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Results for “Synthetic Biology”

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At least 631 records · Page 35Linked to original sources

bioRxiv2023

Microbial biosensor for sensing and treatment of intestinal inflammation

Substantial synthetic biology efforts have been made to engineer biosensors to detect intestinal inflammation, however none target the most clinically accepted biomarker, calprotectin. To develop an in situ biosensor for calprotectin, we optimized a zinc uptake regulator (Zur) regulated promoter coupled with a memory circuit that can detect and record intestinal inflammation in vivo. The level of activation strongly correlates with calprotectin levels in the colon of two independent mouse models of colitis. Coupling of the biosensor with the production of the anti-inflammatory cytokine IL-10 allowed for the resolution of chemically induced colitis, demonstrating the ability of the biosensor to sense and respond to disease. This work highlights the utility of developing synthetic organisms for the diagnosis and treatment of intestinal disease using clinically validated biomarkers. One sentence summaryWe have optimized a microbial biosensor to detect and respond to the clinically relevant intestinal inflammation biomarker calprotectin.

synthetic biology↗

bioRxiv2022

An electrogenetic toggle switch design

Synthetic biology uses molecular biology to implement genetic circuits that perform computations. These circuits can process inputs and deliver outputs according to predefined rules that are encoded, often entirely, into genetic parts. However, the field has recently begun to focus on using mechanisms beyond the realm of genetic parts for engineering biological circuits. We analyse the use of electrogenic processes for circuit design and present a model for a merged genetic and electrogenetic toggle switch. Computational simulations explore conditions under which bistability emerges in order to identify the circuit design principles for best switch performance. The results provide a basis for the rational design and implementation of hybrid devices that can be measured and controlled both genetically and electronically.

synthetic biology↗

bioRxiv2025

Chlamydomonas chloroplast genes tolerate compression of the genetic code to just 51 codons

Genome scale engineering has enabled codon compression of the universal genetic code of up to three codons in E. coli, providing the means for genetic code expansion. To go much beyond this number, smaller and simpler genetic systems are needed to avoid significant technical challenges. Chloroplast genomes offer multiple advantages for codon compression and reassignment. Here we report a recoding scheme for the Chlamydomonas reinhardtii chloroplast genome, in which two stop codons and one or more of the codons for arginine, glycine, isoleucine, leucine and serine, all of which have two cognate tRNAs, are absent, compressing the genetic code to 51 codons. Firstly, several recoding strategies were tested on the essential rpoA gene, encoding a subunit of the chloroplast RNA polymerase. A defined compression scheme, which relied on swapping the target codons with the permitted frequent codons, could replace the native protein coding sequence without affecting chloroplast protein expression levels or the strain fitness. The same strategy was successfully used for codon compression of ycf1, encoding a subunit of the chloroplast translocon, psaA and psbA, intron-containing highly expressed genes encoding reaction centres subunits of both photosystems, and an 8.5 kb operon encoding essential and non-essential genes. Finally, we tested degeneracy of the 51-codon genetic code by exploring the combinatorial design for the large subunit of RuBisCO, relying on restoration of photosynthesis in an rbcL mutant strain. More than 70 functional sequences with diverse codon adaptation indices were recovered. In all codon-compressed genes there was no observable penalty on photosynthetic growth. Significance StatementWe demonstrate the use of a chloroplast genome as a synthetic biology platform to test modifications of the universal genetic code. Elimination of 13 of the 64 possible triplet codes (codons) was possible for several essential and/or highly expressed genes without affecting chloroplast function. The technology is mature, paving the way for codon compression of the entire genome and thus enabling further radical rearrangement or expansion of the genetic code.

synthetic biology↗

bioRxiv2026

Spatial Regulation of CAR Signaling Enables Logic-Gated Activity

Chimeric antigen receptors (CARs) can induce T cells to kill cancer cells but also to kill normal cells that express the same antigens1. Designing CARs to recognize combinations of antigens, via Boolean logic, can simultaneously expand the scope of targetable antigens and make CAR T cells more specific to cancer2. For example, one antigen may be expressed on cancer cells and normal bone marrow cells, while a second antigen may be present on the same cancer cells but only in normal lungs. If recognition of both antigens is required for T cell activation, only the cancer cells will be killed. Creating such AND-gated CAR T cells has been challenging given the need to engineer non-natural signaling mechanisms that integrate two ligand binding events into a single T cell activation stimulus3-6. Here, we design a fundamentally new AND-gated receptor called Multi-ANtigen Triggered Immune Synapse (MANTIS), which leverages differences in extracellular receptor dimensions to regulate CAR signaling. MANTIS initially prevents CAR activity by steric blocking with a bulky extracellular domain. Upon engagement of the first antigen, MANTIS sheds this blocking domain, releasing a free CAR that can bind a second antigen and activate the T cell in an AND-gated manner. This work demonstrates how differences in extracellular receptor size can be leveraged to spatially regulate intracellular signaling pathways in response to antigen patterns, paving the way for new applications in synthetic biology and cell engineering. One sentence summaryDifferences in extracellular size can be leveraged to regulate CAR T cell activity for precise recognition of antigen patterns.

