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Results for “Synthetic Biology”

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bioRxiv2022

Inducible CRISPR/Cas9 allows for multiplexed and rapidly segregated single target genome editing in Synechocystis sp. PCC 6803

Establishing various synthetic biology tools is crucial for the development of cyanobacteria for biotechnology use, especially tools that allow for precise and markerless genome editing in a time-efficient manner. Here we describe a riboswitch-inducible CRISPR/Cas9 system, contained on one single replicative vector, for the model cyanobacteria Synechocystis sp. PCC 6803. A theophylline-responsive riboswitch allowed tight control of Cas9 expression, which enabled reliable transformation of the CRISPR/Cas9 vector into Synechocystis. Induction of the CRISPR/Cas9 mediated various types of genomic edits, specifically deletions and insertions of varying size. The editing efficiency varied depending on the target and intended edit; smaller edits overall performed better, reaching e.g. 100% for insertion of a FLAG-tag onto rbcL. Importantly, the single-vector CRISPR/Cas9 system described herein was also shown to mediate multiplexed editing of up to three targets in parallel in Synechocystis. All single-target and several double-target mutants were also fully segregated after the first round of induction, adding to the usefulness of this system. Further, a vector curing system that is separately induced by nickel and contained on the CRISPR/Cas9 vector itself, improved curing efficiencies by roughly 4-fold, enabling the final mutants to become truly markerless.

synthetic biology↗

bioRxiv2020

Cas9-generated auxotrophs of Phaeodactylum tricornutum are characterized by small and large deletions that can be complemented by plasmid-based genes

The model diatom Phaeodactylum tricornutum is an attractive candidate for synthetic biology applications. Development of auxotrophic strains of P. tricornutum would provide alternative selective markers to commonly used antibiotic resistance genes. Here, using CRISPR/Cas9, we show successful editing of genes in the uracil, histidine, and tryptophan biosynthetic pathways. Editing events are characterized by loss of heterozygosity and by the occurrence of large deletions of up to ~2.7-kb centred on the editing site. The uracil and histidine-requiring phenotypes can be complemented by plasmid-based copies of the intact genes after curing of the Cas9-editing plasmid. Growth of uracil auxotrophs on media supplemented with 5-FOA and uracil results in loss of the complementing plasmid, providing a facile method for plasmid curing with potential applications in strain engineering and CRISPR editing. Metabolomic characterization of uracil auxotrophs revealed changes in cellular orotate concentrations consistent with partial or complete loss of orotate phosphoribosyl transferase activity in knockout strains. Our results expand the range of P. tricornutum auxotrophic strains and demonstrate that auxotrophic complementation markers provide a viable alternative to traditionally used antibiotic selection markers. Plasmid-based auxotrophic markers should expand the range of genome engineering applications and provide a means for biocontainment of engineered P. tricornutum strains.

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.

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bioRxiv2024

Engineering a cell-free biosensor signal amplification circuit with polymerase strand recycling

Cell-free systems are powerful synthetic biology technologies because of their ability to recapitulate sensing and gene expression without the complications of living cells. Cell-free systems can perform even more advanced functions when genetic circuits are incorporated as information processing components. Here we expand cell-free biosensing by engineering a highly specific isothermal signal amplification circuit called polymerase strand recycling (PSR) that leverages T7 RNA polymerase off-target transcription to recycle nucleic acid inputs within DNA strand displacement circuits. We develop design rules for PSR circuit components and use these rules to construct modular biosensors that can directly sense different RNA targets with limits of detection in the nM range and high specificity. We then use PSR for signal amplification within allosteric transcription factor-based biosensors for small molecule detection. We use a double equilibrium model of transcription factor:DNA and transcription factor:ligand binding interactions to predict biosensor sensitivity enhancement by PSR, and then demonstrate this approach experimentally by achieving 3.6-4.6-fold decreases in biosensor EC50 to sub micromolar ranges. We believe this work expands the current capabilities of cell-free circuits by incorporating PSR, which we anticipate will have a wide range of uses within biotechnology.

