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bioRxiv2023

Uncovering the hidden mechanisms of giant unilamellar vesicle formation in cDICE

Giant unilamellar vesicles (GUVs) are widely used as in vitro model membranes in biophysics and as cell-sized containers in synthetic biology. Despite their ubiquitous use, there is no one-size-fits-all method for their production. Numerous methods have been developed to meet the demanding requirements of reproducibility, reliability, and high yield, while simultaneously achieving robust encapsulation. Emulsion-based methods are often praised for their apparent simplicity and good yields; hence, methods like continuous droplet interface crossing encapsulation (cDICE) that make use of this principle, have gained popularity. However, the underlying physical principles governing the formation of GUVs in cDICE and related methods remain poorly understood. To this end, we have developed a high-speed microscopy setup that allows us to visualize GUV formation in real-time. Our experiments reveal a complex droplet formation process occurring at the capillary orifice, generating both larger droplets and, likely, GUV-sized satellite droplets. According to existing theoretical models, the oil-water interface should allow for crossing of all droplets, but based on our observations and theoretical modelling of the fluid dynamics within the system, we find a size-selective crossing of GUV-sized droplets only. Finally, we demonstrate that proteins in the inner solution affect GUV formation by increasing the viscosity and altering lipid adsorption kinetics. These results will not only contribute to a better understanding of GUV formation processes in cDICE, but ultimately also aid the development of more reliable and efficient methods for GUV production. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/562183v2_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@9764cforg.highwire.dtl.DTLVardef@15b63c7org.highwire.dtl.DTLVardef@f39e83org.highwire.dtl.DTLVardef@2bb63a_HPS_FORMAT_FIGEXP M_FIG C_FIG We developed a high-speed microscopy setup to study giant unilamellar vesicle formation in cDICE, revealing a complex droplet formation process occurring at the capillary orifice and size-selectivity at the oil-water interface.

synthetic biology↗

bioRxiv2022

Highly Multiplexed Selection of RNA Aptamers Against a Small Molecule Library

Applications of synthetic biology spanning human health, industrial bioproduction, and ecosystem monitoring often require small molecule sensing capabilities, typically in the form of genetically encoded small molecule biosensors. Critical to the deployment of greater numbers of these systems are methods that support the rapid development of such biosensors against a broad range of small molecule targets. Here, we use a previously developed method for selection of RNA biosensors against unmodified small molecules (DRIVER) to perform a selection against a densely multiplexed mixture of small molecules, representative of those employed in high-throughput drug screening. Using a mixture of 5,120 target compounds randomly sampled from a large diversity drug screening library, we performed a 95-round selection and then analyzed the enriched RNA biosensor library using next generation sequencing (NGS). From our analysis, we identified RNA biosensors with at least 2-fold change in signal in the presence of at least 217 distinct target compounds with sensitivities down to 25 nM. Although many of these biosensors respond to multiple targets, clustering analysis indicated at least 150 different small-molecule sensing patterns. We also built a classifier that was able to predict whether the biosensors would respond to a new compound with an average precision of 0.82. Since the target compound library was designed to be representative of larger diversity compound libraries, we expect that the described approach can be used with similar compound libraries to identify aptamers against other small molecules with a similar success rate. The new RNA biosensors (or their component aptamers) described in this work can be further optimized and used in applications such as biosensing, gene control, or enzyme evolution. In addition, the data presented here provide an expanded compendium of new RNA aptamers compared to the 82 small molecule RNA aptamers published in the literature, allowing further bioinformatic analyses of the general classes of small molecules for which RNA aptamers can be found.

synthetic biology↗

bioRxiv2020

Reprogramming acetogenic bacteria with CRISPR-targeted base editing via deamination

