Expanding the Toolkit for Synthetic Biology: Frameworks for Native-like Non-natural Gene Circuits

Synthetic biology combines biological parts from different sources in order to engineer non-native, functional systems. While there is a lot of potential for synthetic biology to revolutionize processes, such as the production of pharmaceuticals, engineering synthetic systems has been challenging. It is oftentimes necessary to explore a large design space to balance the levels of interacting components in the circuit. There are also times where it is desirable to incorporate enzymes that have non-biological functions into a synthetic circuit. Tuning the levels of different components, however, is often restricted to a fixed operating point, and this makes synthetic systems sensitive to changes in the environment. Natural systems are able to respond dynamically to a changing environment by obtaining information relevant to the function of the circuit. This work addresses these problems by establishing frameworks and mechanisms that allow synthetic circuits to communicate with the environment, maintain fixed ratios between components, and potentially add new parts that are outside the realm of current biological function. These frameworks provide a way for synthetic circuits to behave more like natural circuits by enabling a dynamic response, and provide a systematic and rational way to search design space to an experimentally tractable size where likely solutions exist. We hope that the contributions described below will aid in allowing synthetic biology to realize its potential.

光纤光栅调谐技术的进展与应用

阐述了光纤光栅调谐技术近年来发展的情况，简要地说明了光纤光栅调谐的基本原理，并对其在通信和传感领域的应用作了详细的介绍。

Programming chemical kinetics: engineering dynamic reaction networks with DNA strand displacement

Over the last century, the silicon revolution has enabled us to build faster, smaller and more sophisticated computers. Today, these computers control phones, cars, satellites, assembly lines, and other electromechanical devices. Just as electrical wiring controls electromechanical devices, living organisms employ "chemical wiring" to make decisions about their environment and control physical processes. Currently, the big difference between these two substrates is that while we have the abstractions, design principles, verification and fabrication techniques in place for programming with silicon, we have no comparable understanding or expertise for programming chemistry.

In this thesis we take a small step towards the goal of learning how to systematically engineer prescribed non-equilibrium dynamical behaviors in chemical systems. We use the formalism of chemical reaction networks (CRNs), combined with mass-action kinetics, as our programming language for specifying dynamical behaviors. Leveraging the tools of nucleic acid nanotechnology (introduced in Chapter 1), we employ synthetic DNA molecules as our molecular architecture and toehold-mediated DNA strand displacement as our reaction primitive.

Abstraction, modular design and systematic fabrication can work only with well-understood and quantitatively characterized tools. Therefore, we embark on a detailed study of the "device physics" of DNA strand displacement (Chapter 2). We present a unified view of strand displacement biophysics and kinetics by studying the process at multiple levels of detail, using an intuitive model of a random walk on a 1-dimensional energy landscape, a secondary structure kinetics model with single base-pair steps, and a coarse-grained molecular model that incorporates three-dimensional geometric and steric effects. Further, we experimentally investigate the thermodynamics of three-way branch migration. Our findings are consistent with previously measured or inferred rates for hybridization, fraying, and branch migration, and provide a biophysical explanation of strand displacement kinetics. Our work paves the way for accurate modeling of strand displacement cascades, which would facilitate the simulation and construction of more complex molecular systems.

In Chapters 3 and 4, we identify and overcome the crucial experimental challenges involved in using our general DNA-based technology for engineering dynamical behaviors in the test tube. In this process, we identify important design rules that inform our choice of molecular motifs and our algorithms for designing and verifying DNA sequences for our molecular implementation. We also develop flexible molecular strategies for "tuning" our reaction rates and stoichiometries in order to compensate for unavoidable non-idealities in the molecular implementation, such as imperfectly synthesized molecules and spurious "leak" pathways that compete with desired pathways.

We successfully implement three distinct autocatalytic reactions, which we then combine into a de novo chemical oscillator. Unlike biological networks, which use sophisticated evolved molecules (like proteins) to realize such behavior, our test tube realization is the first to demonstrate that Watson-Crick base pairing interactions alone suffice for oscillatory dynamics. Since our design pipeline is general and applicable to any CRN, our experimental demonstration of a de novo chemical oscillator could enable the systematic construction of CRNs with other dynamic behaviors.