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bioRxiv2025

Generative inverse design of RNA structure and function with gRNAde

The design of RNA molecules with bespoke three-dimensional structures and functions is a central goal in synthetic biology and biotechnology. However, progress has been limited by the challenges of designing complex tertiary interactions such as pseudoknots, as well as engineering catalytic functions--problems that have remained largely intractable for automated methods. Here we present a high-throughput generative AI pipeline for inverse design of RNA structure and function. Central to the pipeline is gRNAde, an RNA language model conditioned on 3D backbone structures and sequence constraints. We have validated the gRNAde pipeline in a community-wide, blinded RNA design competition on the Eterna platform, where it proved able to design complex pseudoknotted RNAs at success rates matching that of human experts (95%), while significantly outperforming other physics- and AI-based automated algorithms (70%). We further demonstrate gRNAdes capabilities by generatively designing functional RNA polymerase ribozymes (RPR) with nearly 20% sequence divergence from the wild type RPR, discovering highly active variants at mutational distances inaccessible to rational design or adaptive walks by directed evolution. gRNAde thus provides an experimentally validated, open-source platform for automated design of complex RNA structures and accelerated engineering of complex RNA functions, providing a step towards programmable RNA catalysts and nanostructures. Open-source code: github.com/chaitjo/geometric-rna-design

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bioRxiv2021

Bioremediation of industrial pollutants by insects expressing a fungal laccase

Inadequate management of household and industrial wastes pose major challenges to human and environmental health. Advances in synthetic biology may help address these challenges by engineering biological systems to perform new functions such as biomanufacturing of high-value compounds from low-value waste streams and bioremediation of industrial pollutants. The current emphasis on microbial systems for biomanufacturing, which often require highly pre-processed inputs and sophisticated infrastructure, is not feasible for many waste streams. Concerns about transgene biocontainment have limited the release of engineered microbes or plants for bioremediation. Engineering animals may provide opportunities for utilizing various waste streams that are not suitable for microbial biomanufacturing while effective transgene biocontainment options should enable in situ bioremediation. Here, we engineer the model insect Drosophila melanogaster to express a functional laccase from the fungus Trametes trogii. Laccase expressing flies reduced concentrations of the endocrine disruptor bisphenol A by more than 50% when present in their growth media. A lyophilized powder made from engineered adult flies retained substantial enzymatic activity, degrading more than 90% of bisphenol A and the textile dye indigo carmine in aqueous solutions. Our results demonstrate that transgenic animals may be used to bioremediate environmental contaminants in vivo and serve as novel production platforms for industrial enzymes. These results support further development of insects, and possibly other animals, as bioproduction platforms and their potential use in bioremediation.

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bioRxiv2021

Biosynthesis of linear protein nanoarrays using the flagellar axoneme

Applications in biotechnology and synthetic biology often make use of soluble proteins, but there are many potential advantages to anchoring enzymes to a stable substrate, including stability and the possibility for substrate channeling. To avoid the necessity of protein purification and chemical immobilization, there has been growing interest in bio-assembly of protein-containing nanoparticles, exploiting the self-assembly of viral capsid proteins or other proteins that form polyhedral structures. But these nanoparticle are limited in size which constrains the packaging and the accessibility of the proteins. The axoneme, the insoluble protein core of the eukaryotic flagellum or cilium, is a highly ordered protein structure that can be several microns in length, orders of magnitude larger than other types of nanoparticles. We show that when proteins of interest are fused to specific axonemal proteins and expressed in living cells, they become incorporated into linear arrays which have the advantages of high protein loading capacity, high stability, and single-step purification with retention of biomass. The arrays can be isolated as membrane enclosed vesicle or as exposed protein arrays. The approach is demonstrated for both fluorescent proteins and enzymes, and in the latter case it is found that incorporation into axoneme arrays provides increased stability for the enzyme.