synthetic biology↗

bioRxiv2023

CRISPR-COPIES: An in silico platform for discovery of neutral integration sites for CRISPR/Cas-facilitated gene integration

The CRISPR/Cas system has emerged as a powerful tool for genome editing in metabolic engineering and human gene therapy. However, locating the optimal site on the chromosome to integrate heterologous genes using the CRISPR/Cas system remains an open question. Selecting a suitable site for gene integration involves considering multiple complex criteria, including factors related to CRISPR/Cas-mediated integration, genetic stability, and gene expression. Consequently, identifying such sites on specific or different chromosomal locations typically requires extensive characterization efforts. To address these challenges, we have developed CRISPR-COPIES, a COmputational Pipeline for the Identification of CRISPR/Cas-facilitated intEgration Sites. This tool leverages ScaNN, a state-of-the-art model on the embedding-based nearest neighbor search for fast and accurate off-target search and can identify genome-wide intergenic sites for most bacterial and fungal genomes within minutes. As a proof of concept, we utilized CRISPR-COPIES to characterize neutral integration sites in three diverse species: Saccharomyces cerevisiae, Cupriavidus necator, and a human cell line. In addition, we developed a user-friendly web interface for CRISPR-COPIES (https://biofoundry.web.illinois.edu/copies/). We anticipate that CRISPR-COPIES will serve as a valuable tool for targeted DNA integration and aid in the characterization of synthetic biology toolkits, enable rapid strain construction to produce valuable biochemicals and support human gene and cell therapy applications. Graphical abstractOverview and application of CRISPR-COPIES in the field of biotechnology. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/556564v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@c723a1org.highwire.dtl.DTLVardef@1f42594org.highwire.dtl.DTLVardef@1cf4bd6org.highwire.dtl.DTLVardef@7c1028_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2019

Polychromatic solar energy conversion in pigment-protein chimeras that unite the two kingdoms of (bacterio)chlorophyll-based photosynthesis

Natural photosynthesis can be divided between the chlorophyll-containing plants, algae and cyanobacteria that make up the oxygenic phototrophs and a diversity of bacteriochlorophyll-containing bacteria that make up the anoxygenic phototrophs. Photosynthetic light harvesting and reaction centre proteins from both groups of organisms have been exploited in a wide range of biohybrid devices for solar energy conversion, solar fuel synthesis and a variety of sensing technologies, but the energy harvesting abilities of these devices are limited by each proteins individual palette of (bacterio)chlorophyll, carotenoid and bilin pigments. In this work we demonstrate a range of genetically-encoded, self-assembling photosystems in which recombinant plant light harvesting complexes are covalently locked with reaction centres from a purple photosynthetic bacterium, producing macromolecular chimeras that display mechanisms of polychromatic solar energy harvesting and conversion not present in natural systems. Our findings illustrate the power of a synthetic biology approach in which bottom-up construction of a novel photosystem using naturally disparate but mechanistically complementary components is achieved in a predictable fashion through the genetic encoding of adaptable, plug-and-play covalent interfaces. ToC image O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=168 SRC="FIGDIR/small/565283v2_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@12cc9c3org.highwire.dtl.DTLVardef@172354corg.highwire.dtl.DTLVardef@10948b6org.highwire.dtl.DTLVardef@84d88c_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

Iterative and modular expression of Botryococcus braunii genes enhances isoprenoid production in the diatom Phaeodactylum tricornutum

Isoprenoids are essential natural compounds with high structural and functional diversity. Among them, carotenoids and triterpenoids such as squalene have high biotechnological value but remain challenging to produce sustainably. The green microalga Botryococcus braunii synthesizes large amounts of triterpenoids through specialized methylerythritol phosphate (MEP) pathway configurations involving distinct 1-deoxy-D-xylulose-5-phosphate synthase (DXS) isoforms, a bifunctional squalene synthase (SQS) and squalene synthase-like (SSL) enzymes working together for the synthesis of the compound. However, its slow growth limits industrial application. In this work, we established the fast-growing diatom Phaeodactylum tricornutum as a heterologous chassis for terpenoid production using a synthetic biology Design-Build-Test-Learn (DBTL)-based approach. Episomal uLoop assembly enabled rapid expression and evaluation of B. braunii DXS and SQS variants, enhancing precursor flux and resulting in the co-accumulation of squalene and carotenoids. This work demonstrates the functional transfer of specialized algal terpenoid enzymes into a tractable diatom host and highlight P. tricornutums potential as a versatile platform for sustainable, high-value isoprenoid biosynthesis.

