Auf Boolescher Logik basierende Assays für die Analyse verschiedener Bacillus cereus Toxine

Bacillus cereus is a Gram-positive and spore-forming bacterium of the Bacillus cereus group, sharing a closely related phylogenetic similarity with other group members such as Bacillus anthracis and Bacillus thuringiensis. Bacillus cereus is responsible for both gastrointestinal and non-gastrointestinal syndromes. Of the former ones, emesis and diarrhea are caused by either the emetic toxin (cereulide) or different enterotoxins mainly the non-haemolytic enterotoxin (Nhe), haemolysin BL (Hbl) and cytotoxin K (CytK). In addition, other toxins and virulence factors have been reported in the past few years, e.g., haemolysin II (HlyII), Certhrax, vegetative insecticidal proteins (VIPs), immune inhibitor A1 (InhA1) and sphingomyelinase (SMase). Since the relative role of the individual toxins and the other factors in disease is still unknown, there is an urgent demand to detect these potential targets for either diagnostic or food safety purposes. In the first part of the thesis, we present an OR gate based on monoclonal antibodies for the simultaneous detection of multiple toxins in a single tube. To further simplify the operating procedure, the Boolean rule of simplification was used to guide the selection of a marker toxin among the natural toxin profiles. Furthermore, we developed a cellular logic circuit for deciphering the toxin profiles produced by B. cereus, using readout techniques based on pore formation on the cell membrane. This new assay enabled the simultaneous detection of seven biomarkers in pathogenic strains from various sources. Lastly, a cellular logic system capable of combinatorial and sequential logic operations based on bacterial protein-triggered cytotoxicity was constructed. Advanced devices such as a keypad lock, half-adder and several basic Boolean properties were demonstrated on the cells. This represents a first example of a Boolean logic-based system for assaying multiple bacterial toxins. In addition, the results suggest that toxins and other virulence factors of bacteria can be used as toolkits for biocomputing.Bacillus cereus ist ein Gram-positives und Sporen bildendes Bakterium aus der Bacillus cereus Gruppe, welche auch die phylogenetisch nah verwandten Bacillus anthracis und Bacillus thuringiensis beinhaltet. B. cereus ist sowohl für gastrointestinale als auch nicht-gastrointestinale Syndrome verantwortlich. In ersterem Fall werden Erbrechen und Diarrhö entweder durch das emetische Toxin (Cereulid) oder durch verschiedene Enterotoxine, hauptsächlich durch das nicht-hämolytische Enterotoxin (Nhe), das Hämolysin BL (Hbl) und das Cytotoxin K (CytK) verursacht. Des Weiteren wurde in den letzten Jahren auch von anderen Toxinen und Virulenz Faktoren berichtet: Hämolysin II (HlyII), Certhrax, die vegetativ expremierten insektiziden Proteine (VIPs), der Immune inhibitor A1 (InhA1) und die Sphingomyelinase (SMase). Da das Zusammenspiel der einzelnen Toxine und der anderen Faktoren im Krankheitsfall noch viele Rätsel aufweist, gibt es einen dringenden Bedarf diese potentiellen Targets zu detektieren – sowohl für die Diagnostik als auch für die Lebensmittelsicherheit. Im ersten Teil dieser Dissertation wird die mathematische Logikfunktion des ODER-Gatters, basierend auf monoklonalen Antikörpern für die simultane Detektion von verschiedenen Toxinen in einem Teströhrchen benutzt. Um den Arbeitsvorgang weiter zu simplifizieren wurde die Boolesche Regel der Vereinfachung angewandt, um ein Marker Toxin aus dem Spektrum der natürlichen Toxine auszuwählen. Desweitern wurde eine zelluläre Schaltkreislogik entwickelt, um die Toxinprofile von B. cereus zu entschlüsseln. Dafür benutzten wir als Testsignal die Porenbildung auf der Zellmembran. Dieser neuartige Assay ermöglicht in pathogenen Stämmen verschiedenen Ursprungs die simultane Detektion von sieben verschiedenen Biomarkern. Zuletzt wurde ein zellulärer Logikschaltkreis, basierend auf durch bakterielle Proteine induzierte Cytotoxizität konstruiert, der zu kombinatorischen und sequentiellen logischen Verknüpfungen befähigt ist. Auch fortgeschrittene Elemente wie ein Tastaturschloss, Halb-Addierer und verschiedene elementare Booleschen Eigenschaften wurden auf zellulärer Ebene demonstriert. Diese Arbeit repräsentiert ein erstes Beispiel für ein auf Boolescher Logik basierendes System unter Benutzung verschiedener bakterieller Toxine. Die Resultate deuten darauf hin, dass bakterielle Toxine und andere Virulenzfaktoren als Bausteine für Bio-Datenverarbeitung genutzt werden können