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bioRxiv2018

Creation and multi-omics characterization of a genomically hybrid strain in the nitrogen-fixing symbiotic bacterium Sinorhizobium meliloti

Many bacteria, often associated with eukaryotic hosts and of relevance for biotechnological applications, harbour a multipartite genome composed by more than one replicon. Biotechnologically relevant phenotypes are often encoded by genes residing on the secondary replicons. A synthetic biology approach to developing enhanced strains for biotechnological purposes could therefore involve merging pieces or entire replicons from multiple strains into a single genome. Here we report the creation of a genomic hybrid strain in a model multipartite genome species, the plant-symbiotic bacterium Sinorhizobium meliloti. In particular, we moved the secondary replicon pSymA (accounting for nearly 20% of total genome content) from a donor S. meliloti strain to an acceptor strain. The cis-hybrid strain was screened for a panel of complex phenotypes (carbon/nitrogen utilization phenotypes, intra- and extra-cellular metabolomes, symbiosis, and various microbiological tests). Additionally, metabolic network reconstruction and constraint-based modelling were employed for in silico prediction of metabolic flux reorganization. Phenotypes of the cis-hybrid strain were in good agreement with those of both parental strains. Interestingly, the symbiotic phenotype showed a marked cultivar-specific improvement with the cis-hybrid strains compared to both parental strains. These results provide a proof-of-principle for the feasibility of genome-wide replicon-based remodelling of bacterial strains for improved biotechnological applications in precision agriculture.

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bioRxiv2025

Magneto-Photonic Gene Circuit for Minimally Invasive Control of Gene Expression in Mammalian Cells

Precise control of gene expression is one of the fundamental goals of synthetic biology. Whether the objective is to modify endogenous cellular function or induce the expression of molecules for diagnostic and therapeutic purposes, gene regulation remains a key aspect of biological systems. Over time, advances in protein engineering and molecular biology have led to the creation of gene circuits capable of inducing the expression of specific proteins in response to external stimulus such as light. These optogenetic, or light-activated circuits hold significant potential for gene therapy as a tool for regulating the expression of therapeutic genes within cells. However, the applications of optogenetic systems can be limited by the lack of efficient ways for light delivery inside cells or tissue. Our approach to address this challenge is to harness the power of bioluminescence to produce light directly inside cells using a luminescent enzyme. Combined with a photosensitive transcription factor, we report the development of a fully genetically encoded optogenetic circuit for control of gene expression. Furthermore, we utilized a magneto sensitive protein to engineer a split protein version of this luminescent enzyme, where its reconstitution is driven by a 50mT magnetic stimulus. Thus, resulting in a first-of-its-kind gene circuit activated by a combination of light and magnetic stimulus. We expect this work to advance the implementation of light-controlled systems without the need of external light sources, as well as serve as a basis for the development of future magneto-sensitive tools.

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bioRxiv2019

Evolution-guided engineering of small-molecule biosensors

Allosteric transcription factors (aTFs) have proven widely applicable for biotechnology and synthetic biology as ligand-specific biosensors enabling real-time monitoring, selection and regulation of cellular metabolism. However, both the biosensor specificity and the correlation between ligand concentration and biosensor output signal, also known as the transfer function, often needs to be optimized before meeting application needs. Here, we present a versatile and high-throughput method to evolve and functionalize prokaryotic aTF specificity and transfer functions in a eukaryote chassis, namely bakers yeast Saccharomyces cerevisiae. From a single round of directed evolution of the effector-binding domain (EBD) coupled with various toggled selection regimes, we robustly select aTF variants of the cis, cis-muconic acid-inducible transcription factor BenM evolved for change in ligand specificity, increased dynamic output range, shifts in operational range, and a complete inversion of function from activation to repression. Importantly, by targeting only the EBD, the evolved biosensors display DNA-binding affinities similar to BenM, and are functional when ported back into a non-native prokaryote chassis. The developed platform technology thus leverages aTF evolvability for the development of new host-agnostic biosensors with user-defined small-molecule specificities and transfer functions.

