A domain-level DNA strand displacement reaction enumerator allowing arbitrary non-pseudoknotted secondary structures

Information technologies enable programmers and engineers to design and synthesize systems of startling complexity that nonetheless behave as intended. This mastery of complexity is made possible by a hierarchy of formal abstractions that span from high-level programming languages down to low-level implementation specifications, with rigorous connections between the levels. DNA nanotechnology presents us with a new molecular information technology whose potential has not yet been fully unlocked in this way. Developing an effective hierarchy of abstractions may be critical for increasing the complexity of programmable DNA systems. Here, we build on prior practice to provide a new formalization of ‘domain-level’ representations of DNA strand displacement systems that has a natural connection to nucleic acid biophysics while still being suitable for formal analysis. Enumeration of unimolecular and bimolecular reactions provides a semantics for programmable molecular interactions, with kinetics given by an approximate biophysical model. Reaction condensation provides a tractable simplification of the detailed reactions that respects overall kinetic properties. The applicability and accuracy of the model is evaluated across a wide range of engineered DNA strand displacement systems. Thus, our work can serve as an interface between lower-level DNA models that operate at the nucleotide sequence level, and high-level chemical reaction network models that operate at the level of interactions between abstract species

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

Cell-Free Synthetic Biology Platform for Engineering Synthetic Biological Circuits and Systems

Synthetic biology brings engineering disciplines to create novel biological systems for biomedical and technological applications. The substantial growth of the synthetic biology field in the past decade is poised to transform biotechnology and medicine. To streamline design processes and facilitate debugging of complex synthetic circuits, cell-free synthetic biology approaches has reached broad research communities both in academia and industry. By recapitulating gene expression systems in vitro, cell-free expression systems offer flexibility to explore beyond the confines of living cells and allow networking of synthetic and natural systems. Here, we review the capabilities of the current cell-free platforms, focusing on nucleic acid-based molecular programs and circuit construction. We survey the recent developments including cell-free transcription– translation platforms, DNA nanostructures and circuits, and novel classes of riboregulators. The links to mathematical models and the prospects of cell-free synthetic biology platforms will also be discussed.11Yscopu

Design Of Dna Strand Displacement Based Circuits

DNA is the basic building block of any living organism. DNA is considered a popular candidate for future biological devices and circuits for solving genetic disorders and several other medical problems. With this objective in mind, this research aims at developing novel approaches for the design of DNA based circuits. There are many recent developments in the medical field such as the development of biological nanorobots, SMART drugs, and CRISPR-Cas9 technologies. There is a strong need for circuits that can work with these technologies and devices. DNA is considered a suitable candidate for designing such circuits because of the programmability of the DNA strands, small size, lightweight, known thermodynamics, higher parallelism, and exponentially reducing the cost of synthesizing techniques. The DNA strand displacement operation is useful in developing circuits with DNA strands. The circuit can be either a digital circuit, in which the logic high and logic low states of the DNA strand concentrations are considered as the signal, or it can be an analog circuit in which the concentration of the DNA strands itself will act as the signal. We developed novel approaches in this research for the design of digital, as well as analog circuits keeping in view of the number of DNA strands required for the circuit design. Towards this goal in the digital domain, we developed spatially localized DNA majority logic gates and an inverter logic gate that can be used with the existing seesaw based logic gates. The majority logic gates proposed in this research can considerably reduce the number of strands required in the design. The introduction of the logic inverter operation can translate the dual rail circuit architecture into a monorail architecture for the seesaw based logic circuits. It can also reduce the number of unique strands required for the design into approximately half. The reduction in the number of unique strands will consequently reduce the leakage reactions, circuit complexity, and cost associated with the DNA circuits. The real world biological inputs are analog in nature. If we can use those analog signals directly in the circuits, it can considerably reduce the resources required. Even though analog circuits are highly prone to noise, they are a perfect candidate for performing computations in the resource-limited environments, such as inside the cell. In the analog domain, we are developing a novel fuzzy inference engine using analog circuits such as the minimum gate, maximum gate, and fan-out gates. All the circuits discussed in this research were designed and tested in the Visual DSD software. The biological inputs are inherently fuzzy in nature, hence a fuzzy based system can play a vital role in future decision-making circuits. We hope that our research will be the first step towards realizing these larger goals. The ultimate aim of our research is to develop novel approaches for the design of circuits which can be used with the future biological devices to tackle many medical problems such as genetic disorders

A domain-level DNA strand displacement reaction enumerator allowing arbitrary non-pseudoknotted secondary structures

Information technologies enable programmers and engineers to design and synthesize systems of startling complexity that nonetheless behave as intended. This mastery of complexity is made possible by a hierarchy of formal abstractions that span from high-level programming languages down to low-level implementation specifications, with rigorous connections between the levels. DNA nanotechnology presents us with a new molecular information technology whose potential has not yet been fully unlocked in this way. Developing an effective hierarchy of abstractions may be critical for increasing the complexity of programmable DNA systems. Here, we build on prior practice to provide a new formalization of ‘domain-level’ representations of DNA strand displacement systems that has a natural connection to nucleic acid biophysics while still being suitable for formal analysis. Enumeration of unimolecular and bimolecular reactions provides a semantics for programmable molecular interactions, with kinetics given by an approximate biophysical model. Reaction condensation provides a tractable simplification of the detailed reactions that respects overall kinetic properties. The applicability and accuracy of the model is evaluated across a wide range of engineered DNA strand displacement systems. Thus, our work can serve as an interface between lower-level DNA models that operate at the nucleotide sequence level, and high-level chemical reaction network models that operate at the level of interactions between abstract species

Configurable and Up-Scalable Microfluidic Life Science Platform for Cell Based Assays by Gravity Driven Sequential Perfusion and Diffusion

Microfluidics has the potential to significantly change the way modern biology is performed, but for this potential to be realized several on-chip integration and operation challenges have to be addressed. Critical issues are addressed in this work by first demonstrating an integrated microfluidic tmRNA purification and real time nucleic acid sequence based amplification (NASBA) device. The device is manufactured using soft lithography and a unique silica bead immobilization method for the nucleic acid micro purification column. The integrated device produced a pathogen-specific response in < 3 min from the chip-purified RNA. Further enhancements in the device design and operation that allow the on-chip integration of mammalian cell handling and culturing produced a novel integrated NASBA array. This system demonstrated for the first time that it is possible to combine on a single micro-device cell culture and real time NASBA. In order to expand the cell based assay capabilities of the integrated NASBA array and simplify the device operation novel hydrodynamics and cell sedimentation within trench structures and gravity driven sequential perfusion and diffusion mechanisms were developed. These mechanisms were characterized and implemented within an iCell array device. iCell array can completely integrate cell based assays with bio-analytical read-out. The device is highly scalable and can enable the configurable on-chip integration of procedures such as adherent and non-adherent cell-culture, cellstimulation, cell-lysis, cell-fixing, protein-immunoassays, bright field and fluorescent microscopic monitoring, and real time detection of nucleic acid amplification. The device uses on-board gravity driven flow control which makes it simple and economical to operate with dilute samples (down to 5 cells per reaction), low reagent volumes (50 nL per reaction), highly efficient cell capture (100% capture rates) and single cell protein and gene expression sensitivity. The key results from this work demonstrate a novel technology for versatile, fully integrated microfluidic array platforms. By multiplexing this integrated functionality, the device can be used from routine applications in a biology laboratory to high content screenings