Auf Boolescher Logik basierende Assays für die Analyse verschiedener Bacillus cereus Toxine

Bacillus cereus is a Gram-positive and spore-forming bacterium of the Bacillus cereus group, sharing a closely related phylogenetic similarity with other group members such as Bacillus anthracis and Bacillus thuringiensis. Bacillus cereus is responsible for both gastrointestinal and non-gastrointestinal syndromes. Of the former ones, emesis and diarrhea are caused by either the emetic toxin (cereulide) or different enterotoxins mainly the non-haemolytic enterotoxin (Nhe), haemolysin BL (Hbl) and cytotoxin K (CytK). In addition, other toxins and virulence factors have been reported in the past few years, e.g., haemolysin II (HlyII), Certhrax, vegetative insecticidal proteins (VIPs), immune inhibitor A1 (InhA1) and sphingomyelinase (SMase). Since the relative role of the individual toxins and the other factors in disease is still unknown, there is an urgent demand to detect these potential targets for either diagnostic or food safety purposes. In the first part of the thesis, we present an OR gate based on monoclonal antibodies for the simultaneous detection of multiple toxins in a single tube. To further simplify the operating procedure, the Boolean rule of simplification was used to guide the selection of a marker toxin among the natural toxin profiles. Furthermore, we developed a cellular logic circuit for deciphering the toxin profiles produced by B. cereus, using readout techniques based on pore formation on the cell membrane. This new assay enabled the simultaneous detection of seven biomarkers in pathogenic strains from various sources. Lastly, a cellular logic system capable of combinatorial and sequential logic operations based on bacterial protein-triggered cytotoxicity was constructed. Advanced devices such as a keypad lock, half-adder and several basic Boolean properties were demonstrated on the cells. This represents a first example of a Boolean logic-based system for assaying multiple bacterial toxins. In addition, the results suggest that toxins and other virulence factors of bacteria can be used as toolkits for biocomputing.Bacillus cereus ist ein Gram-positives und Sporen bildendes Bakterium aus der Bacillus cereus Gruppe, welche auch die phylogenetisch nah verwandten Bacillus anthracis und Bacillus thuringiensis beinhaltet. B. cereus ist sowohl für gastrointestinale als auch nicht-gastrointestinale Syndrome verantwortlich. In ersterem Fall werden Erbrechen und Diarrhö entweder durch das emetische Toxin (Cereulid) oder durch verschiedene Enterotoxine, hauptsächlich durch das nicht-hämolytische Enterotoxin (Nhe), das Hämolysin BL (Hbl) und das Cytotoxin K (CytK) verursacht. Des Weiteren wurde in den letzten Jahren auch von anderen Toxinen und Virulenz Faktoren berichtet: Hämolysin II (HlyII), Certhrax, die vegetativ expremierten insektiziden Proteine (VIPs), der Immune inhibitor A1 (InhA1) und die Sphingomyelinase (SMase). Da das Zusammenspiel der einzelnen Toxine und der anderen Faktoren im Krankheitsfall noch viele Rätsel aufweist, gibt es einen dringenden Bedarf diese potentiellen Targets zu detektieren – sowohl für die Diagnostik als auch für die Lebensmittelsicherheit. Im ersten Teil dieser Dissertation wird die mathematische Logikfunktion des ODER-Gatters, basierend auf monoklonalen Antikörpern für die simultane Detektion von verschiedenen Toxinen in einem Teströhrchen benutzt. Um den Arbeitsvorgang weiter zu simplifizieren wurde die Boolesche Regel der Vereinfachung angewandt, um ein Marker Toxin aus dem Spektrum der natürlichen Toxine auszuwählen. Desweitern wurde eine zelluläre Schaltkreislogik entwickelt, um die Toxinprofile von B. cereus zu entschlüsseln. Dafür benutzten wir als Testsignal die Porenbildung auf der Zellmembran. Dieser neuartige Assay ermöglicht in pathogenen Stämmen verschiedenen Ursprungs die simultane Detektion von sieben verschiedenen Biomarkern. Zuletzt wurde ein zellulärer Logikschaltkreis, basierend auf durch bakterielle Proteine induzierte Cytotoxizität konstruiert, der zu kombinatorischen und sequentiellen logischen Verknüpfungen befähigt ist. Auch fortgeschrittene Elemente wie ein Tastaturschloss, Halb-Addierer und verschiedene elementare Booleschen Eigenschaften wurden auf zellulärer Ebene demonstriert. Diese Arbeit repräsentiert ein erstes Beispiel für ein auf Boolescher Logik basierendes System unter Benutzung verschiedener bakterieller Toxine. Die Resultate deuten darauf hin, dass bakterielle Toxine und andere Virulenzfaktoren als Bausteine für Bio-Datenverarbeitung genutzt werden können

