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Results for “Synthetic Biology”

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bioRxiv2025

Computational design of toehold switches in eukaryotes and prokaryotes for efficient post-transcriptional control

The potential to design RNA molecules implementing translational regulated synthetic networks in eukaryotic cells can be harnessed to ramp up the development of various applications in biotechnology, synthetic biology, agriculture and personalized precision therapeutics. In prokaryotes, RNA based translation activation Toehold switches were demonstrated to achieve high fold change in throughput of a desired expression gene in response to different levels of both exogenous and endogenous target trigger RNA molecules. However, low fold changes were reported for such switches applying similar design architecture for eukaryotes. We developed Triggate, a computational pipeline which implements different algorithms for the efficient design and generation of higher on/off ratio optimized toehold switches in prokaryotes and eukaryotes. We also present here a new design architecture for eukaryotic toehold switches which is leaner and simpler. Using our tool and new architecture we report results with a much higher on/off ratio (up to around 13) than achieved before for mRNA toehold switches for eukaryotes. Triggate is available as a web application at http://www.cs.tau.ac.il/~tamirtul/Triggate.

synthetic biology↗

bioRxiv2017

Mobius Assembly: A Versatile Framework For Golden Gate Assembly

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC=\"FIGDIR/small/140095_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (16K):\norg.highwire.dtl.DTLVardef@165d4e4org.highwire.dtl.DTLVardef@7bd8f1org.highwire.dtl.DTLVardef@dfbda4org.highwire.dtl.DTLVardef@11b8db9_HPS_FORMAT_FIGEXP M_FIG C_FIG Golden Gate Assembly is a powerful synthetic biology tool, which utilizes Type IIS enzymes for unidirectional assembly of multiple DNA fragments. The simplicity of its DNA assembly and the exchangeability of standard parts greatly facilitate the generation of combinatorial assembly libraries. Currently there are two popular Golden Gate Assembly frameworks that allow multigene augmentation (MoClo and Golden Braid); they render either high cloning capacity or vector toolkit simplicity. We have developed a new Golden Gate Assembly framework called Mobius Assembly, which combines vector toolkit simplicity with high cloning capacity. Mobius Assembly is based on a two-level approach and embraces the standard overhangs defined by MoClo and Golden Braid to confer exchangeability, but with reduced domestication requirement. Furthermore, we have implemented drop-out cassettes encoding chromogenic proteins for visible cloning screening. As proofs of concept, we have functionally assembled up to 16 transcriptional units of various pigmentation genes in both operon and multigene arrangements.

synthetic biology↗

bioRxiv2019

Balancing cell populations endowed with a synthetic toggle switch via adaptive pulsatile feedback control

Controlling cells endowed with the genetic toggle switch has been suggested as a benchmark problem in synthetic biology. It has been shown that a carefully selected periodic forcing can balance a population of such cells in an undifferentiated state. The effectiveness of these control strategies, however, can be mined by the presence of stochastic perturbations and uncertainties typically observed in biological systems and is therefore not robust. Here, we propose the use of feedback control strategies to enhance robustness and performance of the balancing action by selecting in real-time both the amplitude and the duty-cycle of the inducer molecular signals affecting the toggle switch behavior. We show, via in-silico experiments and realistic agent-based simulations, the effectiveness of the proposed strategies even in presence of uncertainties and stochastic effects. In so doing, we confirm previous observations made in the literature about coherence of the population when pulsatile forcing inputs are used but, contrary to what proposed in the past, we leverage feedback control techniques to endow the balancing strategy with unprecedented robustness and stability properties. We compare via in-silico experiments different control solutions and show their advantages and limitations from an in-vivo implementation viewpoint.

synthetic biology↗

bioRxiv2025

PURE makes PURE: reconstitution of the PURE cell-free system from self-synthesized non-ribosomal proteins

Building a universal biochemical constructor, an autonomously self-replicating bio-chemical system, is a major challenge in synthetic biology. The PURE cell-free system is an ideal starting point for exploring self-regeneration, and its 36 non-ribosomal proteins constitute the primary macromolecular components that must be regenerated. Here, we demonstrate that the PURE system can be reconstituted from proteins synthesized by PURE itself. We first show that each of the 36 non-ribosomal proteins can be individually synthesized in PURE. We then purify the PURE synthesized proteins as pooled subsets and reconstitute a fully functional PURE system by combining the subsets. Finally, we show that all 36 non-ribosomal PURE proteins can be synthesized simultaneously in a single PURE reaction and, after purification, can reconstitute a functional PURE system. Together, these results establish that the non-ribosomal protein components of the PURE system can be self-regenerated, representing a critical step toward the realization of a universal biochemical constructor.

