Reduced and coded sensing methods for x-ray based security

Current x-ray technologies provide security personnel with non-invasive sub-surface imaging and contraband detection in various portal screening applications such as checked and carry-on baggage as well as cargo. Computed tomography (CT) scanners generate detailed 3D imagery in checked bags; however, these scanners often require significant power, cost, and space. These tomography machines are impractical for many applications where space and power are often limited such as checkpoint areas. Reducing the amount of data acquired would help reduce the physical demands of these systems. Unfortunately this leads to the formation of artifacts in various applications, thus presenting significant challenges in reconstruction and classification. As a result, the goal is to maintain a certain level of image quality but reduce the amount of data gathered. For the security domain this would allow for faster and cheaper screening in existing systems or allow for previously infeasible screening options due to other operational constraints. While our focus is predominantly on security applications, many of the techniques can be extended to other fields such as the medical domain where a reduction of dose can allow for safer and more frequent examinations. This dissertation aims to advance data reduction algorithms for security motivated x-ray imaging in three main areas: (i) development of a sensing aware dimensionality reduction framework, (ii) creation of linear motion tomographic method of object scanning and associated reconstruction algorithms for carry-on baggage screening, and (iii) the application of coded aperture techniques to improve and extend imaging performance of nuclear resonance fluorescence in cargo screening. The sensing aware dimensionality reduction framework extends existing dimensionality reduction methods to include knowledge of an underlying sensing mechanism of a latent variable. This method provides an improved classification rate over classical methods on both a synthetic case and a popular face classification dataset. The linear tomographic method is based on non-rotational scanning of baggage moved by a conveyor belt, and can thus be simpler, smaller, and more reliable than existing rotational tomography systems at the expense of more challenging image formation problems that require special model-based methods. The reconstructions for this approach are comparable to existing tomographic systems. Finally our coded aperture extension of existing nuclear resonance fluorescence cargo scanning provides improved observation signal-to-noise ratios. We analyze, discuss, and demonstrate the strengths and challenges of using coded aperture techniques in this application and provide guidance on regimes where these methods can yield gains over conventional methods

Development of prokaryotic cell-free systems for synthetic biology

Prokaryotic cell-free systems are currently heavily used for the production of protein that can be otherwise challenging to produce in cells. However, historically cell-free systems were used to explore natural phenomena before the advent of genetic modification and transformation technology. Recently, synthetic biology has seen a resurgence of this historical use of cell-free systems as a prototyping tool of synthetic and natural genetic circuits. For these cell-free systems to be effective prototyping tools, an understanding of cell-free system mechanics must be established that is not purely protein-expression driven. Here we discuss the development of E. coli-based cell-free systems, with an emphasis on documenting published extract and energy preparation methods into a uniform format. We also discuss additional considerations when applying cell-free systems to synthetic biology

Rapid cell-free forward engineering of novel genetic ring oscillators

While complex dynamic biological networks control gene expression in all living organisms, the forward engineering of comparable synthetic networks remains challenging. The current paradigm of characterizing synthetic networks in cells results in lengthy design-build-test cycles, minimal data collection, and poor quantitative characterization. Cell-free systems are appealing alternative environments, but it remains questionable whether biological networks behave similarly in cell-free systems and in cells. We characterized in a cell-free system the 'repressilator,' a three-node synthetic oscillator. We then engineered novel three, four, and five-gene ring architectures, from characterization of circuit components to rapid analysis of complete networks. When implemented in cells, our novel 3-node networks produced population-wide oscillations and 95% of 5-node oscillator cells oscillated for up to 72 hours. Oscillation periods in cells matched the cell-free system results for all networks tested. An alternate forward engineering paradigm using cell-free systems can thus accurately capture cellular behavior

Linear DNA for Rapid Prototyping of Synthetic Biological Circuits in an Escherichia coli Based TX-TL Cell-Free System

