum sensing systems, this modelling approach may serve as a foundation for model-based tuning of other quorum sensing systems.

synthetic biology↗

bioRxiv2018

HEx: a heterologous expression platform for the discovery of fungal natural products

For decades, fungi have been a source of FDA-approved natural products such as penicillin, cyclosporine, and the statins. Recent breakthroughs in DNA sequencing suggest that millions of fungal species exist on Earth with each genome encoding pathways capable of generating as many as dozens of natural products. However, the majority of encoded molecules are difficult or impossible to access because the organisms are uncultivable or the genes are transcriptionally silent. To overcome this bottleneck in natural product discovery, we developed the HEx (Heterologous EXpression) synthetic biology platform for rapid, scalable expression of fungal biosynthetic genes and their encoded metabolites in Saccharomyces cerevisiae. We applied this platform to 41 fungal biosynthetic gene clusters from diverse fungal species from around the world, 22 of which produced detectable compounds. These included novel compounds with unexpected biosynthetic origins, particularly from poorly studied species. This result establishes the HEx platform for rapid discovery of natural products from any fungal species, even those that are uncultivable, and opens the door to discovery of the next generation of natural products.\n\nSummaryHere we present the largest scale effort reported to date toward the complete refactoring and heterologous expression of fungal biosynthetic gene clusters utilizing HEx, a novel synthetic biology platform.

synthetic biology↗

bioRxiv2023

Lycopene Production in Dedicated Novel Chasses for Lignocellulosic Waste Material Utilisation Capable of Sustained Coculture

Municipal solid waste (MSW) represents tonnes of material that, for the most part, is relegated to landfill. Synthetic biology proposes solutions to many of the challenges faced by humanity today, but many approaches are confined to use in classical chassis organisms. In MSW there are a variety of potentially toxic materials such as glues, dyes, and preservatives that could pose a challenge to its capitalisation when using these commonplace chassis. We have isolated a bank of strains that utilise paper and cardboard waste from a relevant waste environment. From these we have identified three strains that are capable of utilising cellulose as a sole carbon source. We have analysed how they utilise cellulose and hemicelluloses, both alone and in coculture. This revealed insights to how they might be used in synthetic consortia which were then produced under laboratory conditions. Production of complete genome sequences of these strains provides genetic insight to how these processes may be occurring at the metabolic level, and how they could be augmented using synthetic biology. To this end, we have produced protocols for transforming plasmids into these strains and have produced high value metabolites from this material. HighlightsO_LIFully annotated genomes were produced from novel mesophilic aerobic strains isolated from lignocellulosic solid waste C_LIO_LILycopene was produced directly from relevant solid waste substrates by genetically modified variants of these strains C_LIO_LIOptimised carbon source blends influence coculture compositions of specific strains C_LI

synthetic biology↗

bioRxiv2024

Cyanamide-inducible expression of homing nuclease I-SceI for iterative genome engineering and parallel promoter characterisation in Saccharomyces cerevisiae

In synthetic biology, microbial chasses including yeast Saccharomyces cerevisiae are iteratively engineered with increasing complexity and scale. Wet-lab genetic engineering tools are developed and optimised to facilitate strain construction but are often incompatible with each other due to shared regulatory elements, such as the galactose-inducible (GAL) promoter in S. cerevisiae. Here, we prototyped the cyanamide-induced I-SceI-mediated double-strand DNA breaks (DSBs) for selectable marker recycling in yeast metabolic engineering. We further combined cyanamide-induced I-SceI-mediated DSB and maltose-induced MazF-mediated negative selection for plasmid-free in situ promoter replacement, which simplified the molecular cloning procedure for promoter characterisation in S. cerevisiae. We then characterised three tetracycline-inducible promoters of differential strength, a non-leaky {beta}-estradiol-inducible promoter, cyanamide-inducible DDI2 promoter, bidirectional MAL32/MAL31 promoters, and five pairs of bidirectional GAL1/GAL10 promoters. Overall, alternative regulatory controls for genome engineering tools are important for the construction of complexed genotypes in microbial systems for synthetic biology and metabolic engineering applications.