一种新型光纤光栅无啁啾调谐方法

分析了基于弹性梁的光纤光栅调谐原理，引入了轴向直变传递系数的概念，提出利用部分纯弯曲调谐方法，可实现光纤光栅宽带无啁嗽调谐。在实验中，利用部分纯弯曲调谐法，获得了20．1nm的光纤光栅无啁嗽调谐范围，调谐线性拟合度为0．9996，调谐过程中光栅反射谱的3dB带宽变化小于0．07nm，峰值反射率变化小于0．2dB，基本无啁嗽现象产生，实验结果和理论分析一致。

一种闭环控制的光纤光栅动态色散补偿仪

利用啁啾光纤光栅的应变调谐特性，将色散补偿的啁啾光纤光栅斜贴于悬臂梁的侧面，通过应力实现啁啾量调整而改变其色散补偿量的大小，同时利用固定在同一悬臂梁的均匀布拉格光栅传感器，实现了闭环自动控制色散补偿量，研制出了一种新型的光纤光栅动态色散补偿仪。该色散补偿仪工作在1550nm波段，典型性能数据为：色散动态补偿范围-1000-1680ps／nm，插入损耗小于1．5dB，动态调谐步进响应时间小于50ms，基本上能满足10Gb／s光纤通信系统中色散动态补偿的要求。

A Multiwavelength Study of the Intracluster Medium and the Characterization of the Multiwavelength Sub/millimeter Inductance Camera

The first part of this thesis combines Bolocam observations of the thermal Sunyaev-Zel’dovich (SZ) effect at 140 GHz with X-ray observations from Chandra, strong lensing data from the Hubble Space Telescope (HST), and weak lensing data from HST and Subaru to constrain parametric models for the distribution of dark and baryonic matter in a sample of six massive, dynamically relaxed galaxy clusters. For five of the six clusters, the full multiwavelength dataset is well described by a relatively simple model that assumes spherical symmetry, hydrostatic equilibrium, and entirely thermal pressure support. The multiwavelength analysis yields considerably better constraints on the total mass and concentration compared to analysis of any one dataset individually. The subsample of five galaxy clusters is used to place an upper limit on the fraction of pressure support in the intracluster medium (ICM) due to nonthermal processes, such as turbulent and bulk flow of the gas. We constrain the nonthermal pressure fraction at r500c to be less than 0.11 at 95% confidence, where r500c refers to radius at which the average enclosed density is 500 times the critical density of the Universe. This is in tension with state-of-the-art hydrodynamical simulations, which predict a nonthermal pressure fraction of approximately 0.25 at r500c for the clusters in this sample.

The second part of this thesis focuses on the characterization of the Multiwavelength Sub/millimeter Inductance Camera (MUSIC), a photometric imaging camera that was commissioned at the Caltech Submillimeter Observatory (CSO) in 2012. MUSIC is designed to have a 14 arcminute, diffraction-limited field of view populated with 576 spatial pixels that are simultaneously sensitive to four bands at 150, 220, 290, and 350 GHz. It is well-suited for studies of dusty star forming galaxies, galaxy clusters via the SZ Effect, and galactic star formation. MUSIC employs a number of novel detector technologies: broadband phased-arrays of slot dipole antennas for beam formation, on-chip lumped element filters for band definition, and Microwave Kinetic Inductance Detectors (MKIDs) for transduction of incoming light to electric signal. MKIDs are superconducting micro-resonators coupled to a feedline. Incoming light breaks apart Cooper pairs in the superconductor, causing a change in the quality factor and frequency of the resonator. This is read out as amplitude and phase modulation of a microwave probe signal centered on the resonant frequency. By tuning each resonator to a slightly different frequency and sending out a superposition of probe signals, hundreds of detectors can be read out on a single feedline. This natural capability for large scale, frequency domain multiplexing combined with relatively simple fabrication makes MKIDs a promising low temperature detector for future kilopixel sub/millimeter instruments. There is also considerable interest in using MKIDs for optical through near-infrared spectrophotometry due to their fast microsecond response time and modest energy resolution. In order to optimize the MKID design to obtain suitable performance for any particular application, it is critical to have a well-understood physical model for the detectors and the sources of noise to which they are susceptible. MUSIC has collected many hours of on-sky data with over 1000 MKIDs. This work studies the performance of the detectors in the context of one such physical model. Chapter 2 describes the theoretical model for the responsivity and noise of MKIDs. Chapter 3 outlines the set of measurements used to calibrate this model for the MUSIC detectors. Chapter 4 presents the resulting estimates of the spectral response, optical efficiency, and on-sky loading. The measured detector response to Uranus is compared to the calibrated model prediction in order to determine how well the model describes the propagation of signal through the full instrument. Chapter 5 examines the noise present in the detector timestreams during recent science observations. Noise due to fluctuations in atmospheric emission dominate at long timescales (less than 0.5 Hz). Fluctuations in the amplitude and phase of the microwave probe signal due to the readout electronics contribute significant 1/f and drift-type noise at shorter timescales. The atmospheric noise is removed by creating a template for the fluctuations in atmospheric emission from weighted averages of the detector timestreams. The electronics noise is removed by using probe signals centered off-resonance to construct templates for the amplitude and phase fluctuations. The algorithms that perform the atmospheric and electronic noise removal are described. After removal, we find good agreement between the observed residual noise and our expectation for intrinsic detector noise over a significant fraction of the signal bandwidth.

