Biology subjects

Federici, F.

Publications and source records attributed to Federici, F..

13 recordsLinked to original sources

bioRxiv2026

FluOpti: an open-source system for programmable whole-plate optogenetics and multi-fluorescence imaging

Bacteria are able to self-organize into complex spatial communities, such as colonies, biofilms and swarms. Probing cells and gene networks with dynamic stimuli while preserving their spatial organization is therefore essential for understanding how bacterial communities organize across spatial and temporal scales. Optogenetics offers a powerful approach for such a task given the precise control of stimulus delivery without the perturbations normally associated with the addition of liquid inducers. However, most optogenetics devices have been developed to work with liquid cell cultures or to deliver a spatial pattern without the concurrent recording of gene responses and colony growth. Here, we describe FluOpti, an open source, low-cost device for controlling gene expression, through programmable light inputs, and recording multi-fluorescence responses and colony growth across whole petri dishes. White light LEDs are used for brightfield imaging while green and red LEDs are used as optogenetic inputs to activate and inactivate target genes via the CcaS/R two component system in E. coli. Low cost blue and orange acrylic are used as excitation and emission filters for fluorescence imaging, respectively. We developed custom software to analyze colony growth and optogenetic responses from 72-hour time-lapse experiments. A mathematical model was developed to predict gene expression levels in response to green-to-red light ratio stimuli. We use FluOpti to reveal a spatially structured response to optogenetic stimuli, with gene activation rates differing in cells located at the center and edge of colonies. to validate its use at a whole petri dish scale, FluOpti was used to depict how Voronoi territories can act as predictors of colony size, in congruence with previous studies. Together, these results demonstrate the utility of the device for studying gene networks at multiple spatio-temporal scales, from individual colonies to the whole petri dish. Performing optogenetics studies in whole petri dishes while preserving spatial arrangements could offer valuable insights into the spatial biology of gene networks in microbial populations.

bioengineering↗

bioRxiv2025

Detection of zoonotic Cryptosporidium parvum in invasive beavers from southern Tierra del Fuego, Chile

Since their introduction to Tierra del Fuego in 1946, invasive beavers have expanded their population and caused extensive environmental degradation across the subantarctic region. Beaver dams have profoundly impacted peatlands, mature sub-Antarctic forests, and watercourses. Here, we report a pathogen in invasive beavers in Patagonia and discuss their potential role as a reservoir of waterborne zoonotic pathogens. We detected Cryptosporidium through microscopy using the Ziehl-Neelsen staining technique in beaver collected from southern Tierra del Fuego. Out of the 105 beavers examined, three tested positive to optical microscopy, which were then identified molecularly as Cryptosporidium parvum by sequence analysis of the 18S ribosomal DNA (rDNA). Given the potential impacts of Cryptosporidium parvum upon native species, wildlife, and human health, this finding highlights the potential risks associated with the ongoing expansion of beavers into other areas of the sub-Antarctic region. A potential role of invasive beavers as reservoirs of zoonotic Cryptosporidium spp. in Patagonia emphasizes the unknown consequences of human-introduced exotic species on pristine ecosystems.

bioengineering↗

bioRxiv2024

Novel Library Assembly Technique for Developing Nanobodies Targeting IPNv VP2 Protein

Infectious pancreatic necrosis virus (IPNv) poses a significant threat to the global salmon farming industry, causing high mortality and economic losses. Given the limitations of traditional antibody therapies in aquaculture, we explored the development of nanobodies targeting the VP2 protein of IPNv. We developed a novel nanobody library generation method using a streamlined assembly protocol based on Type IIS restriction enzymes. This method enabled quick and efficient bacterial display library generation, and concatenated, multi-level cloning through an ingenious design in which the ligation of the first level generates a new restriction site that can be utilized in the subsequent cloning level, allowing for rapid transfer to other vectors. By combining this assembly approach with density gradient-based enrichment and high-throughput screening, we identified nanobodies that specifically recognize IPNvs VP2 protein. Notably, the P9 clone demonstrated high specificity for IPNv in immunofluorescence assays, highlighting its diagnostic potential. Our method not only accelerates nanobody library generation but also enhances its quality and adaptability, marking a significant advancement in enhancing the responsiveness of nanobody development platforms against emerging pathogen outbreaks.

molecular biology↗

bioRxiv2024

CIFR (Clone-Integrate-Flip-out-Repeat): a toolset for iterative genome and pathway engineering of Gram-negative bacteria

