Αναλυτική έκθεση υπολογιστικών μοντέλων για την προσομοίωση της συμπεριφοράς μικροβιακών κοινοτήτων που σχηματίζουν βιοϋμένια (Π23-Δ5.1 και Δ5.2)

Περιλαμβάνεται ο ακόλουθος πίνακας: 1. Εισαγωγή 2. Υπάρχουσες προσεγγίσεις (State of the art) 2.1. Υπάρχοντα λογισμικά επεξεργασίας κυτταρικών ταινιών 2.2. Μοντελοποίηση 3. Μεθοδολογία Μοντελοποίησης 4. Τεχνική Προσέγγιση 4.1. Ανάλυση κυτταρικών ταινιών 4.2. Μελέτη μεμονωμένων κυττάρων 4.2.1. Βάση δεδομένων για τα κυτταρικά χαρακτηριστικά 4.2.2. Οπτικοποίηση και μελέτη χαρακτηριστικών μεμονωμένων κυττάρων 4.3. Υπολογιστική μοντελοποίηση 4.3.1. Σενάρια μοντελοποίησης 4.3.2. Αποτελέσματα Μοντελοποίησης 5. Σύνοψη Βιβλιογραφί

LsrR Quorum Sensing ‘‘Switch’ ’ Is Revealed by a Bottom- Up Approach

Quorum sensing (QS) enables bacterial multicellularity and selective advantage for communicating populations. While genetic ‘‘switching’ ’ phenomena are a common feature, their mechanistic underpinnings have remained elusive. The interplay between circuit components and their regulation are intertwined and embedded. Observable phenotypes are complex and context dependent. We employed a combination of experimental work and mathematical models to decipher network connectivity and signal transduction in the autoinducer-2 (AI-2) quorum sensing system of E. coli. Negative and positive feedback mechanisms were examined by separating the network architecture into sub-networks. A new unreported negative feedback interaction was hypothesized and tested via a simple mathematical model. Also, the importance of the LsrR regulator and its determinant role in the E. coli QS ‘‘switch’’, normally masked by interfering regulatory loops, were revealed. Our simple model allowed mechanistic understanding of the interplay among regulatory sub-structures and their contributions to the overall native functioning network. This ‘‘bottom up’ ’ approach in understanding gene regulation will serve to unravel complex QS network architectures and lead to the directe

A MATHEMATICAL MODEL TO STUDY THE ROLE OF THE LSR INTERGENIC REGION IN MEDIATION OF AUTOINDUCER-2 QUORUM SENSING IN ESCHERICHIA COLI

Quorum sensing (QS) is a process that allows bacteria to communicate with each other to coordinate collective behavior in response to changes in environmental conditions. Their ability to mediate biofilm formation of biofilms and antibiotic resistance has created challenges on healthcare systems, and an impetus for us to understand QS systems. QS mediated by autoinducer-2 is likely to be the most common of these mechanisms. Recent work has elaborated on the LuxS-regulated (Lsr) system which can mediate and process AI-2 to QS-dependent behaviors, particularly regulatory elements including the lsr intergenic region and the repressor LsrR, the so-called QS"switch". In this thesis, we present a simulation of an example lsr-QS-system to elucidate the role of the lsr intergenic region binding site interactions and how this model integrates with recent literature on LsrR's protein structure to provide further details on the mechanisms of how the switch may operate in real systems