Acetogenic bacteria are rising in popularity as chassis microbes in biotechnology due to their capability of converting inorganic one-carbon (C1) gases to organic chemicals. To fully uncover the potential of acetogenic bacteria, synthetic-biology tools are imperative to either engineer designed functions or to interrogate the physiology. Here, we report a genome-editing tool at a one-nucleotide resolution, namely base editing, for acetogenic bacteria based on CRISPR-targeted deamination. This tool combines nuclease deactivated Cas9 with activation-induced cytidine deaminase to enable cytosine-to-thymine substitution without DNA cleavage, homology-directed repair, and donor DNA, which are generally the bottlenecks for applying conventional CRISPR-Cas systems in bacteria. We designed and validated a modularized base-editing tool in the model acetogenic bacterium Clostridium ljungdahlii. The editing principles were investigated, and an in-silico analysis revealed the capability of base editing across the genome. Moreover, genes related to acetate and ethanol production were disrupted individually by installing premature STOP codons to reprogram carbon flux towards improved acetate production. This resulted in engineered C. ljungdahlii strains with the desired phenotypes and stable genotypes. Our base-editing tool promotes the application and research in acetogenic bacteria and provides a blueprint to upgrade CRISPR-Cas-based genome editing in bacteria in general. SignificanceAcetogenic bacteria metabolize one-carbon (C1) gases, such as industrial waste gases, to produce fuels and commodity chemicals. However, the lack of efficient gene-manipulation approaches hampers faster progress in the application of acetogenic bacteria in biotechnology. We developed a CRISPR-targeted base-editing tool at a one-nucleotide resolution for acetogenic bacteria. Our tool illustrates great potential in engineering other A-T-rich bacteria and links designed single-nucleotide variations with biotechnology. It provides unique advantages for engineering industrially relevant bacteria without creating genetically modified organisms (GMOs) under the legislation of many countries. This base-editing tool provides an example for adapting CRISPR-Cas systems in bacteria, especially those that are highly sensitive to heterologously expressed Cas proteins and have limited ability of receiving foreign DNA.

synthetic biology↗

bioRxiv2024

Establishing remote communication between human operator and engineered intestinal microbe via bio-electronic optical language

Synthetic biology enables microbes to be engineered to detect biomarkers and deliver therapies in situ, offering advantages such as self-replication and localized response and treatment. However, the engineered microbes in the intestine remains a "black box", the status of microbial functionality is inaccessible, and the activation of microbial functions is uncontrollable. There is a demand of a "language" between microbes and human for real-time and in-situ monitoring and controlling engineered microbes in the intestine. Light, due to its advantages of specificity and accuracy in the body, was chosen as the language to establish an interactive communication path between human and the microbes with the features of sophisticated monitoring and controlling. For effectively remote operation of this "language" in the intestine, we introduced an electronic capsule as the intermedia, translating information and instructions understandable for human operator into bacterial regulatory and detectable signals in vivo. In this paper, the electronic capsule and the engineered Escherichia coli Nissle 1917 (EcN) strains were designed in a collaborative manner. An EcN-to-capsule bioluminescent signal transmission was established and testified as the language to report the status of engineered EcN, especially the response of EcN to intestinal environment. Similarly, another capsule-to-EcN optogenetic signal transmission was established and testified as the language to control the function of engineered EcN, especially the secretion of therapeutic substances. On this basis, bidirectional communication between human operator and intestinal microbes was achieved via bidirectional bio-electronic optical language. The communication was established to monitor and regulate engineered EcN community in the intestine in 50-90 kg live pigs. The in vitro and in vivo estimations demonstrated the capabilities of monitoring and regulating the engineered microbes, offering a remotely controllable language-based communication between human operator and internal intestinal microbes.

synthetic biology↗

bioRxiv2023

A SEVA-based, CRISPR-Cas3-assisted genome engineering approach for Pseudomonas with efficient vector curing

