Biology subjects

Stan, G.-B. V.

Publications and source records attributed to Stan, G.-B. V..

13 recordsLinked to original sources

bioRxiv2025

Investigating The Potential of Division of Labour in Synthetic Bacterial Communities for the Production of Violacein

With advancements in synthetic biology and metabolic engineering, microorganisms can now be engineered to perform increasingly complex functions, which may be limited by the resources available in individual cells. Division of labour in synthetic microbial communities offers a promising approach to enhance metabolic efficiency and resilience in bioproduction. By distributing complex metabolic pathways across multiple subpopulations, the resource competition and metabolic burden imposed on an individual cell is reduced, potentially enabling more efficient production of target compounds. Violacein is a high-value pigment with anti-tumour properties that exemplifies such a challenge due to its complex bioproduction pathway, imposing a significant metabolic burden on host cells. In this study we investigated the benefits of division of labour for violacein production by splitting the violacein bioproduction pathway between two subpopulations of Escherichia coli based synthetic communities. We tested several pathway splitting strategies and reported that splitting the pathway into two subpopulations expressing VioABE and VioDC at a final composition of 60:40 yields a 2.5 fold increase in violacein production as compared to a monoculture. We demonstrated that the coculture outperforms the monoculture when both subpopulations exhibit similar metabolic burden levels, resulting in comparable growth rates, and when both subpopulations are present in sufficiently high proportions.

bioengineering↗

bioRxiv2024

Design of a biomolecular adaptive controller to restore sustained periodic behavior

Periodic behavior is a widespread biological phenomenon occurring across various spatiotemporal scales, where upstream stimuli are encoded into dynamic intracellular signals such as oscillations, varying in duration, amplitude, and frequency. Disruptions to this periodicity can lead to a range of pathologies, for which we propose an adaptive feedback controller with an Incoherent Feedforward Loop (IFFL)-like topology, based on chemical reactions, designed to restore sustained oscillations in systems that have lost their periodicity. By approximating the controllers dynamics, we defined the design requirements for the first implementation of a biomolecular adaptive controller and tested its applicability for destabilizing the steady-state behavior of a self-inhibiting gene. Numerical simulations illustrate the adaptive behavior of the controller and its ability to tune both the amplitude and the period of the resulting oscillations.

synthetic biology↗

bioRxiv2024

Microfluidic platform for microbial spore germination studies in multiple growth conditions

Spores are highly resistant dormant cells, adapted for survival and dispersal, that can withstand unfavourable environmental conditions for extended periods of time and later reactivate. Understanding the germination process of microbial spores is important in numerous areas including agriculture, food safety and health, and other sectors of biotechnology. Microfluidics combined with high-resolution microscopy allows to study spore germination at the single-cell level, revealing behaviours that would be hidden in standard population-level studies. Here, we present a microfluidic platform for germination studies where spores are confined to monolayers inside microchambers, allowing the testing of four growth conditions in parallel. This platform can be used with multiple species, including non-model organisms, and is compatible with existing image analysis software. In this study, we focused on three soil dwellers, two prokaryotes and one fungus, and revealed new insights into their germination. We studied endospores of the model bacterium Bacillus subtilis and demonstrated a correlation between spore density and germination in rich media. We then investigated the germination of the obligate-oxalotrophic environmental bacterium Ammoniphilus oxalaticus in a concentration gradient of potassium oxalate, showing that lower concentrations result in more spores germinating compared to higher concentrations. We also used this microfluidic platform to study the soil beneficial filamentous fungus Trichoderma rossicum, showing for the first time that the size of the spores and hyphae increase in response to increased nutrient availability, while germination times remain the same. Our platform allows to better understand microbial behaviour at the single-cell level, under a variety of controlled conditions. One-Sentence SummaryA microfluidic platform developed for spore germination studies in multiple growth conditions provides new insights into the germination of spores at the cellular level from three soil dwellers.

bioengineering↗

bioRxiv2023

Information propagation through enzyme-free catalytic templating of DNA dimerization with weak product inhibition

Information propagation by sequence-specific, template-catalyzed molecular assembly is the source of the biochemical complexity of living systems. Templating allows the production of thousands of sequence-defined proteins from only 20 distinct building blocks. By contrast, exploitation of this powerful chemical motif is rare in non-biological contexts, particularly in enzyme-free environments, where even the template-catalyzed formation of dimers is a significant challenge. The main obstacle is product inhibition: the tendency of products to bind to their templates more strongly than individual monomers, preventing the effective catalytic templating of longer polymers. Here we present a rationally designed enzyme-free system in which a DNA template catalyzes, with weak competitive product inhibition, the production of sequence-specific DNA dimers. We demonstrate the selective templating of 9 different dimers with high specificity and catalytic turnover. Most importantly, our mechanism demonstrates a rational design principle for engineering information propagation by molecular templating of longer polymers.