Bridging the biology-electronics communication gap with redox signaling

Electronic and biological systems both have the ability to sense, respond to, and communicate relevant data. This dissertation aims to facilitate communication between the two and create bio-hybrid devices that can process the breadths of both electronic and biological information. We describe the development of novel methods that bridge this bi-directional communication gap through the use of electronically and biologically relevant redox molecules for controlled and quantitative information transfer. Additionally, we demonstrate that the incorporation of biological components onto microelectronic systems can open doors for improved capabilities in a variety of fields. First, we describe the original use of redox molecules to electronically control the activity of an enzyme on a chip. Using biofabrication techniques, we assembled HLPT, a fusion protein which generates the quorum sensing molecule autoinducer-2, on an electrodeposited chitosan film on top of an electrode. This allows the electrode to controllably oxidize the enzyme in situ through a redox mediator, acetosyringone. We successfully showed that activity decrease and bacterial quorum sensing response are proportional to the input charge. To engineer bio-electronic communication with cells, we first aimed for better characterizing an electronic method for measuring cell response. We engineered Escherichia coli (E.coli) cells to respond to autoinducer-2 by producing the β-galactosidase enzyme. We then investigated an existing electrochemical method for detecting β-galactosidase activity by measuring a redox-active product of the cleavage of the added substrate molecule PAPG. In our novel findings, the product, PAP, was found to be produced at a rate that correlated with the standard spectrophotometric method for measuring β-galactosidase, the Miller assay, in both whole live and lysed cells. Conversely, to translate electronic signals to something cells can understand, we used pyocyanin, a redox drug which oxidizes the E.coli SoxR protein and allows transcription from the soxS promoter. We utilized electronic control of ferricyanide, an electron acceptor, to amplify the production of a reporter from soxS. With this novel method, we show that production of reporter depends on the frequency and amplitude of electronic signals, and investigate the method’s metabolic effects. Overall, the work in this dissertation makes strides towards the greater goal of creating multi-functional bio-hybrid devices

Bioengineered conduits for directing digitized molecular-based information

Molecular recognition is a prevalent quality in natural biological environments: molecules- small as well as macro- enable dynamic response by instilling functionality and communicating information about the system. The accession and interpretation of this rich molecular information leads to context about the system. Moreover, molecular complexity, both in terms of chemical structure and diversity, permits information to be represented with high capacity. Thus, an opportunity exists to assign molecules as chemical portrayals of natural, non-natural, and even non-biological data. Further, their associated upstream, downstream, and regulatory pathways could be commandeered for the purpose of data processing and transmission. This thesis emphasizes molecules that serve as units of information, the processing of which elucidates context. The project first strategizes a biocompatible assembly process that integrates biological componentry in an organized configuration for molecular transfer (e.g. from a cell to a receptor). Next, we have explored the use of DNA for its potential to store data in richer, digital forms. Binary data is embedded within a gene for storage inside a cell carrier and is selectively conveyed. Successively, a catalytic relay is developed to transduce similar data from sequence-based DNA storage to a delineated chemical cue that programs cellular phenotype. Finally, these cell populations are used as mobile information processing units that independently seek and collectively categorize the information, which is fed back as fluorescently ‘binned’ output. Every development demonstrates a transduction process of molecular data that involves input acquisition, refinement, and output interpretation. Overall, by equipping biomimetic networks with molecular-driven performance, their interactions serve as conduits of information flow

PROGRAMMING BACTERIAL CONSORTIA FOR AUTONOMOUS REGULATION AND COORDINATED ACTIVITY

The potential of genetically engineered microbes seems nearly infinite with applications ranging from human health to bioprocessing. However, metabolic burden and unbalanced use of cell resources are frequent challenges when engineering cells to carry out synthetic functions. To work around this challenge, engineers are attempting to use co-cultures or synthetic consortia wherein labor is divided amongst subpopulations that work together. This emerging strategy requires new tools to regulate the composition of subpopulations and to enable robust coordination between subpopulations. Here, we investigated and rewired a native cell-cell communication process, quorum sensing, in order to develop tools to regulate co-cultures. We developed modules for signal regulated cell growth rate and cell-cell communication in bacteria, and we used these modules to construct co-cultures with autonomous composition control. Specifically, we developed a “controller” strain for signal modulated cell growth rate by using quorum sensing signals to regulate levels of HPr, a protein involved in sugar transport. We developed a second “translator” strain that detects the universal quorum sensing signal AI-2 and translates it into a species-specific AI-1 signal. The composition of the resulting co-culture adjusts autonomously in response to AI-2. Importantly, we developed a simple mathematical model based on individual monocultures that predicts behavior of the co-culture. Then, we used our model to explore in silico alternate construct designs operating in varied environments. To complement the co-culture model, which explores behavior due to interactions between strains but does not encompass information about the genetic circuits underlying the quorum sensing process, we then developed a gene circuit model of a dual-input synthetic AI-2 quorum sensing system. Finally, we demonstrate that the strategies developed in our co-culture platform can be used to engineer co-cultures where the culture composition is controlled electrically. We also show that these strategies can be used to change the culture composition of a synthetic co-culture where each population is working together to produce pyocyanin, thereby changing the rate of pyocyanin production in the co-culture. The techniques developed here may enable further use of co-cultures or synthetic consortia by synthetic biologists and metabolic engineers for varied applications