The development of CRISPR-Cas-based engineering technologies has revolutionized the microbial biotechnology field. Over the years, the Class II Type II CRISPR-Cas9 system has become the gold standard for genome editing in many bacterial hosts. However, the Cas9 system does not allow efficient genomic integration in Pseudomonas putida, an emerging Synthetic Biology host, without the assistance of lambda-Red recombineering. In this work, we utilize the alternative Class I Type I-C CRISPR-Cas3 system from Pseudomonas aeruginosa as a highly-efficient genome editing tool for P. putida and P. aeruginosa. This system consists of two vectors, one encoding the Cas genes, CRISPR array and targeting spacer, and a second SEVA-vector, containing the homologous repair template. Both vectors are Golden Gate compatible for rapid cloning and are available with multiple antibiotic markers, for application in various Gram-negative hosts and different designs. By employing this Cas3 system, we successfully integrated an 820-bp cassette in the genome of P. putida and performed several genomic deletions in P. aeruginosa within four days, with an efficiency of >83% for both hosts. Moreover, by introducing a universal self-targeting spacer, the Cas3 system rapidly cures all helper vectors, including itself, from the host strain in a matter of days. As such, this system constitutes a valuable engineering tool for Pseudomonas, to complement the existing range of Cas9-based editing methods and facilitates genomic engineering efforts of this important genus. ImportanceThe CRISPR-Cas3 editing system as presented here facilitates the creation of genomic alterations in P. putida and P. aeruginosa in a straightforward manner. By providing the Cas3 system as a vector set with Golden Gate compatibility and different antibiotic markers, as well as by employing the established SEVA vector set to provide the homology repair template, this system is flexible and can readily be ported to a multitude of Gram-negative hosts. Besides genome editing, the Cas3 system can also be used as an effective and universal tool for vector curing. This is achieved by introducing a spacer that targets the oriT, present on the majority of established (SEVA) vectors. Based on this, the Cas3 system efficiently removes up to three vectors in only a few days. As such, this curing approach may also benefit other genomic engineering methods or remove naturally-occurring plasmids from bacteria.

synthetic biology↗

bioRxiv2020

Generating complex patterns of gene expression without regulatory circuits

Synthetic biology has successfully advanced our ability to design and implement complex, time-varying genetic circuits to control the expression of recombinant proteins. However, these circuits typically require the production of regulatory genes whose only purpose is to coordinate expression of other genes. When designing very small genetic constructs, such as viral genomes, we may want to avoid introducing such auxiliary gene products while nevertheless encoding complex expression dynamics. To this end, here we demonstrate that varying only the placement and strengths of promoters, terminators, and RNase cleavage sites in a computational model of a bacteriophage genome is sufficient to achieve solutions to a variety of basic gene expression patterns. We discover these genetic solutions by computationally evolving genomes to reproduce desired gene expression time-course data. Our approach shows that non-trivial patterns can be evolved, including complex patterns where the relative ordering of genes by abundance changes over time. We find that some patterns are easier to evolve than others, and comparable expression patterns can be achieved via different genetic architectures. Our work opens up a novel avenue to genome engineering via fine-tuning the balance of gene expression and gene degradation rates. Author summaryViruses that infect bacteria, commonly called bacteriophages, typically have small genomes that encode as few as 10 genes. From the perspective of understanding genome design and regulation, these organisms are important model systems. Similar to cellular species, the genes encoded on a phage genome often must be expressed at different levels and at particular times during the phage lifecycle. Given their unique size constraints, it may be advantageous for phages to accomplish differential gene expression without having to produce a variety of dedicated regulatory molecules--which are frequently encoded on the larger and more complex genomes of free-living species. Here, we use a computational simulation of phage infection coupled with an evolutionary selection algorithm to illustrate that phage genomes can encode complex time-dependent gene expression patterns without the need for dedicated regulatory molecules. We anticipate that this simulation framework may additionally aid future phage genome design and engineering efforts.

synthetic biology↗

bioRxiv2024

Engineering a novel probiotic toolkit in Escherichia coli Nissle1917 for sensing and mitigating gut inflammatory diseases

