Person: Guevara Acosta, Flor Govinda
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First Name
Flor Govinda
Last Name
Guevara Acosta
Affiliation
Universidad Complutense de Madrid
Faculty / Institute
Ciencias Biológicas
Department
Bioquímica y Biología Molecular
Area
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5 results
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Item SEVA-Cpf1, a CRISPR-Cas12a vector for genome editing in cyanobacteria(Microbial Cell Factories, 2022) Baldanta Callejo, Sara; Guevara Acosta, Flor Govinda; Navarro Llorens, Juana MaríaBackground Cyanobacteria are photosynthetic autotrophs that have tremendous potential for fundamental research and industrial applications due to their high metabolic plasticity and ability to grow using CO2 and sunlight. CRISPR technology using Cas9 and Cpf1 has been applied to different cyanobacteria for genome manipulations and metabolic engineering. Despite significant advances with genome editing in several cyanobacteria strains, the lack of proper genetic toolboxes is still a limiting factor compared to other model laboratory species. Among the limitations, it is essential to have versatile plasmids that could ease the benchwork when using CRISPR technology. Results In the present study, several CRISPR-Cpf1 vectors were developed for genetic manipulations in cyanobacteria using SEVA plasmids. SEVA collection is based on modular vectors that enable the exchangeability of diverse elements (e.g. origins of replication and antibiotic selection markers) and the combination with many cargo sequences for varied end-applications. Firstly, using SEVA vectors containing the broad host range RSF1010 origin we demonstrated that these vectors are replicative not only in model cyanobacteria but also in a new cyanobacterium specie, Chroococcidiopsis sp., which is different from those previously published. Then, we constructed SEVA vectors by harbouring CRISPR elements and showed that they can be easily assimilated not only by conjugation, but also by natural transformation. Finally, we used our SEVA-Cpf1 tools to delete the nblA gene in Synechocystis sp. PCC 6803, demonstrating that our plasmids can be applied for CRISPR-based genome editing technology. Conclusions The results of this study provide new CRISPR-based vectors based on the SEVA (Standard European Vector Architecture) collection that can improve editing processes using the Cpf1 nuclease in cyanobacteria.- Project number: 163
Item The Phantom Menace: Cómo salvar el mundo de una pandemia mediante Ingeniería Genética cooperativa(2021) Navarro LLorens, Juana María; Baldanta Callejo, Sara; Bénitez Prian, Mario; Blázquez Ortiz, Cristina; Bruñen Alfaro, Francisco; Cañadas Benito, Olga; Chinarro Sánchez, Adrián; García de la Camacha Selgas, Nuria; García-Fojeda García-Valdecasas, María Belén; Guevara Acosta, Flor Govinda; Leaño Hinojosa, Ariana; López Conejo, Maria Teresa; Lorente Pérez, Maria del Mar; Nogués Vera, Laura; Penalba Iglesias, Diana; Raisman, Andrea; Ranz Valdecasa, Maria Regina; Ruiz Ortega, Marta; Sánchez-Escalonilla Relea, Jose luis; Velasco Díez, Guillermo; Sánchez Torralba, AntonioEl año pasado se llevó a cabo un proyecto de innovación (PIMCD-2019 174) basado en el modelo pedagógico de la clase invertida o “Flipped Classroom” y en el juego de mesa PANDEMIC (ASMODEE IBÉRICA). Brevemente, este proyecto consistió en una propuesta didáctica sobre los contenidos de FIGG en el que tomando como partida un entorno lúdico, los alumnos de FIGG en equipos preparaban contenidos y reforzaban conocimientos adquiridos en clase. Para llevarlo a cabo, a los alumnos se les planteaba una hipotética situación en la que cuatro enfermedades mortales aparecen en la tierra. Los alumnos divididos en equipos de 5 personas, deben cooperar desarrollando una serie de acciones que les permitan ir conociendo a qué patógeno se enfrentan y desarrollar herramientas que les permita controlar la epidemia dentro de un tiempo dado. Así cada equipo debe frenar el avance de las infecciones y a la vez encontrar una cura para la misma. Curiosamente, todo este escenario fue planteado antes de que la crisis del COVID-19 impactara en nuestra sociedad. En cualquier caso, la situación vivida en los últimos meses ha motivado a los estudiantes de FIGG por lo que en su mayor parte han participado en su desarrollo de manera entusiasta. En este nuevo proyecto, hemos tomado como base el proyecto del curso pasado, pero introduciendo algunos elementos innovadores. Entre estas innovaciones destaca la incorporación de dos alumnos por grupo de FIGG que ya lo hubieran realizado en la edición pasada por cada uno de los grupos de FIGG como mentores. Item Further Studies on the 3-Ketosteroid 9α-Hydroxylase of Rhodococcus ruber Chol-4, a Rieske Oxygenase of the Steroid Degradation Pathway(Microorganisms, 2021) Baldanta Callejo, Sara; Navarro Llorens, Juana María; Guevara Acosta, Flor GovindaThe biochemistry and genetics of the bacterial steroid catabolism have been extensively studied during the last years and their findings have been