synthetic biology↗

bioRxiv2023

Ultra-high throughput mapping of genetic design space

Massively parallel genetic screens have been used to map sequence-to-function relationships for a variety of genetic elements. However, because these approaches only interrogate short sequences, it remains challenging to perform high throughput (HT) assays on constructs containing combinations of multiple sequence elements arranged across multi-kb length scales. Overcoming this barrier could accelerate synthetic biology; by screening diverse gene circuit designs and learning "composition-to-function" mappings that reveal genetic part composability rules and enable rapid identification of behavior-optimized variants. Here, we introduce CLASSIC, a genetic screening platform that combines long- and short-read next-generation sequencing (NGS) modalities to quantitatively assess pools of constructs of arbitrary length containing diverse part compositions. We show that CLAS-SIC can measure expression profiles of >105 gene circuit designs (from 5-20 kb) in a single experiment in human cells. The resulting datasets can be used to train ML models that accurately predict circuit behavior across expansive circuit design landscapes, revealing part composability rules that govern circuit performance. Our work shows that by expanding the throughput of each design-build-test-learn (DBTL) cycle, CLASSIC enhances the pace and scale of synthetic biology and establishes an experimental basis for data-driven design of complex genetic systems.

synthetic biology↗

bioRxiv2026

3D bioprinting of engineered living materials in support slurries for complex free-standing structures

The combination of synthetic biology and additive manufacturing has driven major changes in production of biomaterials, especially through the use of three-dimensional (3D) bioprinting to create engineered living materials. However, current fabrication methods can be limited by prohibitive hardware costs and the inability to maintain structural fidelity in complex, free-form living architectures. This work demonstrates how to build a low-cost, open-source 3D bioprinting platform that can make complicated bacterial structures with complex geometry and high dimensional accuracy. A commercially available, conventional fused deposition modeling 3D printer was modified to create a bioprinting system that is simple to build. The modified bioprinter, which costs around $450, is less expensive than many commercial bioprinters. This 3D-printing technology uses slurry-based support bath methods featuring low-cost gelatin and agarose microparticles, resulting in structures with a high aspect ratio (>8:1) and feature sizes as small as 260 m. The optimization of critical printing settings, including the ability of the bioink to retract during non-print movements, resulted in a reduction of unwanted bacterial deposition by nearly two orders of magnitude. Long-term viability experiments showed that bacteria in the bioprints could survive for at least 28 days with nutrient supplementation. Additionally, 3D-printed engineered biofilms revealed that incubation conditions and extracellular matrix composition significantly impacted the mechanical properties of printed constructs, with tradeoffs between matrix production and mechanical integrity. This study showcases an accessible 3D bioprinting platform for advanced bioprinting technologies, enabling development of engineered living materials with potential applications in synthetic biology, biotechnology, and tissue engineering.

synthetic biology↗

bioRxiv2024

MOSAIC: a highly efficient, one-step recombineering approach to plasmid editing and diversification

The editing of plasmids and construction of plasmid libraries is paramount to the engineering of desired functionalities in synthetic biology. Typically, plasmids with targeted mutations are produced through time- and resource-consuming DNA amplification and/or cloning steps. In this study, we establish MOSAIC, a highly efficient protocol for the editing of plasmids and generation of combinatorial plasmid libraries. This quick protocol employs the efficient single-stranded DNA annealing protein (SSAP) CspRecT to incorporate (libraries of) DNA oligos harboring the desired mutations into a target plasmid in E. coli. In addition to up to 90% single-target plasmid editing efficiency, we demonstrate that MOSAIC enables the generation of a combinatorial plasmid library spanning four different target regions on a plasmid, in a single transformation. Lastly, we integrated a user-friendly validation pipeline using Nanopore sequencing reads, requiring minimal computational experience. We anticipate that MOSAIC will provide researchers with a simple, rapid and resource-effective method to edit plasmids or generate large, diverse plasmid libraries for a wide range of in vivo or in vitro applications in molecular and synthetic biology. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/586135v3_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@929537org.highwire.dtl.DTLVardef@1825039org.highwire.dtl.DTLVardef@60e89aorg.highwire.dtl.DTLVardef@46ef46_HPS_FORMAT_FIGEXP M_FIG GRAPHICAL ABSTRACT C_FIG