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bioRxiv2022

Harnessing cellular perception apparatus for smart metabolic reprogramming

Metabolic reprogramming (MRP) is a fundamental approach in synthetic biology that involves redirecting metabolic flux and remodeling metabolic networks. However, only few approaches have been made in effective metabolic operations, especially at global level of metabolic networks. Naturally existing cellular perception apparatuses (CPAs), such as histidine kinases (HKs), are considered to sit on sensitive nodes of the metabolic network, which can trigger natural MRP upon perceiving environmental fluctuations. We develop a plateform for global MRP by natural environmental stimulation based on the combinational interference of CPAs. The plateform consists of a CRISPRi-mediated dual-gene combinational knockdown (CDCK) strategy and survivorship-based metabolic interaction analysis (SMIA). A total of 35 histidine kinase (HK) genes and 24 glycine metabolism genes were selected as targets to determine effectiveness of our approach for fast-growing chassis Vibrio FA2. Combined knockdown of several genes of HKs and glycine metabolism increased the glycine production. Other other hand, effects of CDCK on bacterial antibiotic resistance were assessed by targeting HKs. Many HKs were identified to be associated with antibiotic resistance in Vibrio FA2, of which combinational knockdown of two HK genes sasA_8 and 04288 reduced the ampicillin resistance. This MRP strategy is powerful and cost-effective, and can be considered as a smart strategy capable of operating a broad range of metabolic networks in microorganisms.

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bioRxiv2024

High-throughput optimisation of protein secretion in yeast via an engineered biosensor.

Secretion of high value proteins and enzymes is fundamental to the synthetic biology economy; allowing continuous fermentation during production and protein purification without cell lysis. Most eukaryotic protein secretion is encoded by an N-terminal signal peptide; however, the strong impact of signal peptide sequence variation on the secretion efficiency of a given protein is not well defined. Despite high natural signal peptide sequence diversity, most recombinant protein secretion systems employ only a few well characterised signal peptides. Additionally, the selection of promoters and terminators can significantly affect secretion efficiency, yet screening numerous genetic constructs for optimal sequences remains inefficient. Here, we have adapted a yeast G-protein coupled receptor biosensor, to measure the concentration of a peptide tag that is co-secreted with any protein of interest. Protein secretion efficiency can thus be quantified via the induction of a fluorescent reporter that is upregulated downstream of receptor activation. This enables high-throughput screening of over 6000 combinations of promoters, signal peptides and terminators, assembled using one-pot Combinatorial Golden Gate cloning. We demonstrate this biosensor can quickly identify best combinations for secretion and quantify secretion levels. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=188 HEIGHT=200 SRC="FIGDIR/small/594099v2_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@87d612org.highwire.dtl.DTLVardef@1f73d24org.highwire.dtl.DTLVardef@cf35a7org.highwire.dtl.DTLVardef@1cb8a3b_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2019

Rapid and assured genetic engineering methods applied to Acinetobacter baylyi ADP1 genome streamlining

One goal of synthetic biology is to improve the efficiency and predictability of living cells by removing extraneous genes from their genomes. We demonstrate improved methods for engineering the genome of the metabolically versatile and naturally transformable bacterium Acinetobacter baylyi ADP1 and apply them to a genome streamlining project. In Golden Transformation, linear DNA fragments constructed by Golden Gate Assembly are directly added to cells to create targeted deletions, edits, or additions to the chromosome. We tested the dispensability of 55 regions of the ADP1 chromosome using Golden Transformation. The 19 successful multiple-gene deletions ranged in size from 21 to 183 kilobases and collectively accounted for 24.6% of its genome. Deletion success could only be partially predicted on the basis of a single-gene knockout strain collection and a new Tn-Seq experiment. We further show that ADP1s native CRISPR/Cas locus is active and can be retargeted using Golden Transformation. We reprogrammed it to create a CRISPR-Lock, which validates that a gene has been successfully removed from the chromosome and prevents it from being reacquired. These methods can be used together to implement combinatorial routes to further genome streamlining and for more rapid and assured metabolic engineering of this versatile chassis organism.