Inflammatory Bowel Disease (IBD) is characterized by chronic intestinal inflammation with no cure and limited treatment options that often have systemic side effects. In this study, we developed a target-specific system to potentially treat IBD by engineering the probiotic bacterium Escherichia coli Nissle 1917 (EcN). Our modular system comprises three components: a transcription factor-based sensor (NorR) capable of detecting the inflammation biomarker nitric oxide, a type 1 hemolysin secretion system, and a therapeutic cargo consisting of a library of humanized anti-TNF nanobodies. Despite a reduction in sensitivity, our system demonstrated a concentration-dependent response to nitric oxide, successfully secreting functional nanobodies with binding affinities comparable to the commonly used drug Adalimumab, as confirmed by ELISA and in vitro assays. This newly validated nanobody library expands EcN therapeutic capabilities. The adopted secretion system, also characterized for the first time in EcN, can be further adapted as a platform for screening and purifying proteins of interest. Additionally, we provided a mathematical framework to assess critical parameters in engineering probiotic systems, including the production and diffusion of relevant molecules, bacterial colonization rates, and particle interactions. This integrated approach expands the synthetic biology toolbox for EcN-based therapies, providing novel parts, circuits, and a model for tunable responses at inflammatory hotspots. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=166 HEIGHT=200 SRC="FIGDIR/small/599326v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@173d556org.highwire.dtl.DTLVardef@1bb2ae1org.highwire.dtl.DTLVardef@fc1fcborg.highwire.dtl.DTLVardef@8b5f7f_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical Table of Contents. The engineered probiotic system: Inflamed intestinal cells release the inflammatory regulator TNF (depicted as red squares), which promotes inflammation through a positive feedback loop. Concurrently, these cells produce large amounts of nitric oxide (NO, represented by yellow circles) during inflammation. Our custom-engineered EcN biosensor can detect NO using a NorR-based sensor (in purple) and subsequently trigger the production of nanobodies (in turquoise). These nanobodies are then released into the extracellular environment via a specially engineered secretion system in the bacterial host (shown in dark blue). Once outside the cell, the nanobodies attach to TNF, effectively sequestering them and reducing inflammation. The graph at the bottom of this panel illustrates the general behavior of our system: nanobody production starts upon reaching a certain NO concentration threshold and continues in an NO-dependent fashion. As nanobodies are produced, they capture TNF, leading to a reduction in inflammation and a decrease in NO production. This decrease in NO then halts the nanobody production. SignificanceProbiotics can be engineered to detect and act upon extracellular disease indicators, optimizing therapeutic outcomes. Particularly, self-regulating sense-and-respond genetic circuits have the potential to enhance the accuracy, efficacy, and adaptability of treatment interventions. In this study, we developed and characterized a new integrated and modular toolkit that detects a gut inflammation biomarker, specifically nitric oxide, and responds to it in an inducible manner by secreting humanized nanobodies targeting the pro-inflammatory molecule TNF. We also develop a coarse-grained mathematical framework for modelling engineered probiotic activity in the gut. This novel system contributes to current efforts to develop new engineered probiotic systems and holds promise for inspiring new treatments for gut inflammation associated with various autoimmune diseases.

synthetic biology↗

bioRxiv2024

The phase separation landscape of genome-wide genetic perturbations

Biomolecular organization is central to cell function. While phase separation is a key mechanism orchestrating this organization, we lack a comprehensive view of genes that can globally influence this process in vivo. To identify such genes, we combined functional genomics and synthetic biology. We developed a bioorthogonal system that can identify changes in the intracellular milieu that globally tune phase separation. We measured in vivo phase diagrams of a synthetic system across >25 million cells in 2,888 yeast knockouts, and identified 68 genes whose deletion alters the phase boundaries of the synthetic system, an unexpected result given the systems bioorthogonal design. Genes involved in TORC1 signaling and metabolism, particularly carbohydrate-, amino acid- and nucleotide synthesis were enriched. The mutants that changed phase separation also showed high pleiotropy, suggesting that phase separation interrelates with many aspects of biology. Highlights- A synthetic protein system reveals the genetic and environmental tunability of protein phase separation - Genetic knockouts affecting phase separation are highly pleiotropic - Carbohydrate, amino acid, and nucleotide metabolism contribute to modulating phase separation potential - Protein phase separation is a globally tunable property of the intracellular environment O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/620319v1_ufig1.gif" ALT="Figure 1"> View larger version (81K): org.highwire.dtl.DTLVardef@4b8403org.highwire.dtl.DTLVardef@1c80503org.highwire.dtl.DTLVardef@c10bf2org.highwire.dtl.DTLVardef@1f76c71_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2026