synthetic biology↗

bioRxiv2020

A Lysate Proteome Engineering Strategy for Enhancing Cell-Free Metabolite Production

Cell-free systems present a significant opportunity to harness the metabolic potential of diverse organisms. Removing the cellular context provides the ability to produce biological products without the need to maintain cell viability and enables metabolic engineers to explore novel chemical transformation systems. Crude extracts maintain much of a cells capabilities. However, only limited tools are available for engineering the contents of the extracts used for cell-free systems. Thus, our ability to take full advantage of the potential of crude extracts for cell-free metabolic engineering is limited. Here, we employ Multiplex Automated Genomic Engineering (MAGE) to tag proteins for selective removal from crude extracts so as to specifically direct chemical production. Specific edits to central metabolism are possible without significantly impacting cell growth. Selective removal of pyruvate degrading enzymes are demonstrated that result in engineered crude lysates that are capable of 10 to 20-fold increases of pyruvate production when compared to the non-engineered extract. The described approach melds the tools of systems and synthetic biology to develop cell-free metabolic engineering into a practical platform for both bioprototyping and bioproduction. HighlightsO_LIA novel method of engineering crude cell lysates for enhancing specific metabolic processes is described. C_LIO_LIMultiplex Automated Genomic Engineering (MAGE) can be used to engineer donor strains for improving cell-free metabolite production with minimal impact on cell-growth. C_LIO_LIThe described lysate engineering strategy can specifically direct metabolic flux and create metabolic states not possible in living cells. C_LIO_LIPooling of the central precursor pyruvate was significantly improved through use of this lysate proteome engineering strategy. C_LI

synthetic biology↗

bioRxiv2016

Biosynthesis of the Antibiotic Nonribosomal Peptide Penicillin in Baker’s Yeast

Fungi are a valuable source of enzymatic diversity and therapeutic natural products including antibiotics. By taking genes from a filamentous fungus and directing their efficient expression and subcellular localisation, we here engineer the bakers yeast Saccharomyces cerevisiae to produce and secrete the antibiotic penicillin, a beta-lactam nonribosomal peptide. Using synthetic biology tools combined with long-read DNA sequencing, we optimise productivity by 50-fold to produce bioactive yields that allow spent S. cerevisiae growth media to have antibacterial action against Streptococcus bacteria. This work demonstrates that S. cerevisiae can be engineered to perform the complex biosynthesis of multicellular fungi, opening up the possibility of using yeast to accelerate rational engineering of nonribosomal peptide antibiotics.

Synthetic Biology↗

bioRxiv2023

Robust differentiation in a synthetic stem-cell circuit

Differentiation is a process fundamental to multicellularity. In its simplest form, differentiation converts self-renewing stem cells into non-proliferative cells with specified function. This process is inherently susceptible to mutant takeover -- mutant stem cells that never differentiate produce excess proliferative daughter cells, driving cancer-like expansion and decreasing the availability of differentiated cells to the organism. It has been proposed that coupling differentiation to an essential trait can select against these mutants by producing a biphasic fitness curve. This would provide mutant stem cells that do not differentiate with a selective disadvantage. However, this theory has yet to be tested experimentally. Here we use "fitness landscape engineering" to design and construct a synthetic biological model of stem cell differentiation in Escherichia coli with biphasic fitness. We find that this circuit is robust to mutations as predicted. Surprisingly, its optimal differentiation rate is robust to a wide range of environmental pressures. This environmental robustness is driven by transit-amplifying cells that differentiate and proliferate irrespective of environment. These results provide new interpretations for natural differentiation mechanisms and suggest strategies for engineering robust, complex multicellular consortia.

synthetic biology↗

bioRxiv2020

A chemical strategy to control protein networks in vivo

Direct control of protein interaction by chemically induced protein proximity (CIPP) holds great potential for cell- and synthetic biology as well as therapeutic applications. However, toxicity, low cell-permeability and lack of orthogonality currently limit the use of available chemical inducers of proximity (CIP). We present Mandi, a novel CIP and demonstrate its applicability in cell culture systems as well as living organisms for protein translocation, protein network shuttling and manipulation of endogenous proteins.