Engineering biocomputers in mammalian cells

Endowing cells with enhanced decision-making capacities is essential for creating smarter therapeutics and for dissecting phenotypes. Implementation of synthetic gene circuits affords a means for enhanced cellular control and genetic processing; however, genetic circuits for mammalian cells often require extensive fine-tuning to perform as intended. Here, a robust, general, and scalable system, called 'Boolean logic and arithmetic through DNA excision' (BLADE) is presented that is used to engineer genetic circuits with multiple inputs and outputs in mammalian cells with minimal optimization. The reliability of BLADE arises from its reliance on site-specific recombinases that regulate genes under the control of a single promoter that integrates circuit signals on a single transcriptional layer. Using BLADE, >100 circuits were tested in human embryonic kidney and Jurkat T cells and a quantitative metric was used to evaluate their performance. The circuits include a 3-input, two-output full adder; a 6-input, one-output Boolean logic look-up table; and circuits that incorporate CRISPR–Cas9 to regulate endogenous genes. Moreover, a large library of over 15 small-molecule, light and temperature-inducible recombinases has been established for fine-tuned control. BLADE enables execution of sophisticated cellular computation in mammalian cells, with applications in cell and tissue engineering

Systematic Transfer of Prokaryotic Sensors and Circuits to Mammalian Cells

Prokaryotic regulatory proteins respond to diverse signals and represent a rich resource for building synthetic sensors and circuits. The TetR family contains >10[superscript 5] members that use a simple mechanism to respond to stimuli and bind distinct DNA operators. We present a platform that enables the transfer of these regulators to mammalian cells, which is demonstrated using human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells. The repressors are modified to include nuclear localization signals (NLS) and responsive promoters are built by incorporating multiple operators. Activators are also constructed by modifying the protein to include a VP16 domain. Together, this approach yields 15 new regulators that demonstrate 19- to 551-fold induction and retain both the low levels of crosstalk in DNA binding specificity observed between the parent regulators in Escherichia coli, as well as their dynamic range of activity. By taking advantage of the DAPG small molecule sensing mediated by the PhlF repressor, we introduce a new inducible system with 50-fold induction and a threshold of 0.9 μM DAPG, which is comparable to the classic Dox-induced TetR system. A set of NOT gates is constructed from the new repressors and their response function quantified. Finally, the Dox- and DAPG- inducible systems and two new activators are used to build a synthetic enhancer (fuzzy AND gate), requiring the coordination of 5 transcription factors organized into two layers. This work introduces a generic approach for the development of mammalian genetic sensors and circuits to populate a toolbox that can be applied to diverse applications from biomanufacturing to living therapeutics.United States. Defense Advanced Research Projects Agency (DARPA-BAA-11-23)National Institutes of Health (U.S.) (P50GM098792)Life Technologies, Inc. (Research Contract A114510)United States. Office of Naval Research. Multidisciplinary University Research Initiative (N00014-13-1-0074)National Institute of General Medical Sciences (U.S.) (Award R01 GM095765