Advances in genome engineering enable precise and customizable modifications of bacterial species. Toolsets for metabolic engineering that exhibit broad-host compatibility are particularly valued owing to their portability. Tn5 transposon vectors have been widely used to establish random integrations of desired DNA sequences into bacterial genomes. However, the iteration of the procedure remains challenging because of the limited availability of selection markers. Here, we present CIFR, a mini-Tn5 integration system for iterative genome engineering. The pCIFR vectors incorporate attP and attB sites flanking an antibiotic resistance marker used to select for the insertion. Subsequent removal of these antibiotic determinants is facilitated by the Bxb1 integrase, and a user-friendly counter-selection marker, both encoded in auxiliary plasmids. Hence, CIFR delivers engineered strains harboring stable DNA insertions and free of any antibiotic resistance cassette, allowing for the reusability of the tool. The system was validated in Pseudomonas putida, Escherichia coli, and Cupriavidus necator, underscoring its portability across diverse industrially-relevant hosts. The CIFR toolbox was calibrated through combinatorial integrations of chromoprotein genes in P. putida, generating strains displaying a diverse color palette. Next, we introduced a carotenoid biosynthesis pathway in P. putida in a two-step engineering process, showcasing the potential of the tool for pathway balancing. The broad utility of the toolbox expands the synthetic biology toolkit for metabolic engineering, allowing for the construction of complex phenotypes, while opening new possibilities in bacterial genetic manipulations.

synthetic biology↗

bioRxiv2024

qByte: Open-source isothermal fluorimeter for democratizing analysis of nucleic acids, proteins and cells

Access to affordable and reliable scientific instrumentation remains a significant barrier to the democratization of healthcare and scientific research. In the field of biotechnology, in particular, the complexity, cost, and infrastructure requirements of many instruments continue to limit their accessibility, especially in resource-limited environments. Despite the recent increase in the development of open-source tools, driven by advances in digital fabrication and electronic prototyping, few of these projects have reached large-scale implementation or validation in real-world settings. Here, we present qByte, an open-source, 8-tube isothermal fluorimeter designed to overcome these barriers by offering a cost-effective ($60) yet production-ready solution. qByte leverages standard digital manufacturing and Printed Circuit Board (PCB) assembly techniques and is designed to be portable, making it ideal for both laboratory and field use. The device has been benchmarked against commercial real-time thermocyclers and spectrophotometers, showing comparable results across four key applications: nucleic acid amplification and detection, analysis of protein activity and stability, genetic construct characterization, and bacterial viability tests. Further testing including the on-site diagnosis of human parasites in Kumasi, Ghana, and field work in Patagonia, Chile, validated qBytes reliability in real-world conditions. Taken together, our results proved qByte as flexible and reliable equipment for a variety of biological tests and applications, while its affordability and open-source design simplify further development and allow adaptation to the needs of future users.

bioengineering↗

bioRxiv2024

Open Educational Resources for distributed hands-on teaching in molecular biology

The urgent need to develop a more equitable bioeconomy has positioned biotechnology capacity building at the forefront of international priorities. However, in many educational institutions, particularly in low-and middle-income countries, this remains a major challenge due to limited access to reagents, equipment, and technical documentation. In this work, we describe Open Educational Resources (OER) composed of locally produced biological reagents, open source hardware and free software to teach fundamental techniques in biotechnology such as LAMP DNA amplification, RT-PCR RNA detection, enzyme kinetics and fluorescence imaging. The use of locally produced reagents and devices reduces costs by up to one order of magnitude. During the pandemic lockdowns, these tools were distributed nationwide to students homes as a lab-in-a-box for remote teaching of molecular biology. To test their performance, a total of 93 undergraduate students tested these resources during a biochemistry practical course. 27 out of 31 groups ([~]87%) successfully achieved the objectives of the PCR activity, while 28 out of 31 groups ([~]90%) correctly identified the target using LAMP reactions. To assess the potential application in secondary school, we organized three workshops for high school teachers from different institutions across Chile and performed an anonymous questionnaire, obtaining a strong agreement on how these OER broaden teachers perspectives on the techniques and facilitate the teaching of molecular biology topics. This effort was made possible through a close collaboration with open source technology advocates and members of DIYbio communities, whose work is paving the way for low-cost training in biology. All the protocols and design files are available under open source licenses.

