Rapporto tecnico sulla ottimizzazione del processo di biosintesi di una Green Fluorescent Protein ricombinante estratta da A. sulcata

L’espressione di geni eterologhi in Escherichia coli rappresenta uno dei metodi più veloci, semplici ed economici per la produzione di ampie quantità di proteine target. Tuttavia, meccanismi di folding e le modifiche post traduzionali inducono a volte un non corretto ripiegamento delle proteine nella conformazione nativa, con successiva aggregazione in quelli che vengono definiti corpi di inclusione. Nel nostro caso, l’attenzione è stata focalizzata su una Green Fluorescent Protein (GFP) di 228 aa estratta da Anemonia sulcata, contenente un fluoroforo composto da tre amminoacidi Gln63, Tyr64, Gly65 all'interno di una struttura a barile. Il corretto folding della proteina era correlato strettamente alla funzionalità del fluoroforo. Il nostro obiettivo è stato quello, quindi, di ottimizzare il processo di biosintesi della GFP espressa in E. coli, ovviando alla formazione di corpi di inclusione contenenti la proteina (non funzionale), definendo e standardizzando inoltre, le condizioni che consentivano di produrre la più alta percentuale di GFP correttamente ripiegata (in condizioni non denaturanti) e quindi funzionale

"Boron Effect" on the Thermal Decomposition of Light Metal Borohydrides MBH4(M = Li, Na, Ca)

The hydrogen release rate of thermal decomposition, after the melting, for the borohydride of lithium (Li), sodium (Na), and calcium (Ca) and their boron mixtures, at selected molar ratios, is investigated under 1 bar hydrogen pressure and nonisothermal conditions. The reaction is studied by means of manometric measurements. The maximum hydrogen release rate for all pure borohydrides is 8 7 10 -3 bar/min. By adding boron to the borohydride systems, the hydrogen release rate is affected and, generally, is lowered. For the decomposition process of LiBH 4+B, maximum rate is 2 7 10 -3 bar/min. On the opposite, hydrogen rate is suppressed in boron mixtures of NaBH 4. The addition of boron changed slightly the maximum rate of hydrogen release for Ca(BH 4) 2+B for first and second decompositio

Self-Assembly of molecules on nanostructured graphene

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid. Fecha de lectura: 27-7-2014

Thermal study on decomposition of LiBH4 at non-isothermal and non-equilibrium conditions

Thermal decomposition measurements for lithium borohydride (LiBH4) are performed at non-isothermal and non-equilibrium conditions by means of differential thermal analysis (DTA). A simplified alternative procedure is introduced for evaluating thermodynamic and kinetic parameters simultaneously using a single set of measurements. Rate constant (k) and enthalpy (??H = ???102.1 \ub1 0.7 kJ mol???1 LiBH4) are archived. Temperature dependence for activation energy (E a) is found taking advantage of Guggenheim???Arrhenius method; the mean activation energy is E?????????a 93.9 \ub1 0.9 kJ mol???1 LiBH4 in the range of heating rate ?? 1???50 K min???1

