Browsing by Author "Federici, Fernan"

Now showing 1 - 9 of 9
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    Accurate characterization of dynamic microbial gene expression and growth rate profiles
    (2022) Vidal, Gonzalo; Vidal-Cespedes, Carlos; Silva, Macarena Munoz; Castillo-Passi, Carlos; Feliu, Guillermo Yanez; Federici, Fernan; Rudge, Timothy J.
    Genetic circuits are subject to variability due to cellular and compositional contexts. Cells face changing internal states and environments, the cellular context, to which they sense and respond by changing their gene expression and growth rates. Furthermore, each gene in a genetic circuit operates in a compositional context of genes which may interact with each other and the host cell in complex ways. The context of genetic circuits can, therefore, change gene expression and growth rates, and measuring their dynamics is essential to understanding natural and synthetic regulatory networks that give rise to functional phenotypes. However, reconstruction of microbial gene expression and growth rate profiles from typical noisy measurements of cell populations is difficult due to the effects of noise at low cell densities among other factors. We present here a method for the estimation of dynamic microbial gene expression rates and growth rates from noisy measurement data. Compared to the current state-of-the-art, our method significantly reduced the mean squared error of reconstructions from simulated data of growth and gene expression rates, improving the estimation of timing and magnitude of relevant shapes of profiles. We applied our method to characterize a triple-reporter plasmid library combining multiple transcription units in different compositional and cellular contexts in Escherichia coli. Our analysis reveals cellular and compositional context effects on microbial growth and gene expression rate dynamics and suggests a method for the dynamic ratiometric characterization of constitutive promoters relative to an in vivo reference.
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    An Open One-Step RT-qPCR for SARS-CoV-2 detection
    (Public Library Science, 2024) Cerda Rojas, Ariel Patricio; Rivera, Maira; Armijo, Grace; Ibarra-Henríquez, Catalina; Reyes, Javiera; Blázquez Sánchez, Paula; Avilés, Javiera; Arce, Anibal; Seguel, Aldo; Brown, Alexander J.; Vásquez, Yesseny; Cortez-San Martín, Marcelo; Cubillos, Francisco A.; García, Patricia; Ferrés, Marcela; Ramírez Sarmiento, César Antonio; Federici, Fernan; Gutiérrez, Rodrigo A.
    The COVID-19 pandemic has resulted in millions of deaths globally, and while several diagnostic systems were proposed, real-time reverse transcription polymerase chain reaction (RT-PCR) remains the gold standard. However, diagnostic reagents, including enzymes used in RT-PCR, are subject to centralized production models and intellectual property restrictions, which present a challenge for less developed countries. With the aim of generating a standardized One-Step open RT-qPCR protocol to detect SARS-CoV-2 RNA in clinical samples, we purified and tested recombinant enzymes and a non-proprietary buffer. The protocol utilized M-MLV RT and Taq DNA pol enzymes to perform a Taqman probe-based assay. Synthetic RNA samples were used to validate the One-Step RT-qPCR components, demonstrating sensitivity comparable to a commercial kit routinely employed in clinical settings for patient diagnosis. Further evaluation on 40 clinical samples (20 positive and 20 negative) confirmed its comparable diagnostic accuracy. This study represents a proof of concept for an open approach to developing diagnostic kits for viral infections and diseases, which could provide a cost-effective and accessible solution for less developed countries.
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    An Open One-Step RT-qPCR for SARS-CoV-2 detection
    (2024) Cerda, Ariel; Rivera, Maira; Armijo, Grace; Ibarra-Henriquez, Catalina; Reyes, Javiera; Blazquez-Sanchez, Paula; Aviles, Javiera; Arce, Anibal; Seguel, Aldo; Brown, Alexander J.; Vasquez, Yesseny; Cortez-San Martin, Marcelo; Cubillos, Francisco A.; Garcia, Patricia; Ferres, Marcela; Ramirez-Sarmiento, Cesar A.; Federici, Fernan; Gutierrez, Rodrigo A.
    The COVID-19 pandemic has resulted in millions of deaths globally, and while several diagnostic systems were proposed, real-time reverse transcription polymerase chain reaction (RT-PCR) remains the gold standard. However, diagnostic reagents, including enzymes used in RT-PCR, are subject to centralized production models and intellectual property restrictions, which present a challenge for less developed countries. With the aim of generating a standardized One-Step open RT-qPCR protocol to detect SARS-CoV-2 RNA in clinical samples, we purified and tested recombinant enzymes and a non-proprietary buffer. The protocol utilized M-MLV RT and Taq DNA pol enzymes to perform a Taqman probe-based assay. Synthetic RNA samples were used to validate the One-Step RT-qPCR components, demonstrating sensitivity comparable to a commercial kit routinely employed in clinical settings for patient diagnosis. Further evaluation on 40 clinical samples (20 positive and 20 negative) confirmed its comparable diagnostic accuracy. This study represents a proof of concept for an open approach to developing diagnostic kits for viral infections and diseases, which could provide a cost-effective and accessible solution for less developed countries.
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    Building an Open-Source DNA Assembler Device
    (2023) Orostica, Boris; Nunez, Isaac; Matute, Tamara; Nunez, Felipe; Federici, Fernan
    This article introduces an open-source thermal cycling machine designed specifically for Golden Gate DNA assembly. The prototype device can achieve efficiency similar to a commercial PCR thermocycler.
