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Mechanistic Engineering Strategies to Design and Create Synthetic Cellular Systems & Synthetic Biology Tools
No full textA mechanistic understanding of living cells and the complex interplay between their biochemical and molecular components is a crucial cornerstone in the path towards rational and reproducible bioengineering of cellular and sub cellular systems in synthetic biology. Bioengineering approaches that couple such understanding with traditional synthetic biology tools can be exploited to create reconstituted complex molecular networks, artificial cells, and semi-living cellular systems with the desired traits of engineered biomaterials, but with the adaptability of living cells.The design and creation of such bioengineered systems will need the implementation of a holistic perspective that accounts for and exploits the crosstalk between metabolic networks occurring inside living cells, synthetic circuits, and synthetic materials. In the research presented in this dissertation, I use an integrative approach involving knowledge in a variety of disciplines such as cell, molecular, systems, and synthetic biology to rationally design synthetic systems using a holistic approach. Using this holistic approach, during my doctorate research I was able to create novel strategies to reconstitute metabolic networks in vitro, and biomimetic systems with different synthetic materials and engineered capabilities.
My thesis research is comprised of four different projects, all within the central topic of developing engineering strategies that exploit the mechanistic understanding of bacteria and their modular components to develop new technologies that solve challenges in synthetic biology. The first part of my dissertation focuses on the use of a synthetic microbial consortia approach and a single co-purification step as a new strategy to reconstitute complex metabolic pathways ex vivo. Using this approach, we pioneered the rapid reconstitution of the 34-protein core translation machinery from Escherichia coli.
The next project tackled a persistent problem in cellular engineering, the limited performance of synthetic gene circuits in different chemical contexts outside living cells. My strategy aimed to minimize the context dependency of gene circuits by using artificial cells, which are biological mimics engineered from the bottom-up by encapsulating specific cellular components and gene networks inside a synthetic liposome. The resulting artificial cellular systems are capable of robust gene expression, chemical communication, and detecting and killing bacteria in different chemical contexts.
In the following project, I developed a new approach to tackle the decreased functionality of orthogonal synthetic circuits caused by crosstalk and noise within a cell. Using a holistic approach that integrates systems and synthetic biology concepts, I demonstrated how orthogonal synthetic circuits can be used to reprogram the host proteome and in turn enhance the function of the synthetic orthogonal circuit. I exploited this previously unknown approach to produce a superior cell-free system as a proof of concept aimed to highlight the potential of this new positive feedback loop approach.
The last part of my dissertation research builds on my previous projects and incorporates methodologies from materials science to develop a new class of cell-derived semi-living organism. I developed an approach that uses a physically restrictive method to restrict bacterial growth without compromising other cellular processes. This results in the creation of metabolically active, non-growing, and stress resistant cell chassis that we termed Cyborg Cells with potential applications as living therapeutics, gene delivery vectors, live attenuated vaccines, and biosensors.
Each one of the projects comprising my dissertation research has opened new research avenues in areas such as cancer research, biosensing within bacterial biofilms, and living therapeutics. Also, they have the potential to inspire and become the foundation of new technologies that can help us tackle challenges as complex as the precise engineering of mammalian cells, and the real-time detection & control of disease development
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RoMI: Robotic-microfluidic interface for biomedical analyses
Full text linkIn the past decades, a variety of accurate and reliable analytical technologies have been developed for the quantitative measurements of target analytes (e.g., proteins, metabolites, nucleic acids, unnatured drugs) in biological samples. Nowadays, the new era of personalized medicine and healthcare intelligence is further promoting the development of analytical technologies, especially calling for the replacement of traditional manual analysis with automated and high-throughput machine operations. The emerging field of microfluidics offers a potential solution to miniaturizing and automating traditional analytical methods, where frequently repeated liquid handling operations (e.g., metering, mixing, extraction) can be programmed and processed on a microfluidic device. However, laboratory automation of these delicate devices is hampered by a lack of world-to-chip interfaces and the complexity of external infrastructure settings. In this dissertation, we introduced a universal robotic-fluidic interface (RoMI) and modular microfluidic design for fully automated microfluidics in biomedical applications. Our research projects include: 1) establishing and validating the RoMI platform through a droplet dispensing module; 2) developing a multiplexed strategy for protein network studies in vitro using the RoMI platform and a customized droplet dispensing module; 3) developing a human-free sample-to-answer ELISA system using the RoMI platform and a hybrid microfluidic module. With its modular connectivity, high adaptability, and multitasking capacity, RoMI can be used to drive a variety of microfluidic modules in a simply programmable manner with minimal external infrastructure requirements. Overall, the newly developed RoMI has displayed significant promise in promoting the usability and flexibility of microfluidic technology in biomedical analyses, allowing for new opportunities and insights in the next generations of laboratory automation
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Integrating Synthetic Biology and Microfluidics for High-Throughput Membrane Protein Synthesis
No full textMembrane proteins underlie numerous human pathologies and are critical components of various drug-delivery vehicles, including nanoparticles and exosomes. However, the study and use of these proteins remain limited because of our inability to synthesize them. Current methods rely on brute force synthesis using living cells, which often fail despite relentless optimization. To circumvent the shortcomings of cell-based synthesis, we sought to create a novel high-throughput platform to rapidly synthesize and insert membrane proteins into liposomes using cell-free protein synthesis.We first set out to develop a novel cell-free protein synthesis system that would enhance our ability to produce proteins. Our objective was to bias the resource allocation of the host cells used in the whole-cell extract preparation to prioritize the synthesis of proteins. We took a holistic approach to our engineering efforts, wherein we exploited the natural crosstalk between the metabolic network in the cells and the artificial genetic modules that we designed. Specifically, we show that local modules expressing translation machinery can reprogram the bacterial proteome, changing the expression levels of more than 700 proteins. The resultant feedback generates a cell-free system that can synthesize fluorescent reporters, protein nanocages, and the gene-editing nuclease Cas9, with up to 5-fold higher expression than classical cell-free systems. Our work demonstrates a holistic approach that integrates synthetic and systems biology concepts to achieve outcomes not possible by only local, orthogonal circuits.