Self-assmebly of molecules on nanostructured graphene

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Física Teórica de la Materia Condensada. Fecha de lectura: 27-07-2014In this thesis the growth and morphological characterisation for the self-assembly of various electron acceptor molecules were investigated by mean of Scanning Tunnelling Microscope (STM) in Ultra High Vacuum. Molecules, from the quinone and xanthine classes, such as 11,11, 12,12 - tetracyano-9,10-anthraquino dimethane (TCAQ), 13,13,14,14 - tetracyano-5,12 -tetracenequino dimethane (TCTQ), 15,15, 16,16 -tetracyano -6,13-pentacenequino dimethane (TCPQ) and 1,3,7-Trimethyl xanthine (caffeine), were chosen. Two different substrate, graphene/Ru(0001) and Cu (111), were used to study the influence of the substrate molecule interaction in the self-assembly processes. The metallic substrate allows the hybridisation of the molecular states and the metallic DOS. On the other hand, the graphene decouples the molecules from the metal providing a good playground to study the moleculemolecule interactions. These molecules aggregate and eventually self-assembly on the surface and the aggregation process is influenced by the number of the rings in the backbone of the molecule, the interaction with the substrate and the coverage of the surface. Basically, the TCxQ molecules show on Cu(111) a very limited aggregation character with the formation of small clusters, such as chains or dimers, and eventually, disordered structures. A qualitative explanation for the cluster formation can be given using the molecular potential maps, where the negative potential, associated with the cyano groups, interacts with the positive potential, located on the hydrogens in the molecular backbone, forming a hydrogen bond network. Analysing the apparent size in the STM images on the energetics in theoretical calculations it can be concluded that all TCxQ molecules adopt the socalled X-configuration upon adsorption on Cu(111). Unlike, the deposition of those molecules on graphene/Ru(0001) brings to a very different structure which is characterized by a formation of an extensive hydrogen bond network and a very weak interaction with the substrate. Each of the quinone derived molecules self-assemblies in a different way depending on the number of the aromatic rings in the backbone of the molecule and the coverage of the surface. At low coverage, only symmetric molecules stay on the surface and occupy the low area of the graphene moiré with unordered clusters. By increasing the coverage TCAQ molecules self-assembly into representative “railroad tracks” structure covering low and top area of the moiré . By contrast, TCTQ and TCPQ molecules formed a porous network filling the low area only. When the monolayer conditions are reached, a compact self-assembly structure is obtained only for the symmetric molecules. To investigate the influence of the rigid molecular structure on the surface, caffeine molecules were deposited on Cu(111). Caffeine is basically adsorbed flat-lying on Cu(111) and forms dimers arranged into parallel rows. The molecules are easy to move indicating the absence of a preferential adsorption position and the important of the intermolecular interactions in the formation of the self-assembled structure.En esta tesis fueron investigadas el crecimiento y la caracterización morfológica para el self-assembly de diferentes moléculas aceptoras de electrones por medio de Scanning Tunnelling Microscope (STM) en Ultra Alto Vacío. Fueron elegidas moléculas que van desde las clases quinona y xantina, tales como 11,11, 12,12 - tetraciano - 9,10 - Dimethane anthraquino ( TCAQ ), 13,13,14,14 - tetraciano - 5, Dimethane 12 - tetracenequino ( TCTQ ), 15,15, 16,16 tetraciano Dimethane -6,13 - pentacenequino ( TCPQ ) a la 1,3,7- trimetil xantina (cafeína). Para estudiar la influencia de la interacción molécula-sustrato en los procesos de self-assembly han sido utilizados dos diferentes sustratos, grafeno / Ru(0001) y Cu(111). El sustrato metálico permite la hibridación de los estados moleculares y el metálico DOS. Por otro lado, el grafeno desacopla las moléculas del metal proporcionando una buena zona para estudiar las interacciones molécula-molécula. Estas moléculas se agregan y, al final, se autoensamblan en la superficie. En concreto el proceso de agregación está influenciado por el número de anillos en la estructura de la molécula, la interacción con el sustrato y el recubrimiento de la superficie. Básicamente, las moléculas TCxQ muestran en Cu (111) un carácter de agregación muy limitado con la formación de pequeños grupos, tales como cadenas o dímeros, y, al final, estructuras desordenadas. Una explicación cualitativa para la formación del clúster se puede hallar utilizando los mapas de potencial molecular, donde el potencial negativo, asociado con los grupos ciano, interactúa con el potencial positivo, que se encuentra en los átomos de hidrógeno en el esqueleto molecular, formando una red de enlace de hidrógeno. Analizando el tamaño que aparece en las imágenes de STM en la energética en cálculos teóricos, se puede concluir que todas las moléculas TCxQ adoptan la denominada configuración-X de la adsorción en Cu(111). Por otro lado, la deposición de las moléculas en el grafeno / Ru(0001) lleva a una estructura muy diferente, caracterizada da la formación de una extensa red de enlaces de hidrógeno y una muy débil interacción con el sustrato. Cada una de las moléculas quinonas derivadas se autoensabla de manera diferente en función del número de los anillos aromáticos en la cadena principal de la molécula y del recubrimienti de la superficie. En condiciones de bajo recubrimiento, sólo las moléculas simétricas permanecen en la superficie y ocupan la zona baja del moiré de grafeno con clusters desordenados. A medida que aumenta el recubrimiento las moléculas de TCAQ se autoensemblan en estructuras representativas “railroad tracks”cubriendo la parte del área superior y inferior del moiré. Por el contrario, las moléculas de TCTQ y TCPQ forman una red porosa que llena sólo la zona inferior. Se comprobó tambien que en el caso de tener una monocapa, la estructura self-assembly compacta se obtiene sólo para las moléculas simétricas. Por último, fueron depositadas moléculas de cafeína en superficies de Cu(111) para poder investigar la influencia de la estructura molecular rígida en la superficie. La cafeína es básicamente adsorbida en manera planar con respecto al Cu(111) y forma dímeros dispuestos en filas paralelas. En concreto, las moléculas presentan facilidad de movimientos, que indica la ausencia de una posición de adsorción preferencial y la importancia de las interacciones intermoleculares en la formación de la estructura self-assembl

Technical Report on the reduced graphene oxide biosynthesis protocol for biological applications

The interest of scientific community on carbon-based smart materials is growing and, especially focus on graphene oxide (GO) and reduced graphene oxide (rGO). An increasing number of bio-applications such as biological applications as bacterial inhibition, drug delivery and photothermal therapy aims the use of GO and rGO. For this reason, the methods used for the synthesis of graphene materials are more important because same of those procedures imply chemical reactions that involve hazardous and toxic reagents. In fact, the biocompatibility and toxicological activity of graphene-related materials is related to the methodologies employed for the synthesis that determine the carbon/oxygen (C/O) ratio of graphene oxide species. In this technical report, we focused on the synthesis of GO by means of that lead to a biocompatible GO form with a lower oxygen content. Thus, the synthesis of rAsGFP-rGO with the green fluorescent protein allowed us to obtain a biocompatible materials, without using hazardous and toxic reagents. This biocompatibility is the most important prerogative for the use of GO in biological activity assays as reported in several publications

The structural change of graphene oxide in a methanol dispersion

Graphene oxide is a derivative of graphene which contains oxygen domains. Its debatable structure depends on the specific functional groups bonded to the graphene basal plane which have an impact on its reactivity. Here, we report the influence of methanol which affects the functionalization of pristine graphene differently to water