molecular biology↗

bioRxiv2023

Spatial biology of Ising-like synthetic genetic networks

BackgroundUnderstanding how spatial patterns of gene expression emerge from the interaction of individual gene networks is a fundamental challenge in biology. Developing a synthetic experimental system with a common theoretical framework that captures the emergence of short- and long-range spatial correlations (and anti-correlations) from interacting gene networks could serve to uncover generic scaling properties of these ubiquitous phenomena. ResultsHere, we combine synthetic biology, statistical mechanics models and computational simulations to study the spatial behavior of synthetic gene networks (SGNs) in Escherichia coli quasi-2D colonies growing on hard agar. Guided by the combined mechanisms of the contact process lattice simulation and two-dimensional Ising model (CPIM), we describe the spatial behavior of bi-stable and chemically-coupled SGNs that self-organize into patterns of long-range correlations with power-law scaling or short-range anti-correlations. These patterns, resembling ferromagnetic and anti-ferromagnetic configurations of the Ising model near critical points, maintain their scaling properties upon changes in growth rate and cell shape. ConclusionsOur findings shed light on the spatial biology of coupled and bistable gene networks in growing cell populations. This emergent spatial behavior could provide insights into the study and engineering of self-organizing gene patterns in eukaryotic tissues and bacterial consortia.

synthetic biology↗

bioRxiv2021

Decentralizing cell-free RNA sensing with the use of low-cost cell extracts

Cell-free gene expression systems have emerged as a promising platform for field-deployed biosensing and diagnostics. When combined with programmable toehold switch-based RNA sensors, these systems can be used to detect arbitrary RNAs and freeze-dried for room temperature transport to the point-of-need. These sensors, however, have been implemented using reconstituted PURE cell-free protein expression systems that are difficult to source in the Global South due to their high commercial cost and cold-chain shipping requirements. Here, we describe the implementation of RNA toehold switch-based sensors using E. coli cell lysate-based cell-free protein expression systems, which can be produced locally and reduce the cost of sensors by two orders of magnitude. We then demonstrate that these in-house cell lysates provide sensor performance comparable to commercial PURE cell-free systems. We further optimize use of these lysates with a CRISPRi strategy to enhance the stability of linear DNAs, enabling the direct use of PCR products for fast screening of new designs. As a proof-of-concept, we develop novel toehold sensors for the plant pathogen Potato Virus Y (PVY), which dramatically reduces the yield of this important staple crop. The local implementation of low-cost cell-free toehold sensors could enable biosensing capacity at the regional level and lead to more decentralized models for global surveillance of infectious disease.

bioengineering↗

bioRxiv2020

Flapjack: a data management and analysis tool for genetic circuit characterization

Characterization is fundamental to the design, build, test, learn (DBTL) cycle for engineering synthetic genetic circuits. Components must be described in such a way as to account for their behavior in a range of contexts. Measurements and associated metadata, including part composition, constitute the test phase of the DBTL cycle. These data may consist of measurements of thousands of circuits, measured in hundreds of conditions, in multiple assays potentially performed in different labs and using different techniques. In order to inform the learn phase this large volume of data must be filtered, collated, and analyzed. Characterization consists of using this data to parameterize models of component function in different contexts, and combining them to predict behaviors of novel circuits. Tools to store, organize, share, and analyze large volumes of measurement and metadata are therefore essential to linking the test phase to the build and learn phases, closing the loop of the DBTL cycle. Here we present such a system, implemented as a web app with a backend data registry and analysis engine. An interactive frontend provides powerful querying, plotting and analysis tools, and we provide a REST API and Python package for full integration with external build and learn software. All measurements are associated to circuit part composition via SBOL. We demonstrate our tool by characterizing a range of genetic components and circuits according to composition and context.

synthetic biology↗

bioRxiv2019

Universal Loop assembly (uLoop): open, efficient, and species-agnostic DNA fabrication

Standardised Type IIS DNA assembly methods are becoming essential for biological engineering and research. Although a common syntax has been proposed to enable higher interoperability between DNA libraries, Golden Gate (GG)-based assembly systems remain specific to target organisms. Furthermore, these GG assembly systems become laborious and unnecessarily complicated beyond the assembly of 4 transcriptional units. Here, we describe \"universal Loop\" (uLoop) assembly, a simple system based on Loop assembly that enables hierarchical fabrication of large DNA constructs (> 30 kb) for any organism of choice. uLoop comprises two sets of four plasmids that are iteratively used as odd and even levels to compile DNA elements in an exponential manner (4n-1). The elements required for transformation/maintenance in target organisms are also assembled as standardised parts, enabling customisation of host-specific plasmids. Thus, this species-agnostic method decouples efficiency of assembly from the stability of vectors in the target organism. As a proof-of-concept, we show the engineering of multi-gene expression vectors in diatoms, yeast, plants and bacteria. These resources will become available through the OpenMTA for unrestricted sharing and open-access.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=144 SRC=\"FIGDIR/small/744854v2_ufig.gif\" ALT=\"Figure 1\">\nView larger version (31K):\norg.highwire.dtl.DTLVardef@c36f4forg.highwire.dtl.DTLVardef@c0b007org.highwire.dtl.DTLVardef@64c810org.highwire.dtl.DTLVardef@8a94b0_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2019