bioengineering↗

bioRxiv2023

Overcoming the speed limit of four-way DNA branch migration with bulges in toeholds

Dynamic DNA nanotechnology involves the use of DNA strands to create programmable reaction networks and nanodevices. The key reaction in dynamic DNA nanotechnology is the exchange of DNA strands between different molecular species, which is achieved through three-way and four-way DNA exchange reactions. While both of these reactions have been widely used to build reaction circuits, the four-way exchange reaction has traditionally been slower and less efficient than the three-way reaction. In this paper, we describe a new mechanism to optimise the kinetics of the four-way DNA exchange reaction by adding bulges to the toeholds of the four-way DNA complexes involved in the reaction. These bulges facilitate an alternative branch migration mechanism and destabilise the four-way DNA junction, increasing the branch migration rate and unbinding rate of the four-way exchange reaction, bringing it closer to the kinetics of the three-way reaction. This new mechanism has the potential to expand the field of dynamic DNA nanotechnology by enabling efficient four-way DNA exchange reactions for in vivo applications.

biophysics↗

bioRxiv2023

Host-aware RNA-based control of synthetic microbial consortia

Microbial consortia have been utilised for centuries to produce fermented foods and have great potential in applications such as therapeutics, biomaterials, fertilisers, and biobased production. Working together, microbes become specialized and perform complex tasks more efficiently, strengthening both cooperation and stability of the microbial community. However, imbalanced proportions of microbial community members can lead to unoptimized and diminished yields in biotechnology. To address this, we developed a burden-aware RNA-based multicellular feedback control system that stabilises and tunes coculture compositions. The system consists of three modules: a quorum sensing-based communication module to provide information about the densities of cocultured strains, an RNA-based comparator module to compare the ratio of densities of both strains to a pre-set desired ratio, and a customisable growth module that relies either on heterologous gene expression or on CRISPRi knockdowns to tune growth rates. We demonstrated that heterologous expression burden could be used to stabilise composition in a two-member E. coli coculture. This is the first coculture composition controller that does not rely on toxins or syntrophy for growth regulation and uses RNA sequestration to stabilise and control coculture composition. This work provides a fundamental basis to explore burden-aware multicellular feedback control strategies for robust stabilisation of synthetic community compositions.

synthetic biology↗

bioRxiv2022

When synthetic biology fails: a modular framework for modelling genetic stability in engineered cell populations

Predicting the evolution of engineered cell populations is a highly soughtafter goal in biotechnology. While models of evolutionary dynamics are far from new, their application to synthetic systems is scarce where the vast combination of genetic parts and regulatory elements creates a unique challenge. To address this gap, we herein present a framework that allows one to connect the DNA design of varied genetic devices with mutation spread in a growing cell population. Users can specify the functional parts of their system and the degree of mutation heterogeneity to explore, after which our model generates hostaware transition dynamics between different mutation phenotypes over time. We show how our framework can be used to generate insightful hypotheses across broad applications, from how a devices components can be tweaked to optimise longterm protein yield and genetic shelf life, to generating new design paradigms for gene regulatory networks that improve their functionality.

synthetic biology↗

bioRxiv2021

Building an RNA-based Toggle Switch using Inhibitory RNA Aptamers

Synthetic RNA systems offer unique advantages such as faster response, increased specificity, and programmability compared to conventional protein-based networks. Here, we demonstrate an in-vitro RNA-based toggle switch using RNA aptamers capable of inhibiting the transcriptional activity of T7 or SP6 RNA polymerases. The activities of both polymerases are monitored simultaneously by using Broccoli and Malachite green light-up aptamer systems. In our toggle switch, a T7 promoter drives the expression of SP6 inhibitory aptamers, and an SP6 promoter expresses T7 inhibitory aptamers. We show that the two distinct states originating from the mutual inhibition of aptamers can be toggled by adding DNA sequences to sequester the RNA inhibitory aptamers. Finally, we assessed our RNA-based toggle switch in cell-like conditions by introducing controlled degradation of RNAs using a mix of RNases. Our results demonstrate that the RNA-based toggle switch could be used as a control element for nucleic acid networks in synthetic biology applications. Graphical TOC Entry O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/468831v1_ufig1.gif" ALT="Figure 1"> View larger version (12K): org.highwire.dtl.DTLVardef@5df108org.highwire.dtl.DTLVardef@17b653borg.highwire.dtl.DTLVardef@33315borg.highwire.dtl.DTLVardef@ed81c8_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2021

A linear programming-based strategy to save pipette tips in automated DNA assembly