3' Exonuclease-mediated DNA assembly at room temperature and below

DNA assembly is a cornerstone of synthetic biology, enabling the construction of bespoke genetic systems for applications ranging from metabolic engineering to DNA nanotechnology. Conventional Gibson Assembly (GA), the most widely used method, relies on 5' exonucleolytic resection and elevated temperatures ([~]50 {degrees}C), which together prevent the retention of 5' modifications and restrict compatibility with temperature-sensitive functionalities. Here, we report a DNA assembly strategy, 3 exonuclease-mediated low-temperature DNA assembly (3LTDA), which generates complementary 5' overhangs while preserving 5' end integrity. This approach enables the efficient assembly of blunt-ended, 5'-functionalised DNA fragments into both linear and circular constructs at ambient temperature (21 {degrees}C), with some assembly observed at temperatures as low as 4{degrees}C. We systematically optimise reaction conditions and demonstrate that this method supports efficient plasmid re-circularisation and multi-fragment assembly, including the construction of a [~]12.5 kbp plasmid from multiple DNA components. Comparative analysis across several DNA substrates shows that, under their respective optimal conditions, this approach matches or exceeds GA performance, improving assembly efficiency by up to 12.8%. Sequence analysis confirms high fidelity with no detectable base-pairing errors across assembled junctions. Crucially, this method preserves chemically functionalised 5' termini, enabling downstream conjugation and biochemical functionality. Retention of azide and biotin modifications was verified through fluorescence imaging, bead-based co-localisation, and enzymatic activity in ELISA-based assays. This is in contrast to GA-assembled controls, which showed complete loss of functionality under comparable conditions. We further assembled 5 kbp dsDNA using 3LTDA from four independent segments, three with different fluorescence reporters, and the fourth containing a biotin group for microparticle conjugation, each on the 5 end. Under fluorescence illumination, bead-bound DNA with all three fluorescence markers were detected. Conventional GA assembled constructs, on the other hand, failed to retain the reporter groups and the fluorescent images did not show the presence of any fluorescent markers. In addition to enhanced performance, the method could also reduce reagent cost and eliminate the need for elevated temperatures, simplifying workflows and expanding the applicability of multi-functionalised DNA constructs. Collectively, this work establishes 3LTDA as a robust, low-temperature alternative to conventional GA, with advantages for applications requiring precise chemical modification, temperature-sensitive components, or deployment outside conventional laboratory environments.

synthetic biology↗

bioRxiv2025

Automated, modular assembly of reconstituted cell-free systems from in vitro-produced components

High-performance cell-free protein synthesis has transformative potential for synthetic biology, yet the prohibitive costs of PURE kits and the labor intensity of in-house preparation have restricted accessibility and scalability. We developed i-POPFLEX (Purified components OPtimized for FLEXible protein expression), a modular cell-free protein synthesis system in which 34 translation factors, individually synthesized in vitro, are assembled using automated liquid handling. This workflow minimizes manual input and supports parallelized production, generating complete, ready-to-use systems within two days. i-POPFLEX achieves up to 5-fold higher protein yields and a 95 % cost reduction (20-fold lower cost) compared with commercial kits. Its flexible architecture also enables selective component inclusion for genetic code reprogramming and site-specific incorporation of non-canonical amino acids. By coupling modular design with automation, i-POPFLEX provides an accessible, customizable, and economically viable platform for next-generation biomanufacturing workflows. Technology readinessi-POPFLEX is a reconstituted cell-free system (CFS) composed of 34 translational components that are individually synthesized in vitro and assembled by automation. This system achieves up to 5-fold higher protein yields with a 95 % cost reduction (20-fold lower; <USD 0.06 {micro}L-1) compared with commercial PURE kits (USD 1.36 {micro}L-1) and reduces preparation time from four days to two. The workflow has been validated across diverse protein and peptide synthesis applications, including genetic code reprogramming and site-specific incorporation of non-canonical amino acids, and integrates seamlessly with benchtop automated liquid-handling platforms. These capabilities place i-POPFLEX at Technology Readiness Level (TRL) 4-5: integrated components validated in a laboratory setting and advancing toward pilot-scale readiness. For full-scale deployment, several key challenges remain. Matching crude lysate costs (USD 0.03 {micro}L-1) will require bulk reagent production, optimized procurement strategies, and specialized equipment for high-throughput component synthesis. Enabling milliliter- and liter-scale production will demand improvements in upstream synthesis, downstream purification, and quality control to ensure consistent performance at industrial throughput. Overcoming these barriers would enable production of thousands to millions of fully assembled i-POPFLEX systems for biofoundry-scale enzyme engineering, metabolic pathway prototyping, and large-scale variant screening, democratizing reconstituted CFS technology, and accelerating adoption in next-generation biomanufacturing pipelines.