synthetic biology↗

bioRxiv2021

Data Representation in the DARPA SD2 Program

1Modern scientific enterprises are often highly complex and multidisciplinary, particularly in areas like synthetic biology where the subject at hand is itself inherently complex and multidisciplinary. Collaboration across many organizations is necessary to efficiently tackle such problems [6, 15], but remains difficult. The challenge is further amplified by automation that increases the pace at which new information can be produced, and particularly so for matters of fundamental research, where concepts and definitions are inherently fluid and may rapidly change as an investigation evolves [7]. The DARPA program Synergistic Discovery and Design (SD2) aimed to address these challenges by organizing the development of data-driven methods to accelerate discovery and improve design robustness, with one of the key domains under study being synthetic biology. The program was specifically organized such that teams provided complementary types of expertise and resources, and without any team being in a dominant organizational position, such that subject-matter investigations would necessarily require peer-level collaboration across multiple team boundaries. With more than 100 researchers across more than 20 organizations, several of which ran experimental facilities with high-throughput automation, participants were forced to confront challenges around effective data sharing. The default architecture for scientific collaboration is essentially one of anarchy, with ad-hoc bilateral relations between pairs of collaborators or experimental phases (Figure 1(a)). This was by necessity the case during early phases of the SD2 program as well, in which incorporating new tools into pipelines was ad-hoc and time-consuming, and data was generally disconnected from genetic designs and experimental plans. The other typical approach for collaboration is one of "command and control", in which a dominant organization determines the data sharing content and format for all participants (Figure 1(b)). This can be efficient, but tends to be limited in flexibility and extensibility, rendering it unsuitable for research collaboration, as indeed was found when we attempted this approach during the first year of the SD2 program. We addressed these problems with the application of distributed standards to create a "flexible rendezvous" model of collaboration (Figure 1(c)), enabling information flow to track evolving collaborative relationships, improving the sharing and utility of information across the community and supporting accelerated rates of experimentation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=71 SRC="FIGDIR/small/460644v1_fig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@fc7371org.highwire.dtl.DTLVardef@1ff2733org.highwire.dtl.DTLVardef@66af53org.highwire.dtl.DTLVardef@1809640_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1:C_FLOATNO Architectures for data sharing: bilateral relations (a), command and control (b), and flexible rendezvous (c). C_FIG

synthetic biology↗

bioRxiv2023

A modular plasmid toolkit applied in marine Proteobacteria reveals functional insights during bacteria-stimulated metamorphosis

A conspicuous roadblock to studying marine bacteria for fundamental research and biotechnology is a lack of modular synthetic biology tools for their genetic manipulation. Here, we applied, and generated new parts for, a modular plasmid toolkit to study marine bacteria in the context of symbioses and host-microbe interactions. To demonstrate the utility of this plasmid system, we genetically manipulated the marine bacterium Pseudoalteromonas luteoviolacea, which stimulates the metamorphosis of the model tubeworm, Hydroides elegans. Using these tools, we quantified constitutive and native promoter expression, developed reporter strains that enable the imaging of host-bacteria interactions, and used CRISPR interference (CRISPRi) to knock down a secondary metabolite and a host-associated gene. We demonstrate the broader utility of this modular system for rapidly creating and iteratively testing genetic tractability by modifying marine bacteria that are known to be associated with diverse host-microbe symbioses. These efforts enabled the successful transformation of twelve marine strains across two Proteobacteria classes, four orders and ten genera. Altogether, the present study demonstrates how synthetic biology strategies enable the investigation of marine microbes and marine host-microbe symbioses with broader implications for environmental restoration and biotechnology.