Firing Patterns of Cerebellar Purkinje Cells During Locomotion and Sleep

The cerebellum is a major supraspinal center involved in the coordination of movement. The principal neurons of the cerebellar cortex, Purkinje cells, receive excitatory synaptic input from two sources: the parallel and climbing fibers. These pathways have markedly different effects: the parallel fibers control the rate of simple sodium spikes, while the climbing fibers induce characteristic complex spike bursts, which are accompanied by dendritic calcium transients and play a key role in regulating synaptic plasticity. While many studies using a variety of species, behaviors, and cerebellar regions have documented modulation in Purkinje cell activity during movement, few have attempted to record from these neurons in unrestrained rodents. In this dissertation, we use chronic, multi-tetrode recording in freely-behaving rats to study simple and complex spike firing patterns during locomotion and sleep. Purkinje cells discharge rhythmically during stepping, but this activity is highly variable across steps. We show that behavioral variables systematically influence the step-locked firing rate in a step-phase-dependent way, revealing a functional clustering of Purkinje cells. Furthermore, we find a pronounced disassociation between patterns of variability driven by the parallel and climbing fibers, as well as functional differences between cerebellar lobules. These results suggest that Purkinje cell activity not only represents step phase within each cycle, but is also shaped by behavior across steps, facilitating control of movement under dynamic conditions. During sleep, we observe an attenuation of both simple and complex spiking, relative to awake behavior. Although firing rates during slow wave sleep (SWS) and rapid eye movement sleep (REM) are similar, simple spike activity is highly regular in SWS, while REM is characterized by phasic increases and pauses in simple spiking. This phasic activity in REM is associated with pontine waves, which propagate into the cerebellar cortex and modulate both simple and complex spiking. Such a temporal coincidence between parallel and climbing fiber activity is known to drive plasticity at parallel fiber synapses; consequently, pontocerebellar waves may provide a mechanism for tuning synaptic weights in the cerebellum during active sleep.

The Physiology and Computation of Pyramidal Neurons

A variety of neural signals have been measured as correlates to consciousness. In particular, late current sinks in layer 1, distributed activity across the cortex, and feedback processing have all been implicated. What are the physiological underpinnings of these signals? What computational role do they play in the brain? Why do they correlate to consciousness? This thesis begins to answer these questions by focusing on the pyramidal neuron. As the primary communicator of long-range feedforward and feedback signals in the cortex, the pyramidal neuron is set up to play an important role in establishing distributed representations. Additionally, the dendritic extent, reaching layer 1, is well situated to receive feedback inputs and contribute to current sinks in the upper layers. An investigation of pyramidal neuron physiology is therefore necessary to understand how the brain creates, and potentially uses, the neural correlates of consciousness. An important part of this thesis will be in establishing the computational role that dendritic physiology plays. In order to do this, a combined experimental and modeling approach is used.

This thesis beings with single-cell experiments in layer 5 and layer 2/3 pyramidal neurons. In both cases, dendritic nonlinearities are characterized and found to be integral regulators of neural output. Particular attention is paid to calcium spikes and NMDA spikes, which both exist in the apical dendrites, considerable distances from the spike initiation zone. These experiments are then used to create detailed multicompartmental models. These models are used to test hypothesis regarding spatial distribution of membrane channels, to quantify the effects of certain experimental manipulations, and to establish the computational properties of the single cell. We find that the pyramidal neuron physiology can carry out a coincidence detection mechanism. Further abstraction of these models reveals potential mechanisms for spike time control, frequency modulation, and tuning. Finally, a set of experiments are carried out to establish the effect of long-range feedback inputs onto the pyramidal neuron. A final discussion then explores a potential way in which the physiology of pyramidal neurons can establish distributed representations, and contribute to consciousness.