Phase space characterization for gene circuit design

Genetic circuit design requires characterization of the dynamics of synthetic gene expression. This is a difficult problem since gene expression varies in complex ways over time and across different contexts. Here we present a novel method for characterizing the dynamics of gene expression with a few parameters that account for changes in cellular context (host cell physiology) and compositional context (adjacent genes). The dynamics of gene circuits were characterized by a trajectory through a multi-dimensional phase space parameterized by the expression levels of each of their constituent transcriptional units (TU). These trajectories followed piecewise linear dynamics, with each dynamical regime corresponding to different growth regimes, or cellular contexts. Thus relative expression rates were changed by transitions between growth regimes, but were constant in each regime. We present a plausible two-factor mathematical model for this behavior based on resource consumption. By analyzing different combinations of TUs, we then showed that relative expression rates were significantly affected by the neighboring TU (compositional context), but maintained piecewise linear dynamics across cellular and compositional contexts. Taken together these results show that TU expression dynamics could be predicted by a reference TU up to a context dependent scaling factor. This model provides a framework for design of genetic circuits composed of TUs. A common sharable reference TU may be chosen and measured in the cellular contexts of interest. The output of each TU in the circuit may then be predicted from a simple function of the output of the reference TU in the given cellular context. This will aid in genetic circuit design by providing simple models for the dynamics of gene circuits and their constituent TUs.

synthetic biology↗

bioRxiv2018

Loop Assembly: a simple and open system for recursive fabrication of DNA circuits

High efficiency methods for DNA assembly are based on sequence overlap between fragments or Type IIS restriction endonuclease cleavage and ligation. These have enabled routine assembly of synthetic DNAs of increased size and complexity. However, these techniques require customisation, elaborate vector sets and serial manipulations for the different stages of assembly. We present Loop assembly, based on a recursive approach to DNA fabrication. Alternate use of two Type IIS restriction endonucleases and corresponding vector sets allows efficient and parallel assembly of large DNA circuits. Plasmids containing standard Level 0 parts can be assembled into circuits containing 1, 4, 16 or more genes by looping between the two vector sets. The vectors also contain modular sites for hybrid assembly using sequence overlap methods. Loop assembly provides a simple generalised solution for DNA construction with standardised parts. The cloning system is provided under an OpenMTA license for unrestricted sharing and open access.

synthetic biology↗

bioRxiv2017

Low cost and open source multi-fluorescence imaging system for teaching and research in biology and bioengineering

The advent of easy-to-use open source microcontrollers, off-the-shelf electronics and customizable manufacturing technologies has facilitated the development of inexpensive scientific devices and laboratory equipment. In this study, we describe an imaging system that integrates low-cost and open-source hardware, software and genetic resources. The multi-fluorescence imaging system consists of readily available 470 nm LEDs, a Raspberry Pi camera and a set of filters made with low cost acrylics. This device allows imaging in scales ranging from single colonies to entire plates. We developed a set of genetic components (e.g. promoters, coding sequences, terminators) and vectors following the standard framework of Golden Gate, which allowed the fabrication of genetic constructs in a combinatorial, low cost and robust manner. In order to provide simultaneous imaging of multiple wavelength signals, we screened a series of long stokes shift fluorescent proteins that could be combined with cyan/green fluorescent proteins. We found CyOFP1, mBeRFP and sfGFP to be the most compatible set for 3-channel fluorescent imaging. We developed open source Python code to operate the hardware to run time-lapse experiments with automated control of illumination and camera and a Python module to analyze data and extract meaningful biological information. To demonstrate the potential application of this integral system, we tested its performance on a diverse range of imaging assays often used in disciplines such as microbial ecology, microbiology and synthetic biology. We also assessed its potential for STEM teaching in a high school environment, using it to teach biology, hardware design, optics, and programming. Together, these results demonstrate the successful integration of open source hardware, software, genetic resources and customizable manufacturing to obtain a powerful, low cost and robust system for STEM education, scientific research and bioengineering. All the resources developed here are available under open source licenses.

synthetic biology↗