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Cell-Free Expression of M2Kir6.2 ICCR for Direct Reconstitution into Micropipette Suspended Black Lipid MembranesSousa, Vanessa Rose January 2015 (has links)
Serving as key players in cell signaling, nearly all cells in the human body contain GPCRs. As the largest and most diverse superfamily of proteins in the human body, GPCRs are linked to some of the most prevalent current disease states including cardiovascular disease, type II diabetes, and various types of cancers. The development of new biosensors capable of simple, specific, sensitive, high-throughput screenings of the ligand-binding events of GPCRs are crucial to the diagnosis and maintenance of such diseases. To this end, this research is focused on the development of a novel biosensor platform incorporating ICCRs reconstituted into BLMs. Although ICCRs have been expressed previously in oocytes and HEK293 cells, no occurrence of cell-free expression has yet been performed. The advantages of such a platform include the specificity and real-time measurement capabilities of GPCRs, the innate sensitivity of electrophysiological ion channel flux measurements, and the simplified cellular mimicking of the BLM and cell-free expression. The majority of the presented research was based in the molecular cloning of M2Kir6.2, an ICCR incorporating a muscarinic acetylcholine receptor (M2), from Xenopus oocyte vector pGH2 into cell-free expression vector pT7CFE1-CGST-HA-His. Much optimization of the cloning procedure (PCR, digestion, and ligation) was necessary involving studies into polymerase fidelity, inclusion of DpnI for degradation of methylated DNA, and ligation parameter alterations in time, temperature, and insert:vector ratios. It was discovered that Deep VentR polymerase was beneficial to preventing mutations within the sequence of M2Kir6.2 during PCR, DpnI was capable of degrading unwanted residual M2Kir6.2 pGH2, and ligation performance was optimal using a 1:1 (insert:vector) ratio and reaction time and temperature of 18 h and 4 °C, respectively. With the successful ligation of M2Kir6.2 into cell-free expression vector pT7, expression of the ICCR via cell-free expression lysate kit was performed with direct reconstitution into a micropipette suspended BLM attempted. Five reconstitution trials were performed with electrophysiological single-channel recording results suggesting ICCR insertion based on ion channel currents of ~ 3 pA and mean open-times of ~ 3 ms observed corresponding to literature values for native Kir6.2 channels. Additionally, a Western blot analysis of the cell-free expression mixture contained products with molecular weights corresponding to monomer (~ 100 kDa) and tetramer (~ 400 kDa) constructs of M2Kir6.2. With the successful optimization of the M2Kir6.2 pT7 cloning procedure, this procedure can be used in future cloning attempts with similar ICCR constructs, such as D2Kir6.2. Although preliminary electrophysiological results suggest ICCR expression and BLM reconstitution, further work needs to be done in controlling the amount of ICCR insertion and optimizing BLM stability. Additionally, in order to confirm the functionality of both M2 and Kir6.2 ligand dose response curves must be performed. The evidence supporting ICCR expression and direct reconstitution into suspended BLMs via cell-free protein expression is both exciting and promising. Not only has this research involved the first cell-free expression of M2Kir6.2 but also has great benefits to the further development of such novel ligand-binding biosensor platforms.
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SPICE Modeling of TeraHertz Heterojunction bipolar transistors / Modélisation compacte des transistors bipolaires fonctionnant dans la gamme TeraHertzStein, Félix 16 December 2014 (has links)
Les études qui seront présentées dans le cadre de cette thèse portent sur le développement et l’optimisation des techniques pour la modélisation compacte des transistors bipolaires à hétérojonction (TBH). Ce type de modélisation est à la base du développement des bibliothèques de composants qu’utilisent les concepteurs lors de la phase de simulation des circuits intégrés. Le but d’une technologie BiCMOS est de pouvoir combiner deux procédés technologiques différents sur une seule et même puce. En plus de limiter le nombre de composants externes, cela permet également une meilleure gestion de la consommation dans les différents blocs digitaux, analogiques et RF. Les applications dites rapides peuvent ainsi profiter du meilleur des composants bipolaires et des transistors CMOS. Le défi est d’autant plus critique dans le cas des applications analogiques/RF puisqu’il est nécessaire de diminuer la puissance consommée tout en maintenant des fréquences de fonctionnement des transistors très élevées. Disposer de modèles compacts précis des transistors utilisés est donc primordial lors de la conception des circuits utilisés pour les applications analogiques et mixtes. Cette précision implique une étude sur un large domaine de tensions d’utilisation et de températures de fonctionnement. De plus, en allant vers des nœuds technologiques de plus en plus avancés, des nouveaux effets physiques se manifestent et doivent être pris en compte dans les équations du modèle. Les règles d’échelle des technologies plus matures doivent ainsi être réexaminées en se basant sur la physique du dispositif. Cette thèse a pour but d’évaluer la faisabilité d’une offre de modèle compact dédiée à la technologie avancée SiGe TBH de chez ST Microelectronics. Le modèle du transistor bipolaire SiGe TBH est présenté en se basant sur le modèle compact récent HICUMversion L2.3x. Grâce aux lois d’échelle introduites et basées sur le dessin même des dimensions du transistor, une simulation précise du comportement électrique et thermique a pu être démontrée.Ceci a été rendu possible grâce à l’utilisation et à l’amélioration des routines et méthodes d’extraction des paramètres du modèle. C’est particulièrement le cas pour la détermination des éléments parasites extrinsèques (résistances et capacités) ainsi que celle du transistor intrinsèque. Finalement, les différentes étapes d’extraction et les méthodes sont présentées, et ont été vérifiées par l’extraction de bibliothèques SPICE sur le TBH NPN Haute-Vitesse de la technologie BiCMOS avancée du noeud 55nm, avec des fréquences de fonctionnement atteignant 320/370GHz de fT = fmax. / The aim of BiCMOS technology is to combine two different process technologies intoa single chip, reducing the number of external components and optimizing power consumptionfor RF, analog and digital parts in one single package. Given the respectivestrengths of HBT and CMOS devices, especially high speed applications benefit fromadvanced BiCMOS processes, that integrate two different technologies.For analog mixed-signal RF and microwave circuitry, the push towards lower powerand higher speed imposes requirements and presents challenges not faced by digitalcircuit designs. Accurate compact device models, predicting device behaviour undera variety of bias as well as ambient temperatures, are crucial for the development oflarge scale circuits and create advanced designs with first-pass success.As technology advances, these models have to cover an increasing number of physicaleffects and model equations have to be continuously re-evaluated and adapted. Likewiseprocess scaling has to be verified and reflected by scaling laws, which are closelyrelated to device physics.This thesis examines the suitability of the model formulation for applicability to production-ready SiGe HBT processes. A derivation of the most recent model formulationimplemented in HICUM version L2.3x, is followed by simulation studies, whichconfirm their agreement with electrical characteristics of high-speed devices. Thefundamental geometry scaling laws, as implemented in the custom-developed modellibrary, are described in detail with a strong link to the specific device architecture.In order to correctly determine the respective model parameters, newly developed andexisting extraction routines have been exercised with recent HBT technology generationsand benchmarked by means of numerical device simulation, where applicable.Especially the extraction of extrinsic elements such as series resistances and parasiticcapacitances were improved along with the substrate network.The extraction steps and methods required to obtain a fully scalable model library wereexercised and presented using measured data from a recent industry-leading 55nmSiGe BiCMOS process, reaching switching speeds in excess of 300GHz. Finally theextracted model card was verified for the respective technology.
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