Optomechanical Inertial Sensors and Feedback Cooling

The optomechanical interaction is an extremely powerful tool with which to measure mechanical motion. The displacement resolution of chip-scale optomechanical systems has been measured on the order of 1⁄10th of a proton radius. So strong is this optomechanical interaction that it has recently been used to remove almost all thermal noise from a mechanical resonator and observe its quantum ground-state of motion starting from cryogenic temperatures.

In this work, chapter 1 describes the basic physics of the canonical optomechanical system, optical measurement techniques, and how the optomechanical interaction affects the coupled mechanical resonator. In chapter 2, we describe our techniques for realizing this canonical optomechanical system in a chip-scale form factor.

In chapter 3, we describe an experiment where we used radiation pressure feedback to cool a mesoscopic mechanical resonator near its quantum ground-state from room-temperature. We cooled the resonator from a room temperature phonon occupation of <n> = 6.5 million to an occupation of <n> = 66, which means the resonator is in its ground state approximately 2% of the time, while being coupled to a room-temperature thermal environment. At the time of this work, this is the closest a mesoscopic mechanical resonator has been to its ground-state of motion at room temperature, and this work begins to open the door to room-temperature quantum control of mechanical objects.

Chapter 4 begins with the realization that the displacement resolutions achieved by optomechanical systems can surpass those of conventional MEMS sensors by an order of magnitude or more. This provides the motivation to develop and calibrate an optomechanical accelerometer with a resolution of approximately 10 micro-g/rt-Hz over a bandwidth of approximately 30 kHz. In chapter 5, we improve upon the performance and practicality of this sensor by greatly increasing the test mass size, investigating and reducing low-frequency noise, and incorporating more robust optical coupling techniques and capacitive wavelength tuning. Finally, in chapter 6 we present our progress towards developing another optomechanical inertial sensor - a gyroscope.

On a fiber grating sensor system with the capacity of cross-sensitivity discrimination

A novel fiber Bragg grating (FBG) sensor system based on an interrogating technique by two parallel matched gratings was designed and theoretically discussed. With an interrogation grating playing the role of temperature compensation grating simultaneously, the wavelength drifts induced by temperature and strain were discriminated. Additionally, the expressions of temperature and strain were deduced for our solution, and dual-value problem and cross sensitivity were solved synchronously through data processing. The influence of the FBG's parameters on the dynamic range and precision was discussed. Besides, the change of environment temperature cannot influence the dynamic range of the sensor system through temperature tuning. The system proposed in this paper will be of great significance to accelerate the real engineering applications of FBG sensing techniques. (c) 2007 Elsevier GmbH. All rights reserved.

The ionic basis of frequency selectivity in hair cells of the bullfrog's sacculus

Hair cells from the bull frog's sacculus, a vestibular organ responding to substrate-borne vibration, possess electrically resonant membrane properties which maximize the sensitivity of each cell to a particular frequency of mechanical input. The electrical resonance of these cells and its underlying ionic basis were studied by applying gigohm-seal recording techniques to solitary hair cells enzymatically dissociated from the sacculus. The contribution of electrical resonance to frequency selectivity was assessed from microelectrode recordings from hair cells in an excised preparation of the sacculus.

Electrical resonance in the hair cell is demonstrated by damped membrane-potential oscillations in response to extrinsic current pulses applied through the recording pipette. This response is analyzed as that of a damped harmonic oscillator. Oscillation frequency rises with membrane depolarization, from 80-160 Hz at resting potential to asymptotic values of 200-250 Hz. The sharpness of electrical tuning, denoted by the electrical quality factor, Q_e, is a bell-shaped function of membrane voltage, reaching a maximum value around eight at a membrane potential slightly positive to the resting potential.

In whole cells, three time-variant ionic currents are activated at voltages more positive than -60 to -50 mV; these are identified as a voltage-dependent, non-inactivating Ca current (I_ca), a voltage-dependent, transient K current (I_a), and a Ca-dependent K current (I_c). The C channel is identified in excised, inside-out membrane patches on the basis of its large conductance (130-200 pS), its selective permeability to Kover Na or Cl, and its activation by internal Ca ions and membrane depolarization. Analysis of open- and closed-lifetime distributions suggests that the C channel can assume at least two open and three closed kinetic states.