synthetic biology↗

bioRxiv2021

Inexpensive and easy method for 6 fragment Golden Gate Assembly of a modular S/MARs mammalian expression vector and its variants

BackgroundA basic requirement for synthetic biology is the availability of efficient DNA assembly methods. Numerous methods have been previously reported to accomplish this task. One such method has been reported, which allows parallel assembly of multiple DNA fragments in a one-tube reaction, called Golden Gate Assembly. This study aims to further simplify that method and make it more suitable for small labs and students. MethodsPrior to amplification of the parental plasmids used in building the modules were domesticated using a variation of SDM (Site Directed Mutagenesis) called SPRIP. After careful design and amplification of the desired modules, using a high-fidelity polymerase, amplified PCR fragments that enter the one-step-one-pot reaction were stored in Zymo DNA/RNA Shield at -20 degrees C and thawed whenever needed to be used as fragments or modules in the assembly. The fragments were designed to posses unique overhangs using NEB Golden Gate assembly tool and Snapgene, amplification of modules was performed using a Q5 high fidelity polymerase from preexisting plasmids or gene fragments, clean-up of the PCR products (fragments) was performed in one tube per assembly using Zymo DNA Clean and Concentrator-5, assembled using BsaI and T4 ligase, DpnI digestion performed for eliminating the background plasmids that remain after the PCR reaction and the resulting assembled product was transformed into competent E.coli cells. Transformants were screened using diagnostic digest, transfected into HEK293T cells and the fluorescence was evaluated using fluorescent microscopy and flow cytometry. ResultsHerein presented is a simple and inexpensive alternate protocol to build modular plasmids using the Golden Gate Assembly method. A total of p37 S/MARs mammalian expression vectors were designed and constructed using 6 modules previously amplified by PCR and stored in the appropriate buffer to eliminate exo- and endonuclease activity and to protect the DNA from freeze thaw cycles. The existing modules were interchangeable and new modules were easily amplified and stored for use when needed. The mammalian expression vectors constructed showed the desired restriction pattern and GFP expression in bacteria and in mammalian cells. A comparison of 7 pNoname variants was conducted using flow cytometry. Interestingly, no pNoname variant harbouring the SV40 promoter showed expression in tested HEK293T cells. It appears that using the Ef1a promoter in combination with the BGH polyA signal provides the best expression in S/MARS vectors harboring the DTS40 region, as measured by flow cytometry. ConclusionsProvided the design steps are respected and the fragments are stored and labeled appropriately, multiple plasmid variants and combinations of the pre-designed modules can be assembled in one day, easier and using less resources than the established protocols, with good efficiency. The simplicity of the design and the affordability of the method could make modular cloning of plasmid constructs more accessible to small labs and students.

synthetic biology↗

bioRxiv2026

Modular Assembly of an Infectious Clone of the Akata strain of EBV using Synthetic Genomics Methods in Yeast

