An in vitro biomolecular breadboard for prototyping synthetic biological circuits

Biomolecular circuit engineering is critical for implementing complex functions in vivo, and is a baseline method in the synthetic biology space. However, current methods for conducting biomolecular circuit engineering are time-consuming and tedious. A complete design-build-test cycle typically takes weeks' to months' time due to the lack of an intermediary between design ex vivo and testing in vivo. In this work, we explore the development and application of a "biomolecular breadboard" composed of an in-vitro transcription-translation (TX-TL) lysate to rapidly speed up the engineering design-build-test cycle. We first developed protocols for creating and using lysates for conducting biological circuit design. By doing so we simplified the existing technology to an affordable ($0.03/uL) and easy to use three-tube reagent system. We then developed tools to accelerate circuit design by allowing for linear DNA use in lieu of plasmid DNA, and by utilizing principles of modular assembly. This allowed the design-build-test cycle to be reduced to under a business day. We then characterized protein degradation dynamics in the breadboard to aid to implementing complex circuits. Finally, we demonstrated that the breadboard could be applied to engineer complex synthetic circuits in vitro and in vivo. Specifically, we utilized our understanding of linear DNA prototyping, modular assembly, and protein degradation dynamics to characterize the repressilator oscillator and to prototype novel three- and five-node negative feedback oscillators both in vitro and in vivo. We therefore believe the biomolecular breadboard has wide application for acting as an intermediary for biological circuit engineering.

Structure-function studies of serine hydrolases: synthesis of a gene for ∝-lytic protease and the purification and characterization of a mutant β-lactamase

The author has constructed a synthetic gene for ∝-lytic protease. Since the DNA sequence of the protein is not known, the gene was designed by using the reverse translation of ∝-lytic protease's amino acid sequence. Unique restriction sites are carefully sought in the degenerate DNA sequence to aid in future mutagenesis studies. The unique restriction sites are designed approximately 50 base pairs apart and their appropriate codons used in the DNA sequence. The codons used to construct the DNA sequence of ∝-lytic protease are preferred codons in E-coli or used in the production of β-lactamase. Codon usage is also distributed evenly to ensure that one particular codon is not heavily used. The gene is essentially constructed from the outside in. The gene is built in a stepwise fashion using plasmids as the vehicles for the ∝-lytic oligomers. The use of plasmids allows the replication and isolation of large quantities of the intermediates during gene synthesis. The ∝-lytic DNA is a double-stranded oligomer that has sufficient overhang and sticky ends to anneal correctly in the vector. After six steps of incorporating ∝-lytic DNA, the gene is completed and sequenced to ensure that the correct DNA sequence is present and that no mutations occurred in the structural gene.

β-lactamase is the other serine hydrolase studied in this thesis. The author used the class A RTEM-1 β- lactamase encoded on the plasmid pBR322 to investigate the roll of the conserved threonine residue at position 71. Cassette mutagenesis was previously used to generate all possible amino acid substitutions at position 71. The work presented here describes the purification and kinetic characterization of a T71H mutant previously constructed by S. Schultz. The mutated gene was transferred into plasmid pJN for expression and induced with IPTG. The enzyme is purified by column chromatography and FPLC to homogeneity. Kinetic studies reveal that the mutant has lower k_(cat) values on benzylpenicillin, cephalothin and 6-aminopenicillanic acid but no changes in k_m except for cephalothin which is approximately 4 times higher. The mutant did not change siginificantly in its pH profile compared to the wild-type enzyme. Also, the mutant is more sensitive to thermal denaturation as compared to the wild-type enzyme. However, experimental evidence indicates that the probable generation of a positive charge at position 71 thermally stabilized the mutant.

Markets and microstructure

This document contains three papers examining the microstructure of financial interaction in development and market settings. I first examine the industrial organization of financial exchanges, specifically limit order markets. In this section, I perform a case study of Google stock surrounding a surprising earnings announcement in the 3rd quarter of 2009, uncovering parameters that describe information flows and liquidity provision. I then explore the disbursement process for community-driven development projects. This section is game theoretic in nature, using a novel three-player ultimatum structure. I finally develop econometric tools to simulate equilibrium and identify equilibrium models in limit order markets.

In chapter two, I estimate an equilibrium model using limit order data, finding parameters that describe information and liquidity preferences for trading. As a case study, I estimate the model for Google stock surrounding an unexpected good-news earnings announcement in the 3rd quarter of 2009. I find a substantial decrease in asymmetric information prior to the earnings announcement. I also simulate counterfactual dealer markets and find empirical evidence that limit order markets perform more efficiently than do their dealer market counterparts.

In chapter three, I examine Community-Driven Development. Community-Driven Development is considered a tool empowering communities to develop their own aid projects. While evidence has been mixed as to the effectiveness of CDD in achieving disbursement to intended beneficiaries, the literature maintains that local elites generally take control of most programs. I present a three player ultimatum game which describes a potential decentralized aid procurement process. Players successively split a dollar in aid money, and the final player--the targeted community member--decides between whistle blowing or not. Despite the elite capture present in my model, I find conditions under which money reaches targeted recipients. My results describe a perverse possibility in the decentralized aid process which could make detection of elite capture more difficult than previously considered. These processes may reconcile recent empirical work claiming effectiveness of the decentralized aid process with case studies which claim otherwise.

In chapter four, I develop in more depth the empirical and computational means to estimate model parameters in the case study in chapter two. I describe the liquidity supplier problem and equilibrium among those suppliers. I then outline the analytical forms for computing certainty-equivalent utilities for the informed trader. Following this, I describe a recursive algorithm which facilitates computing equilibrium in supply curves. Finally, I outline implementation of the Method of Simulated Moments in this context, focusing on Indirect Inference and formulating the pseudo model.