synthetic biology↗

bioRxiv2022

Genetic engineering of Acidithiobacillus ferridurans with CRISPR-Cas9/dCas9 systems

Genus Acidithiobacillus includes a group of Gram-negative Fe/S-oxidizing acidophilic chemolithotrophic bacteria that are extensively studied and used for biomining processes. Synthetic biology approaches are key means to study and improve their biomining performance. However, efficient genetic manipulations in Acidithiobacillus are still major bottlenecks. In this study, we report a simple and efficient pAFi system (CRISPR-dCas9) and a scarless pAF system (CRISPR-Cas9) for genetic manipulations in A. ferridurans JAGS. The pAFi system harboring both dCas9 and sgRNA was constructed based on pBBR1MCS-2 to knockdown HdrA and TusA genes, separately, of which the transcription levels were significantly downregulated by 48% and 93%, separately. The pAF system carrying pCas9-sgRNA-homology arms was constructed based on pJRD215 to delete HdrB3 gene and overexpress Rus gene. Our results demonstrated that the pAF system is a fast and efficient genome editing method with an average rate of 15-20% per transconjugant in one recombination event, compared to 10-3 and then 10-2 in two recombination events by traditional markerless engineering strategy. Moreover, with these two systems, we successfully regulated iron and sulfur metabolisms in A. ferridurans JAGS: the deletion of HdrB3 reduced 48% of sulfate production, and substitution overexpression of Rus promoter showed 8.82-fold of mRNA level and enhanced iron oxidation rate. With these high-efficient genetic tools for A. ferridurans, we will be able to study gene functions and create useful recombinants for biomining applications. Moreover, these systems could be extended to other Acidithiobacillus strains and promote the development of synthetic biology-assisted biomining. HighlightsO_LITwo shuttle vectors were constructed for Acidithiobacillus ferridurans C_LIO_LIAll-in-one pAFi (CRISPR-dCas9) and pAF (CRISPR-Cas9) systems were built up for gene knockdown and genome editing, separately C_LIO_LIThe transcription levels of HdrA and TusA were reduced 48% and 93% using pAFi system and thus suppressed sulfur oxidation C_LIO_LIHdrB3 deletion and Rus overexpression were achieved using pAF system and showed significant effects on sulfur and iron oxidation respectively C_LIO_LIOur pAF system facilitated genome editing in Acidithiobacillus ferridurans with high efficiency (15-20%) in less than 4 weeks C_LI

bioengineering↗

bioRxiv2018

Guiding biomolecular interactions in cells using de novo protein-protein interfaces

An improved ability to direct and control biomolecular interactions in living cells would impact on synthetic biology. A key issue is the need to introduce interacting components that act orthogonally to endogenous proteomes and interactomes. Here we show that low-complexity, de novo designed protein-protein-interaction (PPI) domains can substitute for natural PPIs and guide engineered protein-DNA interactions in Escherichia coli. Specifically, we use de novo homo- and hetero-dimeric coiled coils to reconstitute a cytoplasmic split adenylate cyclase; to recruit RNA polymerase to a promoter and activate gene expression; and to oligomerize both natural and designed DNA-binding domains to repress transcription. Moreover, the stabilities of the heterodimeric coiled coils can be modulated by rational design and, thus, adjust the levels of gene activation and repression in vivo. These experiments demonstrate the possibilities for using designed proteins and interactions to control biomolecular systems such as enzyme cascades and circuits in cells.

synthetic biology↗

bioRxiv2020

A standardized broad host range inverter package for genetic circuitry design in Gram-negative bacteria

Genetically encoded logic gates, especially inverters--NOT gates--are the building blocks for designing circuits, engineering biosensors or decision-making devices in synthetic biology. However, the repertoire of inverters readily available for different species is rather limited. In this work, a large whole of NOT gates that was shown to function previously in a specific strain of Escherichia coli, was recreated as broad host range (BHR) collection of constructs assembled in low, medium and high copy number plasmid backbones of the SEVA (Standard European Vector Architecture) collection. The input/output function of each of the gates was characterized and parameterized in the environmental bacterium and metabolic engineering chassis Pseudomonas putida. Comparisons of the resulting fluorescence cytometry data with those published for the same gates in Escherichia coli provided useful hints on the portability of the corresponding gates. The hereby described BHR inverter package (20 different versions of 12 distinct gates) thus becomes a toolbox of choice for designing genetic circuitries in a variety of Gram-negative species other than E. coli.