Towards the implementation of distributed systems in synthetic biology

The design and construction of engineered biological systems has made great strides over the last few decades and a growing part of this is the application of mathematical and computational techniques to problems in synthetic biology. The use of distributed systems, in which an overall function is divided across multiple populations of cells, has the potential to increase the complexity of the systems we can build and overcome metabolic limitations. However, constructing biological distributed systems comes with its own set of challenges. In this thesis I present new tools for the design and control of distributed systems in synthetic biology. The first part of this thesis focuses on biological computers. I develop novel design algorithms for distributed digital and analogue computers composed of spatial patterns of communicating bacterial colonies. I prove mathematically that we can program arbitrary digital functions and develop an algorithm for the automated design of optimal spatial circuits. Furthermore, I show that bacterial neural networks can be built using our system and develop efficient design tools to do so. I verify these results using computational simulations. This work shows that we can build distributed biological computers using communicating bacterial colonies and different design tools can be used to program digital and analogue functions. The second part of this thesis utilises a technique from artificial intelligence, reinforcement learning, in first the control and then the understanding of biological systems. First, I show the potential utility of reinforcement learning to control and optimise interacting communities of microbes that produce a biomolecule. Second, I apply reinforcement learning to the design of optimal characterisation experiments within synthetic biology. This work shows that methods utilising reinforcement learning show promise for complex distributed bioprocessing in industry and the design of optimal experiments throughout biology

Modular construction of mammalian gene circuits using TALE transcriptional repressors

An important goal of synthetic biology is the rational design and predictable implementation of synthetic gene circuits using standardized and interchangeable parts. However, engineering of complex circuits in mammalian cells is currently limited by the availability of well-characterized and orthogonal transcriptional repressors. Here, we introduce a library of 26 reversible transcription activator–like effector repressors (TALERs) that bind newly designed hybrid promoters and exert transcriptional repression through steric hindrance of key transcriptional initiation elements. We demonstrate that using the input-output transfer curves of our TALERs enables accurate prediction of the behavior of modularly assembled TALER cascade and switch circuits. We also show that TALER switches using feedback regulation exhibit improved accuracy for microRNA-based HeLa cancer cell classification versus HEK293 cells. Our TALER library is a valuable toolkit for modular engineering of synthetic circuits, enabling programmable manipulation of mammalian cells and helping elucidate design principles of coupled transcriptional and microRNA-mediated post-transcriptional regulation.National Institutes of Health (U.S.) (Grant 5R01CA155320-04)National Institutes of Health (U.S.) (Grant P50GM098792)National Institutes of Health (U.S.) (Grant 1R01CA173712-01

Synthesis of Biological and Mathematical Methods for Gene Network Control

abstract: Synthetic biology is an emerging field which melds genetics, molecular biology, network theory, and mathematical systems to understand, build, and predict gene network behavior. As an engineering discipline, developing a mathematical understanding of the genetic circuits being studied is of fundamental importance. In this dissertation, mathematical concepts for understanding, predicting, and controlling gene transcriptional networks are presented and applied to two synthetic gene network contexts. First, this engineering approach is used to improve the function of the guide ribonucleic acid (gRNA)-targeted, dCas9-regulated transcriptional cascades through analysis and targeted modification of the RNA transcript. In so doing, a fluorescent guide RNA (fgRNA) is developed to more clearly observe gRNA dynamics and aid design. It is shown that through careful optimization, RNA Polymerase II (Pol II) driven gRNA transcripts can be strong enough to exhibit measurable cascading behavior, previously only shown in RNA Polymerase III (Pol III) circuits. Second, inherent gene expression noise is used to achieve precise fractional differentiation of a population. Mathematical methods are employed to predict and understand the observed behavior, and metrics for analyzing and quantifying similar differentiation kinetics are presented. Through careful mathematical analysis and simulation, coupled with experimental data, two methods for achieving ratio control are presented, with the optimal schema for any application being dependent on the noisiness of the system under study. Together, these studies push the boundaries of gene network control, with potential applications in stem cell differentiation, therapeutics, and bio-production.Dissertation/ThesisDoctoral Dissertation Biomedical Engineering 201