Nonlinear optics and wavelength translation via cavity-optomechanics

The field of cavity-optomechanics explores the interaction of light with sound in an ever increasing array of devices. This interaction allows the mechanical system to be both sensed and controlled by the optical system, opening up a wide variety of experiments including the cooling of the mechanical resonator to its quantum mechanical ground state and the squeezing of the optical field upon interaction with the mechanical resonator, to name two.

In this work we explore two very different systems with different types of optomechanical coupling. The first system consists of two microdisk optical resonators stacked on top of each other and separated by a very small slot. The interaction of the disks causes their optical resonance frequencies to be extremely sensitive to the gap between the disks. By careful control of the gap between the disks, the optomechanical coupling can be made to be quadratic to first order which is uncommon in optomechanical systems. With this quadratic coupling the light field is now sensitive to the energy of the mechanical resonator and can directly control the potential energy trapping the mechanical motion. This ability to directly control the spring constant without modifying the energy of the mechanical system, unlike in linear optomechanical coupling, is explored.

Next, the bulk of this thesis deals with a high mechanical frequency optomechanical crystal which is used to coherently convert photons between different frequencies. This is accomplished via the engineered linear optomechanical coupling in these devices. Both classical and quantum systems utilize the interaction of light and matter across a wide range of energies. These systems are often not naturally compatible with one another and require a means of converting photons of dissimilar wavelengths to combine and exploit their different strengths. Here we theoretically propose and experimentally demonstrate coherent wavelength conversion of optical photons using photon-phonon translation in a cavity-optomechanical system. For an engineered silicon optomechanical crystal nanocavity supporting a 4 GHz localized phonon mode, optical signals in a 1.5 MHz bandwidth are coherently converted over a 11.2 THz frequency span between one cavity mode at wavelength 1460 nm and a second cavity mode at 1545 nm with a 93% internal (2% external) peak efficiency. The thermal and quantum limiting noise involved in the conversion process is also analyzed and, in terms of an equivalent photon number signal level, are found to correspond to an internal noise level of only 6 and 4 times 10x^-3 quanta, respectively.

We begin by developing the requisite theoretical background to describe the system. A significant amount of time is then spent describing the fabrication of these silicon nanobeams, with an emphasis on understanding the specifics and motivation. The experimental demonstration of wavelength conversion is then described and analyzed. It is determined that the method of getting photons into the cavity and collected from the cavity is a fundamental limiting factor in the overall efficiency. Finally, a new coupling scheme is designed, fabricated, and tested that provides a means of coupling greater than 90% of photons into and out of the cavity, addressing one of the largest obstacles with the initial wavelength conversion experiment.

Tools for the study of dynamical spacetimes

This thesis covers a range of topics in numerical and analytical relativity, centered around introducing tools and methodologies for the study of dynamical spacetimes. The scope of the studies is limited to classical (as opposed to quantum) vacuum spacetimes described by Einstein's general theory of relativity. The numerical works presented here are carried out within the Spectral Einstein Code (SpEC) infrastructure, while analytical calculations extensively utilize Wolfram's Mathematica program.

We begin by examining highly dynamical spacetimes such as binary black hole mergers, which can be investigated using numerical simulations. However, there are difficulties in interpreting the output of such simulations. One difficulty stems from the lack of a canonical coordinate system (henceforth referred to as gauge freedom) and tetrad, against which quantities such as Newman-Penrose Psi_4 (usually interpreted as the gravitational wave part of curvature) should be measured. We tackle this problem in Chapter 2 by introducing a set of geometrically motivated coordinates that are independent of the simulation gauge choice, as well as a quasi-Kinnersley tetrad, also invariant under gauge changes in addition to being optimally suited to the task of gravitational wave extraction.

Another difficulty arises from the need to condense the overwhelming amount of data generated by the numerical simulations. In order to extract physical information in a succinct and transparent manner, one may define a version of gravitational field lines and field strength using spatial projections of the Weyl curvature tensor. Introduction, investigation and utilization of these quantities will constitute the main content in Chapters 3 through 6.

For the last two chapters, we turn to the analytical study of a simpler dynamical spacetime, namely a perturbed Kerr black hole. We will introduce in Chapter 7 a new analytical approximation to the quasi-normal mode (QNM) frequencies, and relate various properties of these modes to wave packets traveling on unstable photon orbits around the black hole. In Chapter 8, we study a bifurcation in the QNM spectrum as the spin of the black hole a approaches extremality.

Development of Microfluidic Devices with the Use of Nanotechnology to Aid in the Analysis of Biological Systems Including Membrane Protein Separation, Single Cell Analysis, and Genetic Markers

Computation technology has dramatically changed the world around us; you can hardly find an area where cell phones have not saturated the market, yet there is a significant lack of breakthroughs in the development to integrate the computer with biological environments. This is largely the result of the incompatibility of the materials used in both environments; biological environments and experiments tend to need aqueous environments. To help aid in these development chemists, engineers, physicists and biologists have begun to develop microfluidics to help bridge this divide. Unfortunately, the microfluidic devices required large external support equipment to run the device. This thesis presents a series of several microfluidic methods that can help integrate engineering and biology by exploiting nanotechnology to help push the field of microfluidics back to its intended purpose, small integrated biological and electrical devices. I demonstrate this goal by developing different methods and devices to (1) separate membrane bound proteins with the use of microfluidics, (2) use optical technology to make fiber optic cables into protein sensors, (3) generate new fluidic devices using semiconductor material to manipulate single cells, and (4) develop a new genetic microfluidic based diagnostic assay that works with current PCR methodology to provide faster and cheaper results. All of these methods and systems can be used as components to build a self-contained biomedical device.