Engineering Transcriptional Control and Synthetic Gene Circuits in Cell Free systems

Engineering gene networks offers an opportunity to harness biological function for biotechnological and biomedical applications. In contrast to cell-based systems, cell free extracts offer a flexible and well-characterized context in which to implement predictable gene circuits. Critical to these efforts is the availability of a library of ligand sensitive gene regulatory systems. Here, I describe efforts to develop molecular tools to control gene expression and implement a negative feedback circuit in E.coli cell extracts. First, a strategy to regulate T7 RNA polymerase using DNA aptamers is detailed. I test the hypothesis that a DNA aptamer, when placed near the transcription start site, interferes with transcription in the presence of the target molecule. A DNA aptamer that binds thrombin is used as a model system for demonstrating feasibility of the approach. I show that for the hybrid T7-aptamer promoter, thrombin addition results in up to a 5-fold reduction in gene expression. I further demonstrate that gene expression be tuned by altering the position of the aptamer relative to the transcription start site. I then devised a mechanism to engineer dual regulation of T7 promoters using LacI and TetR repressor proteins. To achieve this, a LacI binding site (lacO) was positioned 92bp upstream from a T7lacO promoter, which resulted in an increased repression from T7lacO promoters presumably by a looping based mechanism. TetR binding sites were introduced into this framework to disrupt the DNA looping to create T7 promoters that respond to both LacI and TetR. I show that positioning a tetO operator between the upstream lacO and the T7lacO promoter results in relieving lacO mediated repression by TetR. Finally, a negative feedback circuit was realized using T7lacO promoters. To this end, mono-cistronic and bi-cistronic system assembly approaches for system assembly are examined leading to the realization of an inducible negative feedback circuit in cell free systems. Collectively, the tools developed in this work pave the way for expanding the library of ligands that can be used for regulating gene expression, enabling signal integration at T7 promoters and facilitating engineering of gene networks in cell free systems

Roadmap for Optical Tweezers 2023

Optical tweezers are tools made of light that enable contactless pushing, trapping, and manipulation of objects ranging from atoms to space light sails. Since the pioneering work by Arthur Ashkin in the 1970s, optical tweezers have evolved into sophisticated instruments and have been employed in a broad range of applications in life sciences, physics, and engineering. These include accurate force and torque measurement at the femtonewton level, microrheology of complex fluids, single micro- and nanoparticle spectroscopy, single-cell analysis, and statistical-physics experiments. This roadmap provides insights into current investigations involving optical forces and optical tweezers from their theoretical foundations to designs and setups. It also offers perspectives for applications to a wide range of research fields, from biophysics to space exploration

Roadmap for optical tweezers

Artículo escrito por un elevado número de autores, solo se referencian el que aparece en primer lugar, el nombre del grupo de colaboración, si le hubiere, y los autores pertenecientes a la UAMOptical tweezers are tools made of light that enable contactless pushing, trapping, and manipulation of objects, ranging from atoms to space light sails. Since the pioneering work by Arthur Ashkin in the 1970s, optical tweezers have evolved into sophisticated instruments and have been employed in a broad range of applications in the life sciences, physics, and engineering. These include accurate force and torque measurement at the femtonewton level, microrheology of complex fluids, single micro- and nano-particle spectroscopy, single-cell analysis, and statistical-physics experiments. This roadmap provides insights into current investigations involving optical forces and optical tweezers from their theoretical foundations to designs and setups. It also offers perspectives for applications to a wide range of research fields, from biophysics to space explorationEuropean Commission (Horizon 2020, Project No. 812780

Roadmap for optical tweezers

Optical tweezers are tools made of light that enable contactless pushing, trapping, and manipulation of objects, ranging from atoms to space light sails. Since the pioneering work by Arthur Ashkin in the 1970s, optical tweezers have evolved into sophisticated instruments and have been employed in a broad range of applications in the life sciences, physics, and engineering. These include accurate force and torque measurement at the femtonewton level, microrheology of complex fluids, single micro- and nano-particle spectroscopy, single-cell analysis, and statistical-physics experiments. This roadmap provides insights into current investigations involving optical forces and optical tweezers from their theoretical foundations to designs and setups. It also offers perspectives for applications to a wide range of research fields, from biophysics to space exploration.journal articl

Biomolecular Materials. Report of the January 13-15, 2002 Workshop

Twenty-two scientists from around the nation and the world met to discuss the way that the molecules, structures, processes and concepts of the biological world could be used or mimicked in designing novel materials, processes or devices of potential practical significance. The emphasis was on basic research, although the long-term goal is, in addition to increased knowledge, the development of applications to further the mission of the Department of Energy

The design and analysis of quartic double well potential with stochastic resonance for communication systems

Non-linearity and noise are two phenomena that are expected to be essential to future advanced technologies. Although largely abstained, in general, from introduction into current communication systems, the counter-intuitive phenomenon called Stochastic Resonance (SR) can be introduced into communication systems in an innovative form. Therefore, in this thesis, the most prominent dynamical system in the SR field, the double well potential, namely the over-damped Duffing equation with symmetric bistable potential, has been studied in order to reveal its signal processing capabilities for communication systems. Within this thesis, the double well potential was designed in order to detect a binary pulse amplitude modulated (BPAM) signal subject to a background noise. The bit-error-rate (BER) performance was enhanced by adding various resonant signals to the input. In addition, the eye patterns of system output indicated that, while decreasing BER, a resonant causes a strong fluctuation. It was eliminated by a use of two systems coupled in parallel, which provided further performance improvement. The results inferred that the double well potential performs filtering and modulation. Following that, the double well potential was designed as a lowpass filter by determining the DC gain and cut-off frequency. Through simulations, as a filter, its noise suppression performance was shown to be better than that of various orders of Butterworth filters. The analog and digital modulation capabilities of the double well potential have also been investigated. In order to clarify the relation between input signal and modulation parameters, the differential equation driving the output was solved, and thus the output was expressed as a function of modulation parameters. It was shown that the output is a multivariate analog modulated signal. In terms of digital modulation, the output of system processing a PAM signal has been interpreted by means of a Markov chain. The results indicated that this process consists of a convolutional coding and multidimensional modulation. In addition, the presence of noise induced coding was found. Finally, the system was designed to obtain a pulse width position modulated (PWPM) output. Throughout the project, detection, filtering, modulation and coding capabilities have been demonstrated, it has been concluded that the double well potential is an sophisticated signal processing tool