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    CFE Expression and Lyophilization of β-Galactosidase (LacZ) v1
    (2022) Navarro Martínez, Felipe; Arce Medina, Aníbal Andrés; Federici, Fernan
    This protocol includes the expression and lyophilization of the β-Galactosidase enzyme (from the LacZ gene) in a cell-free system. We used it to prepare and conduct experiments for a biochemistry lab course.
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    Constructing Cell-Free Expression Systems for Low-Cost Access
    (2022) Guzman-Chavez, Fernando; Arce, Anibal; Adhikari, Abhinav; Vadhin, Sandra; Antonio Pedroza-Garcia, Jose; Gandini, Chiara; Ajioka, Jim W.; Molloy, Jenny; Sanchez-Nieto, Sobeida; Varner, Jeffrey D.; Federici, Fernan; Haseloff, Jim
    Cell-free systems for gene expression have gained attention as platforms for the facile study of genetic circuits and as highly effective tools for teaching. Despite recent progress, the technology remains inaccessible for many in low- and middle-income countries due to the expensive reagents required for its manufacturing, as well as specialized equipment required for distribution and storage. To address these challenges, we deconstructed processes required for cell-free mixture preparation and developed a set of alternative low-cost strategies for easy production and sharing of extracts. First, we explored the stability of cell-free reactions dried through a low-cost device based on silica beads, as an alternative to commercial automated freeze dryers. Second, we report the positive effect of lactose as an additive for increasing protein synthesis in maltodextrin-based cell-free reactions using either circular or linear DNA templates. The modifications were used to produce active amounts of two high-value reagents: the isothermal polymerase Bst and the restriction enzyme BsaI. Third, we demonstrated the endogenous regeneration of nucleoside triphosphates and synthesis of pyruvate in cell-free systems (CFSs) based on phosphoenol pyruvate (PEP) and maltodextrin (MDX). We exploited this novel finding to demonstrate the use of a cell-free mixture completely free of any exogenous nucleotide triphosphates (NTPs) to generate high yields of sfGFP expression. Together, these modifications can produce desiccated extracts that are 203-424-fold cheaper than commercial versions. These improvements will facilitate wider use of CFS for research and education purposes.
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    Decentralizing Cell-Free RNA Sensing With the Use of Low-Cost Cell Extracts
    (2021) Arce, Anibal; Guzman Chavez, Fernando; Gandini, Chiara; Puig, Juan; Matute, Tamara; Haseloff, Jim; Dalchau, Neil; Molloy, Jenny; Pardee, Keith; Federici, Fernan
    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 mainly 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. Based on preliminary demonstrations of toehold sensors working on lysates, we describe the fast prototyping of RNA toehold switch-based sensors that can be produced locally and reduce the cost of sensors by two orders of magnitude. We demonstrate that these in-house cell lysates provide sensor performance comparable to commercial PURE cell-free systems. We further optimize these lysates with a CRISPRi strategy to enhance the stability of linear DNAs by knocking-down genes responsible for linear DNA degradation. This enables 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.
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    Isolation of the three grape sub-lineages of B-class MADS-box TM6, PISTILLATA and APETALA3 genes which are differentially expressed during flower and fruit development
    (2007) Poupin, Maria Josefina; Federici, Fernan; Medina, Consuelo; Matus, Jose Tomas; Timmermann, Tania; Arce-Johnson, Patricio
    The B class of MADS-box floral homeotic genes specifies petal and stamen identity in angiosperms. While this group is one of the most studied in herbaceous plant species, it has remained largely uncharacterized in woody species such as grapevine. Although the B class PI/GLO and AP3/DEF clades have been extensively characterized in model species, the role of the TM6 subgroup within the AP3 clade is not completely understood, since it is absent in Arabidopsis thaliana. In this study, the coding regions of VvTM6 and VvAP3 and the genomic sequence of VvPI, were cloned. VvPI and AtPI were confirmed to be functional homologues by means of complementation of the pi Arabidopsis mutant. Expression analysis revealed that VvPI and VvAP3 transcripts are restricted almost exclusively to inflorescences, although VvPI was detected at low levels in leaves and roots. VvTM6 expresses throughout the plant, with higher levels in flowers and berries. A detailed chronological study of grape flower progression by light microscopy and temporal expression analysis throughout early and late developmental stages, revealed that VvPI expression increases during pollen maturation and decreases between the events of pollination and fertilization, before the cap fall. On the other hand, VvTM6 is expressed in the last stage of anther development. Specific expression of VvAP3 and VvPI was detected in petals and stamens within the flower, while VvTM6 was also expressed in carpels. Moreover, this work provides the first evidence for expression of a TM6-like gene throughout fruit growth and ripening. Even if these genes belong to the same genetic class they could act in different periods and/or tissues during reproductive organ development. (C) 2007 Elsevier B.V. All rights reserved.
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    qByte: Open-source isothermal fluorimeter for democratizing analysis of nucleic acids, proteins and cells
    (2024) Quero, Francisco J.; Aidelberg, Guy; Vielfaure, Hortense; Huon de Kermadec, Yann; Cazaux Severine, Marianne; Pandi, Amir; Pascual Garrigos, Ana; Arce Medina, Aníbal Andrés; Sakyi, Samuel; Gaudenz, Urs; Federici, Fernan; Molloy, Jennifer C.; Lindner, Ariel B.