synthetic biology↗

bioRxiv2024

Prevention of ribozyme catalysis through cDNA synthesis enables accurate RT-qPCR measurements of context-dependent ribozyme activity

Self-cleaving ribozymes are important tools in synthetic biology, biomanufacturing, and nucleic acid therapeutics. These broad applications deploy ribozymes in many genetic and environmental contexts, which can influence activity. Thus, accurate measurements of ribozyme activity across diverse contexts are crucial for validating new ribozyme sequences and ribozyme-based biotechnologies. Ribozyme activity measurements that rely on RNA extraction, such as RNA sequencing or reverse transcription-quantitative polymerase chain reaction (RT-qPCR), are generalizable to most applications and have high sensitivity. However, the activity measurement is indirect, taking place after RNA is isolated from the environment of interest and copied to DNA. So these measurements may not accurately reflect the activity in the original context. Here we develop and validate an RT-qPCR method for measuring context-dependent ribozyme activity using a set of self-cleaving RNAs for which context-dependent ribozyme cleavage is known in vitro. We find that RNA extraction and reverse transcription conditions can induce substantial ribozyme cleavage resulting in incorrect activity measurements with RT-qPCR. To restore the accuracy of the RT-qPCR measurements, we introduce an oligonucleotide into the sample preparation workflow that inhibits ribozyme activity. We then apply our method to measure ribozyme cleavage of RNAs produced in Escherichia coli (E. coli). These results have broad implications for many ribozyme measurements and technologies.

synthetic biology↗

bioRxiv2020

Predator: A novel method for targeted protein degradation

Protein expression and degradation are fundamental to cell function and physiological status of organisms. Interfering with protein expression not only provides powerful strategies to analyze the function of proteins but also inspires effective treatment methods for diseases caused by protein dysfunction. Recently, harnessing the power of the ubiquitin-proteasome system for targeted protein degradation (TPD) has become the focus of researches. Over the past two decades, TPD technologies, such as E3 ligase modification, PROTACs, and the Trim-Away method, have successfully re-oriented the ubiquitin-proteasome pathway and thus degraded many pathogenic proteins and even "undruggable" targets. However, A low-cost, convenient, and modularized TPD method is currently not available. Herein, we proposed a synthetic biology TPD method, termed Predator, by integrating the classic function of E3 ligase Trim21 and the expression of a bifunctional fusion protein that links Trim21 and the target protein, which leads to the formation of a ternary complex inside mammalian cells and therefore induce the ubiquitination and subsequent proteasome-dependent degradation of the target protein. We first proved this concept by using nanobody and scFv as the targeting module for the Predator system to degrade free GFP and membrane protein ErbB3, respectively. Then, we give an example of how the engineered Predator system can be developed towards biomedical solutions in the context of diabetes mellitus. Ligands-receptor interaction and adenovirus-mediated gene delivery were introduced to the Predator system, and we found this bifunctional fusion protein, in which glucagon was selected to function as the targeting module, downregulated the endogenous glucagon receptor (GCGR) and attenuated glucagon-stimulated glucose production in primary hepatocytes. Although preliminarily, our results showed that this Predator system is a highly modularized and convenient TPD method with good potential for both fundamental researches and clinical usage. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=160 HEIGHT=200 SRC="FIGDIR/small/231787v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@21e152org.highwire.dtl.DTLVardef@4ff069org.highwire.dtl.DTLVardef@309d74org.highwire.dtl.DTLVardef@1b1653d_HPS_FORMAT_FIGEXP M_FIG C_FIG