Laboratory automation and mathematical optimisation are key to improving the efficiency of synthetic biology research. While there are algorithms optimising the construct designs and synthesis strategies for DNA assembly, the optimisation of how DNA assembly reaction mixes are prepared remains largely unexplored. Here, we focus on reducing the pipette tip consumption of a liquid-handling robot as it delivers DNA parts across a multi-well plate where several constructs are being assembled in parallel. We propose a linear programming formulation of this problem based on the capacitated vehicle routing problem, along with an algorithm which applies a linear programming solver to our formulation, hence providing a strategy to prepare a given set of DNA assembly mixes using fewer pipette tips. The algorithm performed well in randomly generated and real-life scenarios concerning several modular DNA assembly standards, proving capable of reducing the pipette tip consumption by up to 61% in large-scale cases. Combining automatic process optimisation and robotic liquid-handling, our strategy promises to greatly improve the efficiency of DNA assembly, either used alone or in combination with other algorithmic methods.

synthetic biology↗

bioRxiv2020

In situ generation of RNA complexes for synthetic molecular strand displacement circuits in autonomous systems

Synthetic molecular circuits implementing DNA or RNA strand-displacement reactions can be used to build complex systems such as molecular computers and feedback control systems. Despite recent advances, application of nucleic acid-based circuits in vivo remains challenging due to a lack of efficient methods to produce their essential components - multi-stranded complexes known as "gates" - in situ, i.e. in living cells or other autonomous systems. Here, we propose the use of naturally occurring self-cleaving ribozymes to cut a single-stranded RNA transcript into a gate complex of shorter strands, thereby opening new possibilities for the autonomous and continuous production of RNA strands in a stoichiometrically and structurally controlled way.

synthetic biology↗

bioRxiv2020

Handhold-mediated strand displacement: a nucleic acid-based mechanism for generating far-from-equilibrium assemblies through templated reactions

Toehold-mediated strand displacement (TMSD) is a nucleic acid-based reaction wherein an invader strand (I) replaces an incumbent strand (N) in a duplex with a target strand (T). TMSD is driven by toeholds, overhanging single-stranded domains in T recognised by I. Although TMSD is responsible for the outstanding potential of dynamic DNA nanotechnology1, 2, TMSD cannot implement templating, the central mechanism by which biological systems generate complex, far-from equilibrium assemblies like RNA or proteins3, 4. Therefore, we introduce handhold-mediated strand displacement (HMSD). Handholds are toehold analogues located in N and capable of implementing templating. We measure the kinetics of 98 different HMSD systems to demonstrate that handholds can accelerate the rate of invader-target (IT) binding by more than 4 orders of magnitude. Furthermore, handholds of moderate length accelerate reactions whilst allowing detachment of the product IT from N. We are thus able to experimentally demonstrate the use of HMSD-based templating to produce highly-specific far-from-equilibrium DNA duplexes.

biophysics↗

bioRxiv2019

A modular RNA interference system for gene regulation and robust dynamic feedback control

The rational design and realisation of simple-to-use genetic control elements that are modular, orthogonal and robust is essential to the construction of predictable and reliable biological systems of increasing complexity. To this effect, we introduce modular Artificial RNA interference (mARi), a rational, modular and extensible design framework that enables robust, portable and multiplexed post-transcriptional regulation of gene expression in Escherichia coli. The regulatory function of mARi was characterised in a range of relevant genetic contexts, demonstrating its independence from other genetic control elements and the gene of interest, and providing new insight into the design rules of RNA based regulation in E. coli, while a range of cellular contexts also demonstrated it to be independent of growth-phase and strain type. Importantly, the extensibility and orthogonality of mARi enables the simultaneous post-transcriptional regulation of multi-gene systems as both single-gene cassettes and poly-cistronic operons. To facilitate adoption, mARi was designed to be directly integrated into the modular BASIC DNA assembly framework. We anticipate that mARi-based genetic control within an extensible DNA assembly framework will facilitate metabolic engineering, layered genetic control, and advanced genetic circuit applications.

synthetic biology↗

bioRxiv2017

Computational Re-Design of Synthetic Genetic Oscillators for Independent Amplitude and Frequency Modulation

Engineering robust and tuneable genetic clocks is a topic of current interest in Systems and Synthetic Biology with wide applications in biotechnology. Synthetic genetic oscillators share a common structure based on a negative feedback loop with a time delay, and generally display only limited tuneability. Recently, the dual-feedback oscillator was demonstrated to be robust and tuneable, to some extent, by the use of chemical inducers. Yet no engineered genetic oscillator currently allows for the independent modulation of amplitude and period. In this work, we demonstrate computationally how recent advances in tuneable synthetic degradation can be used to decouple the frequency and amplitude modulation in synthetic genetic oscillators. We show how the range of tuneability can be increased by connecting additional input dials, e.g. orthogonal transcription factors that respond to chemical, temperature or even light signals. Modelling and numerical simulations predict that our proposed re-designs enable amplitude tuning without period modulation, coupled modulation of both period and amplitude, or period adjustment with near-constant amplitude. We illustrate our work through computational re-designs of both the dual-feedback oscillator and the repressilator, and show that the repressilator is more flexible and can allow for independent amplitude and near-independent period modulation.

synthetic biology↗