Control óptico de la expresión génica en sistemas biológicos mediante nanopartículas de oro: Expresión génica fototérmica en Escherichia coli y silenciamiento génico en Chlamydomonas reinhardtii

Las nanopartículas de oro pueden ser encontrados de diferentes formas, tamaños y que determinan sus características químicas y físicas. Las propiedades físicas y químicas de las nanopartículas metálicas pueden ser moduladas al cambiar su forma, tamaño y la química de su superficie. Por lo tanto, esto ha permitido su uso en una gran variedad de aplicaciones en los sectores industriales y académicos. Una de las características de las nanopartículas metálicas es su habilidad para actuar como convertidos de energía optotérmicos. Esta característica ha sido utilizada en muchas aplicaciones donde las nanopartículas son acopladas con sistemas de respuesta térmica para generar una respuesta óptica. En este estudio, nosotros sintetizamos nanopartículas metálicas que son mayormente esféricas en su forma con un promedio de diámetro de 20.07 nm. En este estudio, nosotrosutilizamos dos fuentes de luz: LED y láser. Diferentes enfoques estadísticos fueron utilizados para medir la potencia y capacidad funcional de la luz láser y LED así como identificar a la variable más necesaria para incrementar la temperatura en una solución de nanopartículas de oro. En este trabajo se realizaron simultáneamente técnicas teóricas y experimentales para evaluar los diferentes factores que afectan la generación de calor en la superficie de nanopartículas cuando son expuestas a una longitud de onda específica por la luz láser y LED. Respecto al láser, los resultados mostraron que los factores que más contribuyeron al cambio de temperatura exhibido en la solución de nanopartículas resultaron ser el poder del láser, la concentración de las nanopartículas de oro, la interacción tiempo × láser y el tiempo de iluminación. Nosotros reportamos un modelo de regresión que permite predecir la generación de calor y cambios de temperatura con errores estándares residuales en menos de 4%. Los resultados son altamente relevantes para diseños futuros y en el desarrollo de aplicaciones donde las aplicaciones de nanopartículas sean incorporadas en los sistemas para inducir un cambio en la temperatura a partir de la exposición de con luz. Respecto al LED, nosotros analizamos estadísticamente la temperatura producida en la superficie de las nanopartículas de oro cuando utilizando LED como fuente de luz. Los resultados mostrados que los efectores principales y las interacciones de todos los factores fueron significativos. Finalmente, basados en el modelo de regresión presentado, los coeficientes de regresión y los resultados de ANOVA nos permiten presentan un poderoso modelo de regresión que muestra las relaciones entre la temperatura de cambio y sus variables. Nosotros simulamos el cambio de generación de nuestras nanopartículas de oro cuando la solución con nanopartículas de oro era iluminada con una fuente de luz LED. Nosotros demostramos que el máximo incremento de temperatura en la solución de nanopartículas (resultados de simulación) cotejaron excelentemente con nuestras observaciones (resultados prácticos). Para evaluar nuestra aplicación fototérmica obtenida a partir de nanopartículas de oro en un sistema biológico en células, evaluamos su factibilidad en la producción de proteína con enfoque fototérmico por primera vez. Para lograr este objetivo, utilizamos luz LED en vez de un dispositivo láser al considerarse como un método nuevo, barato, inofensivo y conmutable para sistemas biológicos vivos. Después de sintetizar las nanopartículas de oro y obtener su perfil de temperatura, nosotros diseñamos un gen sintético, donde el sitio de unión a ribosoma pudiera ser activo y trabajar eficientemente a 37°C. Basado en el modelo de regresión lineal y en análisis de respuesta de superficie de curva, nosotros encontramos el cómo proveer la temperatura necesaria. De esta manera, nosotros mostramos el uso de nanopartículas metálicas y LED como fuente de luz pueden trabajar eficientemente en una estructura tipo stem – loop que contiene un sitio a unión a ribosoma y consecuentemente una alta producción de mCherry es logrado. Además, para mostrar su factibilidad en la desbridamiento de dsDNA unido a nanopartículas metálicas a partir de LED como fuente de luz, nosotros elaboramos conmutadores (nanopartículas de oro acoplados con dsDNA) y finalmente fueron caracterizados. Entonces, nosotros mostramos la factibilidad del desbridamiento del dsDNA unido a nanopartículas de oro (prueba in vitro) utilizando LED como fuente de luz bajo diferentes longitudes de onda. La prueba demostró ser exitosa y se mostró la probabilidad de que el calor generado fototérmica pueda ser utilizado para el silenciamiento de genes por antisentido en células de microalgas vivas

Microalgal co-cultivation -recent methods, trends in omic-studies, applications, and future challenges

The burgeoning human population has resulted in an augmented demand for raw materials and energy sources, which in turn has led to a deleterious environmental impact marked by elevated greenhouse gas (GHG) emissions, acidification of water bodies, and escalating global temperatures. Therefore, it is imperative that modern society develop sustainable technologies to avert future environmental degradation and generate alternative bioproduct-producing technologies. A promising approach to tackling this challenge involves utilizing natural microbial consortia or designing synthetic communities of microorganisms as a foundation to develop diverse and sustainable applications for bioproduct production, wastewater treatment, GHG emission reduction, energy crisis alleviation, and soil fertility enhancement. Microalgae, which are photosynthetic microorganisms that inhabit aquatic environments and exhibit a high capacity for CO2 fixation, are particularly appealing in this context. They can convert light energy and atmospheric CO2 or industrial flue gases into valuable biomass and organic chemicals, thereby contributing to GHG emission reduction. To date, most microalgae cultivation studies have focused on monoculture systems. However, maintaining a microalgae monoculture system can be challenging due to contamination by other microorganisms (e.g., yeasts, fungi, bacteria, and other microalgae species), which can lead to low productivity, culture collapse, and low-quality biomass. Co-culture systems, which produce robust microorganism consortia or communities, present a compelling strategy for addressing contamination problems. In recent years, research and development of innovative co-cultivation techniques have substantially increased. Nevertheless, many microalgae co-culturing technologies remain in the developmental phase and have yet to be scaled and commercialized. Accordingly, this review presents a thorough literature review of research conducted in the last few decades, exploring the advantages and disadvantages of microalgae co-cultivation systems that involve microalgae-bacteria, microalgae-fungi, and microalgae-microalgae/algae systems. The manuscript also addresses diverse uses of co-culture systems, and growing methods, and includes one of the most exciting research areas in co-culturing systems, which are omic studies that elucidate different interaction mechanisms among microbial communities. Finally, the manuscript discusses the economic viability, future challenges, and prospects of microalgal co-cultivation methods