Accelerating the pace of synthetic biology experiments requires new approaches for rapid prototyping of circuits from individual DNA regulatory elements. However, current testing standards require days to weeks due to cloning and in vivo transformation. In this work, we first characterized methods to protect linear DNA strands from exonuclease degradation in an Escherichia coli based transcription-translation cell-free system (TX-TL), as well as mechanisms of degradation. This enabled the use of linear DNA PCR products in TX-TL. We then compared expression levels and binding dynamics of different promoters on linear DNA and plasmid DNA. We also demonstrated assembly technology to rapidly build circuits entirely in vitro from separate parts. Using this strategy, we prototyped a four component genetic switch in under 8 h entirely in vitro. Rapid in vitro assembly has future applications for prototyping multiple component circuits if combined with predictive computational models

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform. This is useful for expressing proteins and genetic circuits in a controlled manner as well as for providing a prototyping environment for synthetic biology. To achieve the latter goal, cell-free expression systems that preserve endogenous Escherichia coli transcription-translation mechanisms are able to more accurately reflect in vivo cellular dynamics than those based on T7 RNA polymerase transcription. We describe the preparation and execution of an efficient endogenous E. coli based transcription-translation (TX-TL) cell-free expression system that can produce equivalent amounts of protein as T7-based systems at a 98% cost reduction to similar commercial systems. The preparation of buffers and crude cell extract are described, as well as the execution of a three tube TX-TL reaction. The entire protocol takes five days to prepare and yields enough material for up to 3000 single reactions in one preparation. Once prepared, each reaction takes under 8 hr from setup to data collection and analysis. Mechanisms of regulation and transcription exogenous to E. coli, such as lac/tet repressors and T7 RNA polymerase, can be supplemented.6 Endogenous properties, such as mRNA and DNA degradation rates, can also be adjusted. The TX-TL cell-free expression system has been demonstrated for large-scale circuit assembly, exploring biological phenomena, and expression of proteins under both T7- and endogenous promoters. Accompanying mathematical models are available. The resulting system has unique applications in synthetic biology as a prototyping environment, or "TX-TL biomolecular breadboard.

Rapidly Characterizing the Fast Dynamics of RNA Genetic Circuitry with Cell-Free Transcription Translation (TX-TL) Systems

RNA regulators are emerging as powerful tools to engineer synthetic genetic networks or rewire existing ones. A potential strength of RNA networks is that they may be able to propagate signals on time scales that are set by the fast degradation rates of RNAs. However, a current bottleneck to verifying this potential is the slow design-build-test cycle of evaluating these networks in vivo. Here, we adapt an Escherichia coli-based cell-free transcription-translation (TX-TL) system for rapidly prototyping RNA networks. We used this system to measure the response time of an RNA transcription cascade to be approximately five minutes per step of the cascade. We also show that this response time can be adjusted with temperature and regulator threshold tuning. Finally, we use TX-TL to prototype a new RNA network, an RNA single input module, and show that this network temporally stages the expression of two genes in vivo

Characterizing and prototyping genetic networks with cell-free transcription–translation reactions

A central goal of synthetic biology is to engineer cellular behavior by engineering synthetic gene networks for a variety of biotechnology and medical applications. The process of engineering gene networks often involves an iterative ‘design–build–test’ cycle, whereby the parts and connections that make up the network are built, characterized and varied until the desired network function is reached. Many advances have been made in the design and build portions of this cycle. However, the slow process of in vivo characterization of network function often limits the timescale of the testing step. Cell-free transcription–translation (TX–TL) systems offer a simple and fast alternative to performing these characterizations in cells. Here we provide an overview of a cell-free TX–TL system that utilizes the native Escherichia coli TX–TL machinery, thereby allowing a large repertoire of parts and networks to be characterized. As a way to demonstrate the utility of cell-free TX–TL, we illustrate the characterization of two genetic networks: an RNA transcriptional cascade and a protein regulated incoherent feed-forward loop. We also provide guidelines for designing TX–TL experiments to characterize new genetic networks. We end with a discussion of current and emerging applications of cell free systems