Sensing and Regulation from Nucleic Acid Devices

abstract: The highly predictable structural and thermodynamic behavior of deoxynucleic acid (DNA) and ribonucleic acid (RNA) have made them versatile tools for creating artificial nanostructures over broad range. Moreover, DNA and RNA are able to interact with biological ligand as either synthetic aptamers or natural components, conferring direct biological functions to the nucleic acid devices. The applications of nucleic acids greatly relies on the bio-reactivity and specificity when applied to highly complexed biological systems. This dissertation aims to 1) develop new strategy to identify high affinity nucleic acid aptamers against biological ligand; and 2) explore highly orthogonal RNA riboregulators in vivo for constructing multi-input gene circuits with NOT logic. With the aid of a DNA nanoscaffold, pairs of hetero-bivalent aptamers for human alpha thrombin were identified with ultra-high binding affinity in femtomolar range with displaying potent biological modulations for the enzyme activity. The newly identified bivalent aptamers enriched the aptamer tool box for future therapeutic applications in hemostasis, and also the strategy can be potentially developed for other target molecules. Secondly, by employing a three-way junction structure in the riboregulator structure through de-novo design, we identified a family of high-performance RNA-sensing translational repressors that down-regulates gene translation in response to cognate RNAs with remarkable dynamic range and orthogonality. Harnessing the 3WJ repressors as modular parts, we integrate them into biological circuits that execute universal NAND and NOR logic with up to four independent RNA inputs in Escherichia coli.Dissertation/ThesisDoctoral Dissertation Biochemistry 201

Programming microbes to treat superbug infection

Superbug infection is one of the greatest public health threat with grave implications across all levels of society. Towards a new solution to combat infection by multi-drug resistant bacteria, this thesis presents an engineering framework and genetic tools applied to repurpose commensal bacteria into “micro-robots” for the treatment of superbug infection. Specifically, a prototype of designer probiotic was developed using the human commensal bacteria Escherichia coli. The engineered commensal was reprogrammed with user-specified functions to sense superbug, produced pathogen-specific killing molecules and released the killing molecules via a lytic mechanism. The engineered commensal was effective in suppressing ~99% of planktonic Pseudomonas and preventing ~ 90% of biofilm formation. To enhance the sensing capabilities of engineered commensal, genetic interfaces comprising orthogonal AND & OR logic devices were developed to mediate the integration and interpretation of binary input signals. Finally, AND, OR and NOT logic gates were networked to generate a myriad of cellular logic operations including half adder and half subtractor. The creation of half adder logic represents a significant advancement of engineering human commensal to be biological equivalent of microprocessor chips in programmable computer with the ability to process input signals into diversified actions. Importantly, this thesis provides exemplary case studies to the attenuation of cellular and genetic context dependent effects through principles elucidated herein, thereby advancing our capability to engineer commensal bacteria.Open Acces

Design and implementation of a mammalian synthetic gene oscillator

The core goal of synthetic biology as a discipline is to design, develop and characterize biological parts in order to precisely control cellular behaviour. Much of the research in this field has been focused on the development of gene regulatory networks, namely switches and oscillators. The study of synthetic gene oscillators has attracted significant attention in the past decade due to their intriguing dynamics and relevance in controlling inflammatory, metabolic and circadian signalling pathways. Additionally, the precise expression dynamics and molecular mechanisms that underlie the mammalian circadian clock structure are not fully understood. The work presented herein regards the design and implementation of a tuneable mammalian synthetic gene oscillator with a novel biological structure. To this end, an approach based on a combination of in silico design and in vivo part validation, in conjunction with a comparative analysis of previously implemented synthetic gene oscillators, was taken when assembling the proposed system. The topology of the system relies on a delayed negative feedback loop, consisting of the coupled regulatory activities of the transcription regulators LacI, tTA, and Gal4. The numerical solution and stability analysis of an ODE-based model describing the dynamics of the system are indicative that the proposed system is capable of generating sustained oscillations across a wide range of parameter values. The biological parts that comprise the system have been monitored and validated in HEK293T cells through time-lapse fluorescence microscopy and image analysis. The in vivo performance of the proposed mammalian synthetic gene oscillator was also assessed in the HEK293T cell line, and monitored using time-lapse fluorescence microscopy. Damped fluorescence oscillations were observed: these could be tuned by a differential IPTG concentration gradient and abolished by doxycycline. The proposed mammalian synthetic gene oscillator provides valuable insight into the gene expression regulatory processes leading to oscillatory behaviour, and has the potential to foster progress in future synthetic biology-based therapies.Open Acces