We have used synthetic biology recombination methods in yeast to build herpes simplex virus type-1 (HSV-1) and human cytomegalovirus (HCMV) genomes from multiple fragments. The genomes were built using transformation-associated recombination (TAR) in yeast, by virtue of overlapping sequences between the different fragments. This study demonstrates the successful assembly of the Epstein-Barr virus (EBV) genome. We used as the model genome, the Akata Burkitts lymphoma genome, specifically the BX1 genome which encodes a neomycin selectable marker and a GFP expression cassette in the BXLF1 region. The 171.3 kb genome was first deconstructed into 11 fragments in silico, each having 80 bp overlapping sequence between the fragments. The 11 fragments (TAR 1 to TAR 11) were cloned using TAR in yeast, analyzed by restriction enzyme analyses and Nanopore sequencing to validate the cloned fragment. The EBV genome was built in two stages: TAR fragments 1 to 6 and TAR fragments 7 to 11 were assembled to generate two half-genomes. The whole genome (TAR 1-11) was then assembled by joining TAR 1-6 with TAR 7-11. Complete EBV genomes were examined by PCR assays and restriction enzyme analyses and then transfected into HEK-293 cells to generate virus producer cell lines. The HEK-293 cell clones were tested for virus production following lytic induction using baculovirus transduction of Zta, Rta and glycoprotein B (BALF4). The supernatants from these induced cells were harvested and used to infect Raji cells. This analysis revealed a significant number of cells displaying strong GFP fluorescence indicative of infectious virus. We used this supernatant virus to infect primary B cells and were able to derive lymphoblastoid cell lines (LCL) indicative of the ability of this virus to transform B cells. We tested this method for engineering different mutations. Two mutations were made, one in Zta and the other in the small capsid protein (BFRF3). Mutations were engineered in the TAR plasmid in which the genes reside and after sequence validation, assembled into the TAR 1-6 half genome and then the TAR 1-11 genome, which was used to generate HEK-293 cell clones. For the {Delta}Zta cell lines, we could detect virus in the supernatants only if baculovirus expressing Zta in trans was included, this {Delta}Zta EBV virus could transform B cells. The small capsid protein (BFRF3) decorates the capsid shell and is required for capsid assembly in a self-assembly system. When the HEK-293 cell clones were induced using co-expression of Zta, Rta and gB, no virus was detected in the culture supernatants. However, if we provided BFRF3 in trans using baculovirus expressing this protein, virus was detected in the supernatants. This provides the first report of the essential role of the small capsid protein in EBV-infected cells.

Synthetic Biology↗

bioRxiv2021

A generic systems-theoretic approach to identify biological networks capable of adaptation

Constructing biological networks capable of performing specific biological functionalities has been of sustained interest in synthetic biology. Adaptation is one such ubiquitous functional property, which enables every living organism to sense a change in its surroundings and return to its operating condition prior to the disturbance. In this paper, we present a generic systems theory-driven method for designing adaptive protein networks. First, we translate the necessary qualitative conditions for adaptation to mathematical constraints using the language of systems theory, which we then map back as design requirements for the underlying networks. We go on to prove that a protein network with different input-output nodes (proteins) needs to be at least of third-order in order to provide adaptation. Next, we show that the necessary design principles obtained for a three-node network in adaptation consist of negative feedback or a feed-forward realization. Interestingly, the design principles obtained by the proposed method remain the same for a network of arbitrary size and connectivity. Finally, we prove that the motifs discovered for adaptation are non-retroactive for a canonical downstream connection. This result explains how complex biological networks achieve robustness while keeping the core motifs unchanged in the context of a particular functionality. We corroborate our theoretical results with detailed and thorough numerical simulations. Overall, our results present a generic, systematic and robust framework for designing various kinds of biological networks.

systems biology↗

bioRxiv2022

Universal structural requirements for maximal robust perfect adaptation in biomolecular networks

Consider a biomolecular reaction network that exhibits robust perfect adaptation to disturbances from several parallel sources. The well-known Internal Model Principle of control theory suggests that such systems must include a subsystem (called the "internal model") that is able to recreate the dynamic structure of the disturbances. This requirement poses certain structural constraints on the network which we elaborate in this paper for the scenario where constant-in-time disturbances maximally affect network interactions and there is model uncertainty and possible stochasticity in the dynamics. We prove that these structural constraints are primarily characterized by a simple linear-algebraic stoichiometric condition which remains the same for both deterministic and stochastic descriptions of the dynamics. Our results reveal the essential requirements for maximal robust perfect adaptation in biology, with important implications for both systems and synthetic biology. We exemplify our results through many known examples of robustly adapting networks and we construct new examples of such networks with the aid of our linear-algebraic characterization.