synthetic biology↗

bioRxiv2023

Biofoundry-assisted Golden Gate cloning with AssemblyTron

Golden Gate assembly is a requisite method in synthetic biology that facilitates critical conventions such as genetic part abstraction and rapid prototyping. However, compared to robotic implementation, manual Golden Gate implementation is cumbersome, error-prone, and inconsistent for complex assembly designs. AssemblyTron is an open-source python package that provides an affordable automation solution using open-source Opentrons OT-2 lab robots. Automating Golden Gate assembly with AssemblyTron can reduce failure-rate, resource consumption, and training requirements for building complex DNA constructs, as well as indexed and combinatorial libraries. Here, we dissect a panel of upgrades to AssemblyTrons Golden Gate assembly capabilities, which include Golden Gate assembly into modular cloning part vectors, error-prone PCR combinatorial mutant library assembly, and modular cloning indexed plasmid library assembly. These upgrades enable a broad pool of users with varying levels of experience to readily implement advanced Golden Gate applications using low-cost, open-source lab robotics.

synthetic biology↗

bioRxiv2026

Engineering a light-responsive, orthogonal Lon protease in E. coli for targeted protein degradation

Optogenetic methods are powerful tools for synthetic biology, allowing light to control cellular processes. While most bacterial optogenetic systems regulate gene expression at the transcriptional level, relatively few enable post-translational control, which can provide faster and growth-independent regulation of protein activity. Here, we describe the development of a post-translational optogenetic tool in Escherichia coli using the Mesoplasma florum Lon (mfLon) protease, an AAA+ protease that is orthogonal to native E. coli degradation machinery. To engineer a light-responsive mfLon, we constructed a large library in which a blue-light responsive domain, Avena sativa LOV2, was introduced into nearly every codon position in the protease using an unbiased molecular approach. We screened 726 mfLon-LOV variants using fluorescence-activated cell sorting and multi-round enrichment campaigns. We identified a novel dark-active variant (mfLon-LOV-534) that degrades target proteins in the dark and is inactivated upon blue-light exposure. Characterization of this variant demonstrates that its proteolytic response can be tuned by varying blue-light intensity and transcriptional expression levels. Furthermore, we show that mfLon-LOV-534 can degrade a target protein in both exponential and stationary growth phases, which addresses the limitations of division-based protein dilution. This work establishes a scalable approach to engineering allosteric control in complex multimeric enzymes and provides a foundation for orthogonal, growth-independent control of protein stability in synthetic circuits.

Synthetic Biology↗

bioRxiv2022

An Optogenetic Toolkit for Light-Inducible Antibiotic Resistance

Antibiotics are a key control mechanism for synthetic biology and microbiology. Resistance genes are used to select desired cells and regulate bacterial populations, however their use to-date has been largely static. Precise spatiotemporal control of antibiotic resistance could enable a wide variety of applications that require dynamic control of susceptibility and survival. Here, we use light-inducible Cre recombinase to activate expression of drug resistance genes in Escherichia coli. We demonstrate light-activated resistance to four antibiotics: carbenicillin, kanamycin, chloramphenicol, and tetracycline. Cells exposed to 465 nm blue light survive in the presence of lethal antibiotic concentrations, while those kept in the dark do not. To optimize resistance induction ranges, we characterize the impact of the promoter, ribosome binding site, and enzyme variant strength using chromosome and plasmid-based constructs. Using time-lapse microscopy, we further show resistance activation dynamics. These optogenetic drug resistance tools pave the way for spatiotemporal control of cell survival.

synthetic biology↗

bioRxiv2020

Light-driven ATP production promotes mRNA biosynthesis inside hybrid multi-compartment artificial protocells