Exposing hair cells to external solutions that inhibit the Ca or C conductances degrades the electrical resonance properties measured under current-clamp conditions, while blocking the A conductance has no significant effect, providing evidence that only the Ca and C conductances participate in the resonance mechanism. To test the sufficiency of these two conductances to account for electrical resonance, a mathematical model is developed that describes I_ca, I_c, and intracellular Ca concentration during voltage-clamp steps. I_ca activation is approximated by a third-order Hodgkin-Huxley kinetic scheme. Ca entering the cell is assumed to be confined to a small submembrane compartment which contains an excess of Ca buffer; Ca leaves this space with first-order kinetics. The Ca- and voltage-dependent activation of C channels is described by a five-state kinetic scheme suggested by the results of single-channel observations. Parameter values in the model are adjusted to fit the waveforms of I_ca and I_c evoked by a series of voltage-clamp steps in a single cell. Having been thus constrained, the model correctly predicts the character of voltage oscillations produced by current-clamp steps, including the dependencies of oscillation frequency and Q_e on membrane voltage. The model shows quantitatively how the Ca and C conductances interact, via changes in intracellular Ca concentration, to produce electrical resonance in a vertebrate hair cell.

一种利用相位掩模版对光栅波长进行微调的光栅刻写方法

提出并实验证实了一种刻写光栅时既能保护相位版又能对光栅的反射波长进行微调的方法．通过调整光纤和相位版之间的距离，利用1550nm单模光纤和掺铒分别实现了0．48nm和2．2nm的光栅反射波长的调节．在相位版和光纤之间的距离保持在3mm的条件下，既可以保护相位版又可以获得高质量的光栅．

Cr~(2+):ZnSe的激光输出和调谐性能

Cr~(2+):ZnSe具有很宽的吸收带和发射带,是中红外波段优秀的可调谐激光材料。从吸收光谱、发射光谱以及角度调谐输出对Cr~(2+):ZnSe晶体的激光输出性能进行了研究。采用真空高温扩散法制备Cr~(2+):ZnSe晶体.获得了高浓度的Cr~(2+)离子掺杂的厚1.7 mm,直径10 mm的薄片ZnSe晶体。使用中心波长2.05μm,最大输出功率8 W的Tm离子掺杂的光纤激光器抽运,使用平凹腔结构搭建谐振腔,获得了最大平均功率1.034 W,中心波长2.367μm,线宽10 nm的连续激光输出。利用角度调谐的方法,对Cr:ZnSe晶体的调谐性能进行了研究,在100 nm范围内获得了调谐输出。

激光二极管阵列的窄线宽、可调谐输出

外腔反馈的激光二极管阵列(LDA)可获得窄线宽、可调谐的光谱输出。外腔由快轴准直镜、准直光学系统和闪耀光栅组成。由于阵列中各发光单元的排列弯曲导致不同波长的光原路返回,引起谱线展宽,在输出光路中加入光谱滤波器,使激光二极管阵列的线宽进一步窄化。这样,激光二极管阵列的输出光谱由自由运转时的2 nm压缩到0.12 nm,在恒定温度23 ℃时,实现了激光在806～818 nm的调谐,调谐范围达12 nm。

Projeto de redes neurais para determinar os ganhos PID de um sistema de posicionamento dinâmico de uma embarcação.

As sintonias dos Controladores PID existentes em um Sistema de Posicionamento Dinâmico, utilizado em embarcações e plataformas a fim de manter uma posição fixa em alto-mar ou de realizar determinada manobra, sempre tem sido um desafio a ser vencido. Trata-se de uma tarefa demorada, dependente das condições ambientais e com um elevado custo financeiro, uma vez que as horas dedicadas do profissional habilitado são caras. Além disso, a embarcação deve-se manter estabilizada durante o período de tempo no qual determinada função é realizada, como por exemplo, perfuração, abastecimento, ou lançamento de dutos. Foi utilizado um software para simular o posicionamento de uma embarcação em alto-mar sob diversas condições de vento e correnteza, com o qual foi possível verificar a influência da sintonia dos parâmetros PID do Controlador no desempenho do sistema de controle. O Sistema dinâmico abordado possui um comportamento não linear e sujeito a fortes distúrbios não medidos, o que são apenas alguns exemplos de questões avaliadas deste trabalho. Neste contexto, foram projetadas Redes Neurais com o intuito de aprimorar a técnica utilizada para determinar os ganhos de um dos Controladores PID de um Sistema de Posicionamento Dinâmico. Os melhores resultados foram obtidos através da avaliação de desempenho de diversas simulações de Redes Neurais que revelam a viabilidade da implementação da sintonia automática de Controladores em Sistemas de Posicionamento Dinâmico.