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

Promoter-Driven Modulation of Flagellin Expression and Motility in Escherichia coli

Synthetic biology offers powerful tools to engineer biological systems for diverse applications. However, key challenges persists before achieving real-world applications like environmental bioremediation or therapeutic microrobots for targeted drug delivery. This study aimed to precisely control bacterial movement by modulating gene expression using engineered promoters in Escherichia coli. We focused on Escherichia coli, a model organism, and manipulated its motility by engineering the expression of flagellin, a crucial protein for bacterial movement. To achieve this, specific genetic promoters were employed to regulate the production of flagellin, thereby dictating the movement capabilities of these bacteria. The promoters enabled targeted adjustments to flagellin expression, which in turn allowed for the enhancement or suppression of bacterial locomotion. Interestingly, the relationship between promoter design parameters and gene expression levels was non-linear, highlighting complex underlying dynamics. Optimal bacterial motility occurred at 30{degrees}C, illustrating the influence of environmental factors. Our findings demonstrate the ability to effectively regulate complex microbial phenotypes like motility using genetic engineering strategies. The results not only extend our understanding of bacterial gene regulation but also highlight the transformative potential of synthetic biology in creating functional and adaptable microbial phenotypes for diverse biotechnological applications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/635866v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@1fca66dorg.highwire.dtl.DTLVardef@12529dorg.highwire.dtl.DTLVardef@ede03aorg.highwire.dtl.DTLVardef@11c2c6c_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2024

Design nonrepetitive and diverse activity single-guide RNA by deep learning

Multiplex and precise control of the gene expression based on CRISPR/Cas9 is important to metabolic regulation in synthetic biology. However, employing single guide RNAs (sgRNAs) that possess repetitive DNA sequences and exhibit uniform activity could detrimentally affect the editing process, undermining both its stability and regulatory potential. In this study, we developed a deep generative model based on a decoder-only Transformer architecture (sgRNAGen) for the de novo generation of a series of nonrepetitive and diverse sgRNAs with activity. To assess the quality of sgRNAs generated by sgRNAGen, we evaluated their activity by targeting essential genes, with the results indicating that 98% of the generated sgRNAs were active in Bacillus subtilis. The generated sgRNAs were further validated for applications in single-gene editing, large fragment knockouts, and multiplex editing. Notably, the efficiency of knocking out long fragments up to 169.5 kb reached 100%, and targeting multiple sites allowed for the creation of strains with various combinations of mutations in a single editing. Furthermore, we developed a CRISPRi system utilizing the designed sgRNAs to regulate gene expression with desired strength and high precision. SgRNAGen offers a method for devising nonrepetitive and diverse activity sgRNAs, enhancing metabolic control and advancing applications within synthetic biology. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/596019v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@f68faaorg.highwire.dtl.DTLVardef@151f5e2org.highwire.dtl.DTLVardef@1e5cb28org.highwire.dtl.DTLVardef@17cd3f8_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

Optimization of heuristic logic synthesis by iteratively reducing circuit substructures using a database of optimal implementations

Minimal synthesis of Boolean functions is an NP-hard problem, and heuristic approaches typically give suboptimal circuits. However, in the emergent field of synthetic biology, genetic logic designs that use even a single additional Boolean gate can render a circuit unimplementable in a cell. This has led to a renewed interest in the field of optimal multilevel Boolean synthesis. For small numbers (1-4) of inputs, an exhaustive search is possible, but this is impractical for large circuits. In this work, we demonstrate that even though it is challenging to build a database of optimal implementations for anything larger than 4-input Boolean functions, a database of 4-input optimal implementations can be used to greatly reduce the number of logical gates required in larger heuristic logic synthesis implementations. The proposed algorithm combines the heuristic results with an optimal implementation database and yields average improvements in fractional gate-count reduction relative to ABC of 5.16% for 5-input circuits and 4.54% for 6-input circuits on outputs provided by the logic synthesis tool ABC. In addition to the gains in the efficiency of the implemented circuits, this work also attests to the importance and practicality of the field of optimal synthesis, even if it cannot directly provide results for larger circuits. The focus of this work is on circuits made exclusively of 2-input NOR gates but the presented results are readily applicable to 2-input NAND circuits as well as (2-input) AND/NOT circuits. The framework proposed here is likely to be adaptable to other types of circuits. Moreover, a small computational pipeline is provided for integration with synthetic biology tools such as Cello. An implementation of the described algorithm, HLM (Hybrid Logic Minimizer), is available at https://github.com/sontaglab/HLM/.

synthetic biology↗