Development of Novel Analytical Methods with the Aim of Forensic Analyte Detection using Ultra-Thin Layer Chromatography, Surface Enhanced Raman Spectroscopy, and Magneto-Elastic Wire Sensing

The purpose of this dissertation is to develop analytical methods that aid in the detection of forensic analytes. Forensic analytes require methods with increased sensitivity and low limit of detection capabilities. Improvements in separation techniques, surface enhanced Raman spectroscopic techniques, and wire-less gas sensing can each assist in the detection of trace evidence. When surface enhanced Raman is coupled with thin-layer chromatography a mixture of compounds can be separated and transferred to a metal substrate to be detected using Raman spectroscopy. Surface enhanced Raman scattering enhances the Raman signal intensity by placing a metal substrate in close proximity to an analyte. The new method gives a chemically specific intensified signal along with a chromatographic separation. A traditional separation is performed on a TLC plate, allowed to dry, wetted with a solvent, placed in contact with a metal substrate, and detected using Raman. More efficient chromatographic platforms can be implemented with this method. New efficient chromatographic platforms are also beneficial to the detection of forensic analytes. Recently, photolithographically nanofabricated open system pillar arrays have proved to be more efficient separation platforms when compared to traditional TLC. These platforms are a form of ultra-thin layer chromatography. This dissertation describes the effects of manipulation on the inter-pillar gap distances with respect to band dispersion. The studies herein manipulate the pillar arrays in order to optimize the separation platform. The third method developed involved gas sensing of volatile organic compounds. An amorphous ferromagnetic micro-wire was coated with a polymer, where the polymer swelled in response to the gas introduced. When the gas caused the polymer to swell a differential stress response was applied on the micro-wire. The fabricated sensor was tested on simple organic gases but has capabilities to detect low concentrations of low vapor pressure forensic analytes. All three projects were significant advancements in analytical method development. The analytes used were either fluorescent dyes or volatile organic compounds to test feasibility of each method. More efficient chromatographic platforms were fabricated, surface enhanced Raman was coupled to TLC, and a micro-wire gas sensor was calibrated for the studies performed in this dissertation

Label-Free Monitoring of Tumor Models by Surface-Enhanced Raman Scattering

184 p.El objetivo general de la presente tesis se ha centrado en la monitorización de modelos celulares mediante la técnica de espectroscopia de Raman aumentada en superficies (SERS). Las tecnologías desarrolladas en la tesis han perseguido, por un lado, mejorar la recreación del ambiente tumoral a escala de laboratorio, y por otra parte, su integración junto con estructuras plasmónicas para el análisis por SERS de los modelos tumorales creados artificialmente. Más en concreto, se han analizado las alteraciones en la concentración relativa de los metabolitos presentes en el medio extracelular como resultado de la reprogramación metabólica característica de los tumores, la cual permite a su vez un crecimiento descontrolado de dichas células.La disposición conjunta de ambas tecnologías (cultivos celulares en 3D y nanoplasmónica) ofrece un marco único para la identificación de aquellos procesos celulares que se encuentran alterados durante el crecimiento de tumores. Hasta la fecha, la mayoría de las técnicas de laboratorio que se habían empleado para caracterizar ambientes celulares en el laboratorio implicaban procesos invasivos, es decir, quemodifican o incluso desintegraban la muestra para poder analizarla. En contraposición, la espectroscopia Raman había permitido adquirir información sobre la composición del medio celular de una manera mínimamente invasiva. Basada en los fenómenos de dispersión inelástica, la técnica de Raman emplea luz monocromática (generalmente de un láser) para irradiar la muestra bajo análisis, de forma que la interacción entre la muestra y el láser provoca un cambio en la energía de los fotones dispersados, específico de los modos vibraciones de las moléculas irradiadas. Por lo tanto, la luz dispersada y recogida por un detector, permite caracterizar el sistema biológico que ha sido previamente iluminado, sin marcaje previo. Sin embargo, las señales detectadas por dispersión Raman son de manera general muy débiles, por lo que se requiere una intensificación de dichas señales para poder detectar la presencia de metabolitos extracelulares (a bajas concentraciones). En esta tesis se decidió implantar la modalidad conocida como SERS, que hace uso de las propiedades plasmónicas de nanopartículas metálicas (principalmente de oro), las cuales dan lugar a campos eléctricos elevados cuando se iluminan en resonancia con los plasmones superficiales. Como resultado, la señal de Raman de las moléculas adsorbidas sobre dichas superficies metálicas se ve amplificada en varios órdenes de magnitud. Sobre esta base, se han desarrollado en la tesis diferentes plataformas destinadas a combinar sustratos plasmónicos, formados por fijación de nanopartículas de oro sobre estructuras rígidas en 2D, o bien embebidas en redes poliméricas, junto con modelos de células tumorales en crecimiento. La finalidad de la tesis ha sido pues, la monitorización de diferentes procesos celulares en dichos dispositivos mediante SERS, y su posterior interpretación biológica en el ámbito del metabolismo tumoral y la mejora del tratamiento.CICbioGUNE; CICbiomaGUN