The construction of energetically autonomous artificial protocells is one of the most urgent and challenging requirements in bottom-up synthetic biology. Here we show a hybrid multi-compartment approach to build Artificial Simplified-Autotroph Protocells (ASAPs) in an effective manner. Chromatophores obtained from Rhodobacter sphaeroides accomplish the photophosphorylation of ADP to ATP functioning as nanosized photosynthetic organellae when encapsulated inside artificial giant phospholipid vesicles. Under continuous illumination chromatophores produce ATP that in turn sustains the transcription of a DNA gene by T7 RNA polymerase inside ASAPs. Cryo-EM and time-resolved spectroscopy were used for characterizing the chromatophore morphology and the orientation of the photophosphorylation proteins, which allow high ATP production rates (up to ~100 ATP/s per ATP synthase). mRNA biosynthesis inside individual vesicles has been determined by confocal microscopy. The hybrid multi-compartment approach here proposed appears at the same time convenient and effective, and thus very promising for the construction of full-fledged artificial protocells.

synthetic biology↗

bioRxiv2021

A modular toolset for electrogenetics

Synthetic biology research and its industrial applications rely on the deterministic spatiotemporal control of gene expression. Recently, electrochemical control of gene expression has been demonstrated in electrogenetic systems (redox-responsive promoters used alongside redox inducers and an electrode), allowing for the direct integration of electronics with complex biological processes for a variety of new applications. However, the use of electrogenetic systems is limited by poor activity, tunability and standardisation. Here, we have developed a variety of genetic and electrochemical tools that facilitate the design and vastly improve the performance of electrogenetic systems. We developed a strong, unidirectional, redox-responsive promoter before deriving a mutant promoter library with a spectrum of strengths. We then constructed genetic circuits with these parts and demonstrated their activation by multiple classes of redox molecules. Finally, we demonstrated electrochemical activation of gene expression in aerobic conditions utilising a novel, modular bioelectrochemical device. This toolset provides researchers with all the elements needed to design and build optimised electrogenetic systems for specific applications.

synthetic biology↗

bioRxiv2026

BxbI-mediated insertion of a 77kb human RET sensitive haplotype into the mouse genome to generate a humanized model of Hirschsprung disease

Hirschsprung disease (HSCR) is a complex developmental disorder of the enteric nervous system, primarily driven by regulatory variants within enhancer elements of the RET gene. To investigate how these variants lead to aganglionosis, we developed a humanized mouse model by inserting an intact 77kb human RET genomic locus into the Rosa26 safe-harbor locus. Utilizing "big DNA" synthetic biology and Bxb1-mediated recombination, we integrated the complete human locus including all exons, introns, and upstream regulatory elements which we validated via nanopore and short-read sequencing. Functional analysis confirmed in vivo human RET expression; however, our initial HSCR-associated "sensitive" haplotype expressed at only 21% of wild-type levels. This significant reduction proved insufficient to rescue the viability when endogenous mouse Ret was deleted. We identified that this deficiency is partially driven by five risk SNPs within established enhancers. Specifically, using CRISPR/Cas9 to restore a conserved Sox10 binding site (converting a sensitive SNP to a protective one) increased RET expression by 1.9-fold and restored transcription factor binding. This study provides a robust framework for modeling human-specific regulatory disorders and demonstrates the critical impact of non-coding variation on disease pathogenesis.

synthetic biology↗

bioRxiv2026

Machine learning guided design of strong transcriptional activation domains

Artificial intelligence has accelerated the design of structured proteins, but it remains difficult to design functional intrinsically disordered proteins because of weak structural constraints and poorly defined sequence-to-function relationships1. Activation domains (ADs), transcription factor regions that recruit coactivators, are an excellent test case for the de novo design of intrinsically disordered sequences. ADs are one of the oldest groups of functional intrinsically disordered sequences2 and designed ADs have applications in synthetic biology, biotechnology, and agriculture. We computationally generated 500,000 candidate sequences that resembled known ADs and predicted AD function with extant machine learning models, including three neural networks. From high-scoring sequences, we selected 18,750 for experimental validation in yeast. Surprisingly, >90% of the 16,770 well-measured sequences function as ADs. Sequences predicted to be active by multiple models were stronger, motivating development of a fast and accurate consensus AD predictor, MADRIGAL. A number of our designed ADs retain functionality across fungi, plants, and human cells. This work illustrates a generalizable paradigm for incorporating sequence-to-function models into the design loop to efficiently generate functional intrinsically disordered sequences.

synthetic biology↗

bioRxiv2024

Integrative multi-omics data elucidating the biosynthesis and regulatory mechanisms of furanocoumarins in Angelica dahurica