synthetic biology↗

bioRxiv2021

A lipid-based parallel processor for chemical signals

A key goal of bottom-up synthetic biology is to construct cell- and tissue-like structures. Underpinning cellular life is the ability to process several external chemical signals, often in parallel. Until now, however, cell- and tissue-like structures have only been constructed with one signalling pathway. Here, we construct a dual-signal processor from the bottom up in a modular fashion. The processor comprises three aqueous compartments bounded by lipid bilayers and operates in an aqueous environment. It can receive two chemical signals from the external environment, process them orthogonally, and then produce a distinct output for each signal. It is suitable for both sensing and enzymatic processing of environmental signals with fluorescence and molecular outputs. In the future, such processors could serve as smart drug delivery vehicles or as modules within synthetic tissues to control their behaviour in response to external chemical signals.

synthetic biology↗

bioRxiv2024

Cascaded Antithetic Integral Feedback Motifs for Robust Stability and Performance Improvement

Precise intracellular regulation and robust perfect adaptation can be achieved using biomolecular integral controllers and it holds enormous potential for synthetic biology applications. In this letter, we consider the cascaded implementation of a class of such integrator motifs. Our cascaded integrators underpin proportional-integral-derivative (PID) control structures, which we leverage to suggest ways to improve dynamic performance. Moreover, we demonstrate how our cascaded strategy can be harnessed to enhance robust stability in a class of uncertain reaction networks. We also discuss the genetic implementation of our controllers and the natural occurrence of their cascaded sequestration pairs in bacterial pathogens.

synthetic biology↗

bioRxiv2026

Long-term stability of soil microbiome structure and function in liquid soil extracts

Soil microbial communities (SMCs) play an important role in various ecological processes, including plant growth, carbon cycling, and greenhouse gas production and consumption. There have been many prior studies of soil microbiome function and structure. However, soil is a complex environment in which to conduct biological studies. Therefore, simplified SMC models, often adapted to liquid culture, have been employed in the laboratory to study specific microbial interactions and individual microbial functions. Specific advantages of these laboratory liquid SMC models include the ability to modulate community membership, control environmental conditions, and employ high-throughput assay techniques. The disadvantages of current laboratory liquid SMC models include long cycles for growing bacteria in vitro, the obligatory use of strains that are culturable in isolation, intricate media requirements, and complex community assembly protocols. To address some limitations of current liquid SMC models, we sought to create a streamlined process for extracting and maintaining a liquid culture of an existing SMC. Soil-Extracted Solubilized Organic Matter (SESOM) was made from four different soil types, including rich organic potting soils and environmental samples, and filtered to maintain the SMC. These SESOM liquid SMC models were cultured for 28 days, and SMC composition was measured by 16S rDNA sequencing. The SESOM SMCs maintain high alpha and beta diversity over time, including strains that are not culturable in isolation, with the greatest stability correlated with higher soil organic carbon. Further, the SESOM SMCs maintain unique signatures of their starting solid soils, suggesting that drift in SMC composition over extended time in liquid culture does not eliminate the defining microbial relationships of a given soil type. Network analysis of SESOM SMCs relative to solid soils suggests the functional roles of bacterial taxa were maintained in the liquid models over time. We further demonstrate that the platform can be applied to monitor the survival and persistence of a model engineered microbe - the common synthetic biology chassis Pseudomonas putida - within a native SMC. We conclude that the SESOM model is a valuable tool for facilitating the study of SMCs in the laboratory.

synthetic biology↗

bioRxiv2023

Using methyl bromide for interspecies cell-cell signaling and as a reporter in a model soil consortium

Soil microbial communities with reduced complexity are emerging as model systems for studying consortia-scale phenotypes. To establish synthetic biology tools for studying these communities in hard-to-image environmental materials, we evaluated whether a single member of a model soil consortium (MSC) can be programmed to report on gene expression without requiring matrix disruption. For these studies, we targeted a five member MSC that includes Dyadobacter fermentans, Ensifer adhaerens, Rhodococcus sp003130705, Streptomyces sp001905665, and Variovorax beijingensis. By coupling the expression of a methyl halide transferase to a constitutive promoter, we show that Variovorax beijingensis can be programmed to synthesize methyl halides that accumulate in the soil headspace at levels that are [≥]24-fold higher than all other MSC members across a range of environmentally-relevant hydration conditions. We find that methyl halide production can report on a MSC promoter that is activated by changes in water potential, and we demonstrate that a synthetic gas signal can be read out directly using gas chromatography and indirectly using a soil-derived Methylorubrum that is programmed to produce a visual output in response to methyl halides. These tools will be useful for future studies that investigate how MSC respond to dynamic hydration conditions, such as drought and flood events induced by climate change, which can alter soil water potential and induce the release of stored carbon.