Biomass and lipid induction strategies in microalgae for biofuel production and other applications

The use of fossil fuels has been strongly related to critical problems currently affecting society, such as: global warming, global greenhouse effects and pollution. These problems have affected the homeostasis of living organisms worldwide at an alarming rate. Due to this, it is imperative to look for alternatives to the use of fossil fuels and one of the relevant substitutes are biofuels. There are different types of biofuels (categories and generations) that have been previously explored, but recently, the use of microalgae has been strongly considered for the production of biofuels since they present a series of advantages over other biofuel production sources: (a) they don’t need arable land to grow and therefore do not compete with food crops (like biofuels produced from corn, sugar cane and other plants) and; (b) they exhibit rapid biomass production containing high oil contents, at least 15 to 20 times higher than land based oleaginous crops. Hence, these unicellular photosynthetic microorganisms have received great attention from researches to use them in the large-scale production of biofuels. However, one disadvantage of using microalgae is the high economic cost due to the low-yields of lipid content in the microalgae biomass. Thus, development of different methods to enhance microalgae biomass, as well as lipid content in the microalgae cells, would lead to the development of a sustainable low-cost process to produce biofuels. Within the last 10 years, many studies have reported different methods and strategies to induce lipid production to obtain higher lipid accumulation in the biomass of microalgae cells; however, there is not a comprehensive review in the literature that highlights, compares and discusses these strategies. Here, we review these strategies which include modulating light intensity in cultures, controlling and varying CO2 levels and temperature, inducing nutrient starvation in the culture, the implementation of stress by incorporating heavy metal or inducing a high salinity condition, and the use of metabolic and genetic engineering techniques coupled with nanotechnology

Development of a Theoretical Model That Predicts Optothermal Energy Conversion of Gold Metallic Nanoparticles

Gold nanoparticles (AuNPs) can be found in different shapes and sizes, which determine their chemical and physical characteristics. Physical and chemical properties of metallic NPs can be tuned by changing their shape, size, and surface chemistry; therefore, this has led to their use in a wide variety of applications in many industrial and academic sectors. One of the features of metallic NPs is their ability to act as optothermal energy converters, where they absorb light at a specific wavelength and heat up their local nanosurfaces. This feature has been used in many applications where metallic NPs get coupled with thermally responsive systems to trigger an optical response. In this study, we synthesized AuNPs that are spherical in shape with an average diameter of 20.07 nm. This work assessed simultaneously theoretical and experimental techniques to evaluate the different factors that affect heat generation at the surface of AuNPs when exposed to a specific light wavelength. The results indicated that laser power, concentration of AuNPs, time × laser power interaction, and time illumination, were the most important factors that contributed to the temperature change exhibited in the AuNPs solution. We report a regression model that allows predicting heat generation and temperature changes with residual standard errors of less than 4%. These results are highly relevant in the future design and development of applications where metallic NPs are incorporated into systems to induce a temperature change triggered by light exposure

LED control of gene expression in a nanobiosystem composed of metallic nanoparticles and a genetically modified E. coli strain

Abstract Background Within the last decade, genetic engineering and synthetic biology have revolutionized society´s ability to mass-produce complex biological products within genetically-modified microorganisms containing elegantly designed genetic circuitry. However, many challenges still exist in developing bioproduction processes involving genetically modified microorganisms with complex or multiple gene circuits. These challenges include the development of external gene expression regulation methods with the following characteristics: spatial–temporal control and scalability, while inducing minimal permanent or irreversible system-wide conditions. Different stimuli have been used to control gene expression and mitigate these challenges, and they can be characterized by the effect they produce in the culture media conditions. Invasive stimuli that cause permanent, irreversible changes (pH and chemical inducers), non-invasive stimuli that cause partially reversible changes (temperature), and non-invasive stimuli that cause reversible changes in the media conditions (ultrasound, magnetic fields, and light). Methods Opto-control of gene expression is a non-invasive external trigger that complies with most of the desired characteristics of an external control system. However, the disadvantage relies on the design of the biological photoreceptors and the necessity to design them to respond to a different wavelength for every bioprocess needed to be controlled or regulated in the microorganism. Therefore, this work proposes using biocompatible metallic nanoparticles as external controllers of gene expression, based on their ability to convert light into heat and the capacity of nanotechnology to easily design a wide array of nanostructures capable of absorbing light at different wavelengths and inducing plasmonic photothermal heating. Results Here, we designed a nanobiosystem that can be opto-thermally triggered using LED light. The nanobiosystem is composed of biocompatible gold nanoparticles and a genetically modified E. coli with a plasmid that allows mCherry fluorescent protein production at 37 °C in response to an RNA thermometer. Conclusions The LED-triggered photothermal protein production system here designed offers a new, cheaper, scalable switchable method, non-destructive for living organisms, and contribute toward the evolution of bioprocess production systems