systems biology↗

bioRxiv2024

Protein design of two-component tubular assemblies like cytoskeletons

Recent advances in protein design have ushered in an era of constructing intricate higher-order structures1. Despite this progress, orchestrating the assembly of diverse protein units into cohesive artificial structures akin to biological assembly systems, especially in tubular forms, remains an elusive goal. To address this, we introduce the Nature-Inspired Protein Assembly Design (NIPAD), a novel methodology that utilises two distinct protein units to create unique tubular structures under carefully designed conditions. These structures demonstrate dynamic flexibility similar to that of actin filaments, with cryo-electron microscopy revealing diverse morphologies, like microtubules. By mimicking actin filaments, helical conformations were incorporated into tubular assemblies, thereby enriching their structural diversity. Remarkably, these assemblies can be reversibly disassembled and reassembled in response to environmental stimuli, such as changes in salt concentration and temperature, mirroring the dynamic behaviour of natural systems. NIPAD combines rational protein design with biophysical insights, leading to the creation of biomimetic, adaptable, and reversible higher-order assemblies. This approach deepens our understanding of protein assembly design and complex biological structures. Concurrently, it broadens the horizons of synthetic biology and material science, holding significant implications for unravelling lifes fundamental processes and pioneering new applications.

biochemistry↗

bioRxiv2024

Making Neurobots and Chimerical Ctenophores

Making living machines using biological materials (cells, tissues, and organs) is one of the challenges in developmental biology and modern biomedicine. Constraints in regeneration potential and immune self-defense mechanisms limit the progress in the field. Here, we present unanticipated features related to self-recognition and ancestral neuro-immune architectures of new emerging reference species - ctenophores or comb jellies. These are descendants of the earliest survival metazoan lineage with unique tissues, organs and independent origins of major animal traits such as neurons, muscles, mesoderm, and through-gut. Thus, ctenophores convergently evolved complex organization, compared to bilaterians. Nevertheless, their neural and immune systems are likely functionally coupled, enabling designs and experimental construction of hybrid neural systems and even entire animals. This report illustrates impressive opportunities to build both chimeric animals and neurobots using ctenophores as models for bioengineering. The obtained neurobots and chimeric animals from three ctenophore species (Bolinopsis, Mnemiopsis, and Pleurobrachia) were able to be autonomous and survive for days. In sum, the unification of biodiversity, cell biology, and neuroscience opens unprecedented opportunities for experimental synthetic biology.

developmental biology↗

bioRxiv2018

Expanding the toolbox of broad host-range transcriptional terminators for Proteobacteria through metagenomics

As the field of synthetic biology moves towards the utilization of novel bacterial chassis, there is a growing need for biological parts with enhanced performance in a wide number of hosts. Is not unusual that biological parts (such as promoters and terminators), initially characterized in the model bacteria Escherichia coli, do not perform well when implemented in alternative hosts, such as Pseudomonas, therefore limiting the construction of synthetic circuits in industrially relevant bacteria. In order to address this limitation, we present here the mining of transcriptional terminators through functional metagenomics to identify novel parts with broad host-range activity. Using a GFP-based terminator trap strategy and a broad host-range plasmid, we identified 20 clones with potential terminator activity in Pseudomonas putida. Further characterization allowed the identification of 4 unique sequences between 58 bp and 181 bp long that efficiently terminates transcription in P. putida, E. coli, Burkholderia phymatum and two Pseudomonas strains isolated from Antarctica. Therefore, this work presents a new set of biological parts useful for the engineering of synthetic circuits in Proteobacteria.

synthetic biology↗

bioRxiv2017

A Synthetic Microbial Operational Amplifier

Synthetic biology has created oscillators, latches, logic gates, logarithmically linear circuits, and load drivers that have electronic analogs in living cells. The ubiquitous operational amplifier, which allows circuits to operate robustly and precisely has not been built with bio-molecular parts. As in electronics, a biological operational-amplifier could greatly improve the predictability of circuits despite noise and variability, a problem that all cellular circuits face. Here, we show how to create a synthetic 3-stage inducer-input operational amplifier with a differential transcription-factor stage, a CRISPR-based push-pull stage, and an enzymatic output stage with just 5 proteins including dCas9. Our Bio-OpAmp expands the toolkit of fundamental circuits available to bioengineers or biologists, and may shed insight into biological systems that require robust and precise molecular homeostasis and regulation.\n\nOne Sentence SummaryA synthetic bio-molecular operational amplifier that can enable robust, precise, and programmable homeostasis and regulation in living cells with just 5 protein parts is described.

synthetic biology↗