Furocoumarins (FCs) are crucial natural products playing a dual role as plant defense molecules and pharmacologically active substances. Angelica dahurica is a renowned herb with diverse and abundant FCs. However, the accumulation pattern over developmental stages, biosynthesis pathway and regulatory mechanisms of FCs in A. dahurica remain elusive, hindering the production of FCs via synthetic biology approaches. Here, we constructed a chromosome-level reference genome for A. dahurica and quantified the content dynamics of 17 coumarins across six developmental stages of its medicinal organ, root. It showed a gradual decrease in FC concentration with root enlargement. The combined analyses of transcriptomic and metabolomic data, together with in vivo enzymatic assay, confirmed that CYP71AZ18 was involved in the biosynthesis of bergaptol, whereas CYP71AZ19 and CYP83F95 contributed to the biosynthesis of xanthotoxol. Notably, CYP71AZ19 originated from a proximal duplication event of CYP71AZ18, specific to A. dahurica, subsequently undergoing neofunctionalization. Accessible chromatin regions (ACRs), especially proximal ACRs, are correlated with higher gene expression levels, including the three validated genes involved in FC biosynthesis, showing potential to regulate metabolite biosynthesis. Our findings provide new insights into the biosynthetic pathway of FCs and the epigenetic regulation of metabolite biosynthesis.

synthetic biology↗

bioRxiv2018

Activation of innate immune responses by a CpG oligonucleotide sequence composed entirely of threose nucleic acid.

Recent advances in synthetic biology have led to the development of nucleic acid polymers with backbone structures distinct from those found in nature, termed xeno-nucleic acids (XNAs). Several unique properties of XNAs make them attractive as nucleic acid therapeutics, most notably their high resistance to serum nucleases and ability to form Watson-Crick base-pairing with DNA and RNA. The ability of XNAs to induce immune responses has not been investigated. Threose nucleic acid (TNA), a type of XNA, is recalcitrant to nuclease digestion and capable of undergoing Darwinian evolution to produce high affinity aptamers; thus, TNA is an attractive candidate for diverse applications, including nucleic acid therapeutics. Here, we evaluated a TNA oligonucleotide derived from a CpG oligonucleotide sequence known to activate TLR9-dependent immune signaling in B cell lines. We observed a slight induction of relevant mRNA signals, robust B cell line activation, and negligible effects on cellular proliferation.

synthetic biology↗

bioRxiv2014

Selection of chromosomal DNA libraries using a multiplex CRISPR system in Saccharomyces cerevisiae

The directed evolution of biomolecules to improve or change their activity is central to many engineering and synthetic biology efforts. However, selecting improved variants from gene libraries in living cells requires plasmid expression systems that suffer from variable copy number effects, or the use of complex marker-dependent chromosomal integration strategies. We developed quantitative gene assembly and DNA library insertion into the Saccharomyces cerevisiae genome by optimizing an efficient single-step and marker-free genome editing system using CRISPR-Cas9. With this Multiplex CRISPR (CRISPRm) system, we selected an improved cellobiose utilization pathway in diploid yeast in a single round of mutagenesis and selection, which increased cellobiose fermentation rates by over ten-fold. Mutations recovered in the best cellodextrin transporters reveal synergy between substrate binding and transporter dynamics, and demonstrate the power of CRISPRm to accelerate selection experiments and discoveries of the molecular determinants that enhance biomolecule function.

Synthetic Biology↗

bioRxiv2015

Engineering Transcriptional Regulator Effector Specificity using Computational Design and In Vitro Rapid Prototyping: Developing a Vanillin Sensor

The pursuit of circuits and metabolic pathways of increasing complexity and ro-bustness in synthetic biology will require engineering new regulatory tools. Feedback control based on relevant molecules, including toxic intermediates and environmental signals, would enable genetic circuits to react appropriately to changing conditions. In this work, variants of qacR, a tetR family repressor, were generated by computational protein design and screened in a cell-free transcription-translation (TX-TL) system for responsiveness to a new targeted effector. The modified repressors target vanillin, a growth-inhibiting small molecule found in lignocellulosic hydrolysates and other industrial processes. Promising candidates from the in vitro screen were further characterized in vitro and in vivo in a gene circuit. The screen yielded two qacR mutants that respond to vanillin both in vitro and in vivo. We believe this process, a combination of the generation of variants coupled with in vitro screening, can serve as a framework for designing new sensors for other target compounds.

Synthetic Biology↗