Next, we shifted to developing a system to test a wide array of different cell-free reactions. This task requires rapid, ultra-low volume handling operations. To address this, we developed a pipette-free robotic-microfluidic interface using a microfluidic-embedded printing cartridge to achieve seamless integration of nanoliter scale liquid handling and robotic automation. Our microfluidic adaptive printing system incorporates on-deck calibrations and real-time monitoring to achieve nanoliter precision in droplet volumes and reproducible droplet generation across thousands of droplets. The optimized system can assemble unique cell-free reactions across a 384-well plate utilizing more than ten different reagents—equivalent to approximately 7,500 droplets—in under an hour.
Finally, we combine our cell-free protein synthesis system and our droplet printing robot to tackle membrane protein synthesis. Specifically, we first create a high-throughput workflow, combining a novel fluorescent reporter, cell-free protein synthesis, and nanoliter droplet printing, to enable 384-well plate-based synthesis of membrane proteins. Based on the high-throughput platform, we generate the first big data on membrane protein synthesis from over 20,000 different synthesis reactions, covering 28 membrane proteins, and varying lipid types, chemical environments, and chaperone proteins. Using this wealth of data, we implement an active-learning algorithm to synthesize membrane proteins that have not been synthesized in the literature. We also identify structural descriptors of each protein that allow for accurate prediction of a protein’s likelihood to be successfully synthesized. Our new platform and dataset of membrane-protein synthesis overcome a major barrier in the field of membrane proteins, enabling rapid study and design of membrane proteins in future work
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AmbuBox: A Facile Design of Automated Ambulatory Ventilator for Emergency Use
Full text linkRespiratory failure has been a common cause of death in patients infected by SARS-CoV-2 during the time of worldwide ventilator shortage. In early 2020, our team have introduced a fast-deployable and low-cost emergency resuscitator system, AmbuBox, in response to the rising demand of ventilators. The AmbuBox system uses a controllable pneumatic enclosure in conjunction with a standard manual resuscitator (or AmbuBag) to achieve the basic functionalities of a modern ventilator. It is a modularized system integrated with off-the-shelf components for ramp-up production capability at a competitive cost. However, the regulatory requirements for emergency use have not been considered. My project focuses on improving the AmbuBox system in accordance with regulatory consensus and documenting the design process in detail. As part of the initial effort to make AmbuBox publicly available, this project further studies the general process and requirements of FDA Emergency Use Authorization for ventilators. Through this project, I am able to implement key functionalities on the AmbuBox system and understand the expectations from FDA for emergency use approval
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Bistability, Synthetic Biology, and Antibiotic Treatment
No full textBistable switches are commonly observed in the regulation of critical processes such as cell cycles and differentiation. The switches possess two fundamental properties: memory and bimodality. Once switched ON, the switches can remember their ON state despite a drastic drop in stimulus levels. Furthermore, at intermediate stimulus levels with cellular noise, the switches can cause a population to exhibit bimodal distribution of cell states. Till date, experimental studies have focused primarily on cellular mechanisms that generate bistable switches and their impact on cellular dynamics. Here, I study emergent bistability due to bacterial interactions with either synthetic gene circuits or antibiotics. A synthetic gene circuit is often engineered by considering the host cell as an invariable "chassis". Circuit activation, however, may modulate host physiology, which in turn can drastically impact circuit behavior. I illustrate this point by a simple circuit consisting of mutant T7 RNA polymerase (T7 RNAP*) that activates its own expression in bacterium Escherichia coli. Although activation by the T7 RNAP* is noncooperative, the circuit caused bistable gene expression. This counterintuitive observation can be explained by growth retardation caused by circuit activation, which resulted in nonlinear dilution of T7 RNAP* in individual bacteria. Predictions made by models accounting for such effects were verified by further experimental measurements. The results reveal a novel mechanism of generating bistability and underscore the need to account for host physiology modulation when engineering gene circuits.In the context of antibiotic treatment, I investigate bistability as the underlying mechanism of inoculum effect. The inoculum effect refers to the decreasing efficacy of an antibiotic with increasing bacterial density. Despite its implication for the design of antibiotic treatment strategies, its mechanism remains poorly understood. Here I show that, for antibiotics that target the core replication machinery, the inoculum effect can be explained by bistable bacterial growth. My results suggest that a critical requirement for this bistability is sufficiently fast turnover of the core machinery induced by the antibiotic via the heat shock response. I further show that antibiotics that exhibit the inoculum effect can cause a "band-pass" response of bacterial growth on the frequency of antibiotic treatment, whereby the treatment efficacy drastically diminishes at intermediate frequencies. The results have implications on optimal design of antibiotic treatment.</p
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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