synthetic biology↗

bioRxiv2018

Design of a Synthetic sRNA-based Feedback Filter Module

Filters are widely used in engineering to reduce noise and/or the magnitude of a signal of interest. Feedback filters, or adaptive filters, are preferred if the signal noise distribution is unknown. One of the main challenges in Synthetic Biology remains the design of reliable constructs but these often fail to work as intended due, e.g. to their inherent stochasticity and burden on the host. Here we design, implement and test experimentally a biological feedback filter module based on small non-coding RNAs (sRNAs) and self-cleaving ribozymes. Mathematical modelling demonstrates that it attenuates noise for a large range of parameters due to negative feedback introduced by the use of ribozymes and sRNA. Our module modifies the steady-state response of the filtered signal, and hence can be used for tuning the feedback strength while also reducing noise. We demonstrated these properties theoretically on the TetR autorepressor, enhanced with our sRNA module.

synthetic biology↗

bioRxiv2022

Tailored synthetic sRNAs dynamically tune multilayer genetic circuits

Predictable and controllable tuning of genetic circuits to regulate gene expression, including modulation of existing circuits or constructs without the need for redesign or rebuilding, is a persistent challenge in synthetic biology. Here, we propose a rational design of de novo small RNAs (sRNAs) to dynamically modulate gene expression within a broad range, from high, medium to low repression, and implemented them in Escherichia coli. We designed multiple multilayer genetic circuits, in which the variable effector element is a transcription factor (TF) controlling downstream the production of a reporter protein. Our approach harnesses the intrinsic RNA-interference pathway in E. coli to exert dynamic and modular control of the multilayer genetic circuits. The sRNAs were designed to target TFs instead of the reporter gene, which allowed for wide range of expression modulation of the reporter protein, including the most difficult to achieve dynamic switch to an OFF state. Out work provides a frame for achieving independent modulation of gene expression, by only including an independent control circuit expressing synthetic sRNAs, without altering the structure of existing genetic circuits.

synthetic biology↗

bioRxiv2020

Multidimensional single-cell benchmarking of inducible promoters for precise dynamic control in budding yeast

For quantitative systems biology, simultaneous readout of multiple cellular processes as well as precise, independent control over different genes activities are essential. In contrast to readout systems such as fluorescent proteins, control systems such as inducible transcription-factor-promoter systems have only been characterized in an ad hoc fashion, impeding precise system-level manipulations of biological systems and reliable modeling. We designed and performed systematic benchmarks involving easy-to-communicate units to characterize and compare inducible transcriptional systems. We built a comprehensive single-copy library of inducible systems controlling standardized fluorescent protein expression in budding yeast, including GAL1pr, GALL, MET3pr, CUP1pr, PHO5pr, tetOpr, terminator-tetOpr, Z3EV system, the blue-light optogenetic systems El222-LIP, El222-GLIP and the red-light inducible PhyB-PIF3 system. To analyze these systems dynamic properties, we performed high-throughput time-lapse microscopy. The analysis of >100 000 cell images was made possible by the recently developed convolutional neural network YeaZ. We report key kinetic parameters, scaling of noise levels, impacts on growth, and, crucially, the fundamental leakiness of each system. Our multidimensional benchmarking additionally uncovers unexpected disadvantages of widely used tools, e.g., nonmonotonic activity of the MET3 and GALL promoters, slow off kinetics of the doxycycline and estradiol-inducible systems tetOpr and Z3EV, and high variability of PHO5pr and red-light activated PhyB-PIF3 system. We introduce two new tools for controlling gene expression: strongLOV, a more light-sensitive El222 mutant, and ARG3pr that functions as an OR gate induced by the lack of arginine or presence of methionine. To demonstrate the ability to finely control genetic circuits, we experimentally tuned the time between cell cycle Start and mitotic entry in budding yeast, artificially simulating near-wild-type timing. The characterizations presented here define the compromises that need to be made for quantitative experiments in systems and synthetic biology. To calibrate perturbations across laboratories and to allow new inducible systems to be benchmarked, we deposited single-copy reporter yeast strains, plasmids, and computer analysis code in public repositories. Furthermore, this resource can be accessed and expanded through the website https://promoter-benchmark.epfl.ch/.

synthetic biology↗