essential to the development of biotechnological applications. For instance, metabolic engineering of the steroid-eater strains has allowed to obtain intermediaries of industrial value. However, there are still some drawbacks that must be overcome, such as the redundancy of the steroid catabolism genes in the genome and a better knowledge of its genetic regulation. KshABs and KstDs are key enzymes involved in the aerobic breakage of the steroid nucleus. Rhodococcus ruber Chol-4 contains three kshAs genes, a single kshB gene and three kstDs genes within its genome. In the present work, the growth of R. ruber ΔkshA strains was evaluated on different steroids substrates; the promoter regions of these genes were analyzed; and their expression was followed by qRT-PCR in both wild type and ksh mutants. Additionally, the transcription level of the kstDs genes was studied in the ksh mutants. The results show that KshA2B and KshA1B are involved in AD metabolism, while KshA3B and KshA1B contribute to the cholesterol metabolism in R. ruber. In the kshA single mutants, expression of the remaining kshA and kstD genes is re-organized to survive on the steroid substrate. These data give insight into the fine regulation of steroid genes when several isoforms are present.Item First characterization of cultivable extremophile Chroococcidiopsis isolates from a solar panel(Frontiers in Microbiology, 2023) Baldanta Callejo, Sara; Arnal, Raquel; Blanco-Rivero, Amaya; Guevara Acosta, Flor Govinda; Navarro Llorens, Juana MaríaIntroduction: Microorganisms colonize a wide range of natural and artificial environments. Even though most of them are unculturable in laboratory conditions, some ecosystems are ideal niches for bioprospecting extremophiles with unique properties. Up today, there are few reports concerning microbial communities found on solar panels, a widespread, artificial, extreme habitat. Microorganisms found in this habitat belong to drought-, heat- and radiation-adapted genera, including fungi, bacteria, and cyanobacteria. Methods: Here we isolated and identified several cyanobacteria from a solar panel. Then, some strains isolated were characterizated for their resistance to desiccation, UV-C exposition, and their growth on a range of temperature, pH, NaCl concentration or diverse carbon and nitrogen sources. Finally, gene transfer to these isolates was evaluated using several SEVA plasmids with different replicons to assess their potential in biotechnological applications. Results and discussion: This study presents the first identification and characterization of cultivable extremophile cyanobacteria from a solar panel in Valencia, Spain. The isolates are members of the genera Chroococcidiopsis, Leptolyngbya, Myxacorys, and Oculatella all genera with species commonly isolated from deserts and arid regions. Four of the isolates were selected, all of them Chroococcidiopsis, and characterized. Our results showed that all Chroococcidiopsis isolates chosen were resistant up to a year of desiccation, viable after exposition to high doses of UV-C, and capable of being transformed. Our findings revealed that a solar panel is a useful ecological niche in searching for extremophilic cyanobacteria to further study the desiccation and UV-tolerance mechanisms. We conclude that these cyanobacteria can be modified and exploited as candidates for biotechnological purposes, including astrobiology applications.Item Characterization of Limnospira platensis PCC 9108 R-M and CRISPR-Cas systems(Microbiological Research, 2023) Castillo López, María; Guevara Acosta, Flor Govinda; Baldanta Callejo, Sara; Suárez Rodríguez, Patricia; Agudo, Lucía; Nogales, Juan; Díaz Carrasco, Asunción; Arribas-Aguilar, Fernando; Pérez-Pérez, Julián; García, Jose Luis; Galán, Beatriz; Navarro Llorens, Juana MaríaThe filamentous cyanobacterium Limnospira platensis, formerly known as Arthrospira platensis or spirulina, is one of the most commercially important species of microalgae. Due to its high nutritional value, pharmacological and industrial applications it is extensively cultivated on a large commercial scale. Despite its widespread use, its precise manipulation is still under development due to the lack of effective genetic protocols. Genetic transformation of Limnospira has been attempted but the methods reported have not been generally reproducible in other laboratories. Knowledge of the transformation defense mechanisms is essential for understanding its physiology and for broadening their applications. With the aim to understand more about the genetic defenses of L. platensis, in this work we have identified the restriction-modification and CRISPR-Cas systems and we have cloned and characterized thirteen methylases. In parallel, we have also characterized the methylome and orphan methyltransferases using genome-wide analysis of DNA methylation patterns and RNA-seq. The identification and characterization of these enzymes will be a valuable resource to know how this strain avoids being genetically manipulated and for further genomics studies.