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bioRxiv2019

Mathematical models of protease-based enzymatic biosensors

An important goal of synthetic biology is to build biosensors and circuits with well-defined input-output relationships that operate at speeds found in natural biological systems. However, for molecular computation, most commonly used genetic circuit elements typically involve several steps from input detection to output signal production: transcription, translation, and post-translational modifications. These multiple steps together require up to several hours to respond to a single stimulus, and this limits the overall speed and complexity of genetic circuits. To address this gap, molecular frame-works that rely exclusively on post-translational steps to realize reaction networks that can process inputs at a timescale of seconds to minutes have been proposed. Here, we build mathematical models of fast biosensors capable of producing Boolean logic functionality. We employ protease-based chemical and light-induced switches, investigate their operation, and provide selection guidelines for their use as on-off switches. We then use these switches as elementary blocks, developing models for biosensors that can perform OR and XOR Boolean logic computation while using reaction conditions as tuning parameters. We use sensitivity analysis to determine the time-dependent sensitivity of the output to proteolytic and protein-protein binding reaction parameters. These fast protease-based biosensors can be used to implement complex molecular circuits with a capability of processing multiple inputs controllably and algorithmically. Our framework for evaluating and optimizing circuit performance can be applied to other molecular logic circuits.

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bioRxiv2026

In vitro evolution of DNA operators enables multivalency protein-DNA interactions: towards programmable transcription factor regulation

In vitro transcription (IVT) systems regulated by allosteric transcription factors (aTFs) are central to emerging cell-free biosensing and synthetic biology platforms, yet their performance is often limited by suboptimal protein-DNA interactions and the need for well-characterized regulatory elements. Here, we report an in vitro evolution strategy to engineer DNA operator sequences that enables tunable aTF-DNA interactions without requiring prior detailed knowledge of the native operator or regulatory mechanism. Using a SELEX-based approach with integrated positive and counter-selection steps, we evolved non-natural operators for the sulfane sulfur-responsive transcriptional repressor SqrR. The selected sequences preserve ligand-responsive allostery, with some sequences exhibiting enhanced binding affinity and reducing transcriptional leakage. Notably, we identify operator with binding behaviors consistent with cooperative recruitment of multiple SqrR dimers, suggesting that sequence architecture can modulate aTF-DNA interactions beyond affinity alone. Incorporation of these operators into IVT circuits improves transcriptional control and dynamic range, enabling the development of ROSALIND-based sensors for sulfane sulfur species, achieving sensitive and selective detection in a fully cell-free format. More broadly, this work establishes operator evolution as a programmable strategy to optimize transcription factor-DNA interactions and expand the design space of transcription-based biosensors, including for systems lacking well-characterized genetic components.

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bioRxiv2022

Golden Standard: A complete standard, portable, and interoperative MoClo tool for model and non-model bacterial hosts.

Modular cloning assembly has become a benchmark technology in synthetic biology. However, there is a mismatch between its impressive development and the standardization required to promote interoperability between the different systems available. The full development of the field is thus hampered by a surge of oftentimes incompatible organism-specific systems. To overcome these issues, we present Golden Standard (GS), a Type IIS assembly method underpinned by the Standard European Vector Architecture (SEVA). GS unlocks modular cloning applications with any type of microorganism and delivers consistent combinatorial multi-part assembly of standardized genetic elements to create genetic circuits of up to twenty transcription units. Reliance on the Golden Gate syntax renders GS fully compatible with many existing tools and it sets the path towards efficient reusability of available part libraries and assembled TUs. GS was fully validated in terms of DNA assembly performance, portability and phenotype engineering in model and non-model bacteria. In order to facilitate the widespread adoption and future community-driven development of GS, we provide a web-portal featuring: i) a repository of parts and vectors, ii) a SBOLHub for exchange and analysis of constructs and iii) Wizard and Setup tools to guide the design of constructs using stored and user-specific parts.

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