Synthetic Biology: A Bridge between Artificial and Natural Cells.

Artificial cells are simple cell-like entities that possess certain properties of natural cells. In general, artificial cells are constructed using three parts: (1) biological membranes that serve as protective barriers, while allowing communication between the cells and the environment; (2) transcription and translation machinery that synthesize proteins based on genetic sequences; and (3) genetic modules that control the dynamics of the whole cell. Artificial cells are minimal and well-defined systems that can be more easily engineered and controlled when compared to natural cells. Artificial cells can be used as biomimetic systems to study and understand natural dynamics of cells with minimal interference from cellular complexity. However, there remain significant gaps between artificial and natural cells. How much information can we encode into artificial cells? What is the minimal number of factors that are necessary to achieve robust functioning of artificial cells? Can artificial cells communicate with their environments efficiently? Can artificial cells replicate, divide or even evolve? Here, we review synthetic biological methods that could shrink the gaps between artificial and natural cells. The closure of these gaps will lead to advancement in synthetic biology, cellular biology and biomedical applications

Chemical communication between synthetic and natural cells: a possible experimental design

The bottom-up construction of synthetic cells is one of the most intriguing and interesting research arenas in synthetic biology. Synthetic cells are built by encapsulating biomolecules inside lipid vesicles (liposomes), allowing the synthesis of one or more functional proteins. Thanks to the in situ synthesized proteins, synthetic cells become able to perform several biomolecular functions, which can be exploited for a large variety of applications. This paves the way to several advanced uses of synthetic cells in basic science and biotechnology, thanks to their versatility, modularity, biocompatibility, and programmability. In the previous WIVACE (2012) we presented the state-of-the-art of semi-synthetic minimal cell (SSMC) technology and introduced, for the first time, the idea of chemical communication between synthetic cells and natural cells. The development of a proper synthetic communication protocol should be seen as a tool for the nascent field of bio/chemical-based Information and Communication Technologies (bio-chem-ICTs) and ultimately aimed at building soft-wet-micro-robots. In this contribution (WIVACE, 2013) we present a blueprint for realizing this project, and show some preliminary experimental results. We firstly discuss how our research goal (based on the natural capabilities of biological systems to manipulate chemical signals) finds a proper place in the current scientific and technological contexts. Then, we shortly comment on the experimental approaches from the viewpoints of (i) synthetic cell construction, and (ii) bioengineering of microorganisms, providing up-to-date results from our laboratory. Finally, we shortly discuss how autopoiesis can be used as a theoretical framework for defining synthetic minimal life, minimal cognition, and as bridge between synthetic biology and artificial intelligence.Comment: In Proceedings Wivace 2013, arXiv:1309.712

Abstract Machines of Systems Biology (Extended Abstract)

Living cells are extremely well-organized autonomous systems, consisting of discrete interacting components. Key to understanding and modelling their behavior is modelling their system organization, which can be described as a collection of distinct but interconnected abstract machines. Biologists have invented a number of notations attempting to describe, abstractly, these abstract machines and the processes that they implement. Systems biology aims to understand how these abstract machines work, separately and together

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

Roadmap on semiconductor-cell biointerfaces.

This roadmap outlines the role semiconductor-based materials play in understanding the complex biophysical dynamics at multiple length scales, as well as the design and implementation of next-generation electronic, optoelectronic, and mechanical devices for biointerfaces. The roadmap emphasizes the advantages of semiconductor building blocks in interfacing, monitoring, and manipulating the activity of biological components, and discusses the possibility of using active semiconductor-cell interfaces for discovering new signaling processes in the biological world

Bio-Computation using Holliday junctions

We present a design for a novel computing machine composed of an artificial arrangement of DNA and proteins. We characterise the computational power of this construction by proving that its prediction problem is P-Complete

Bio-Computation using Holliday junctions

We present a design for a novel computing machine composed of an artificial arrangement of DNA and proteins. We characterise the computational power of this construction by proving that its prediction problem is P-Complete

Permanent genetic memory with >1-byte capacity

Genetic memory enables the recording of information in the DNA of living cells. Memory can record a transient environmental signal or cell state that is then recalled at a later time. Permanent memory is implemented using irreversible recombinases that invert the orientation of a unit of DNA, corresponding to the [0,1] state of a bit. To expand the memory capacity, we have applied bioinformatics to identify 34 phage integrases (and their cognate attB and attP recognition sites), from which we build 11 memory switches that are perfectly orthogonal to each other and the FimE and HbiF bacterial invertases. Using these switches, a memory array is constructed in Escherichia coli that can record 1.375 bytes of information. It is demonstrated that the recombinases can be layered and used to permanently record the transient state of a transcriptional logic gate.United States. Defense Advanced Research Projects Agency (DARPA CLIO N66001-12-C-4016)United States. Defense Advanced Research Projects Agency (DARPA CLIO N66001-12-C-4018)United States. Office of Naval Research. Multidisciplinary University Research Initiative (N00014-13-1-0074)National Institutes of Health (U.S.) (GM095765)National Institute of General Medical Sciences (U.S.) (P50 GM098792)National Science Foundation (U.S.). Synthetic Biology Engineering Research Center (SynBERC EEC0540879)FA9550-11-C-0028American Society for Engineering Education. National Defense Science and Engineering Graduate Fellowship (32 CFR 168a