Towards Synthetic Life: Establishing a Minimal Segrosome for the Rational Design of Biomimetic Systems

DNA segregation is a fundamental life process, crucial for renewal, reproduction and propagation of all forms of life. Hence, a dedicated segregation machinery, a segrosome, must function reliably also in the context of a minimal cell. Conceptionally, the development of such a minimal cell follows a minimalistic approach, aiming at engineering a synthetic entity only consisting of the essential key elements necessary for a cell to survive. In this thesis, various prokaryotic segregation systems were explored as possible candidates for a minimal segrosome. Such a minimal segrosome could be applied for the rational design of biomimetic systems including, but not limited to, a minimal cell. DNA segregation systems of type I (ParABS) and type II (ParMRC) were compared for ensuring genetic stabilities in vivo using vectors derived from the natural secondary chromosome of Vibrio cholerae. The type II segregation system R1-ParMRC was chosen as the most promising candidate for a minimal segrosome, and it was characterized and reconstituted in vitro. This segregation system was encapsulated into biomimetic micro-compartments and its lifetime prolonged by coupling to ATP-regenerating as well as oxygen-scavenging systems. The segregation process was coupled to in vitro DNA replication using DNA nanoparticles as a mimic of the condensed state of chromosomes. Furthermore, another type II segregation system originating from the pLS20 plasmid from Bacillus subtilis (Alp7ARC) was reconstituted in vitro as a secondary orthogonal segrosome. Finally, a chimeric RNA segregation system was engineered that could be applied for an RNA-based protocell. Overall, this work demonstrates successful bottom-up assemblies of functional molecular machines that could find applications in biomimetic systems and lead to a deeper understanding of living systems

Photo-controlled permeation of spiropyran modified gramicidin A ion channel

Thesis (M.S.) University of Alaska Fairbanks, 2006Biomimetic devices show great potential as being molecular sensors of biological species. Gramicidin A (gA) is a well studied ionophore that can be easily modified at the C-terminus to be incorporated into phosphotidylcholine bilayer membrane systems. Potassium permeation of modified gA attached to spiropyran can be controlled with light. Upon ultra-violet irradiation spiropyran transforms to the more polar form merocyanine. The process back to spiropyran is completely reversible upon irradiation with 550 nm light or thermally. Free bilayer membrane vesicles are employed to describe the characteristics of modified ion channels. Characteristics of gA modified with spiropyran are described herein. A device has been created and characterized using electrochemical impedance spectroscopy to analyze potassium permeation through a tethered bilayer membrane system (tBMS) on a sheet of gold utilizing sulfur anchors. The device consists of a tethered phase and a mobile upper phase. The mobile lipid layer incorporates gA modified with spiropyran. The modification allows for control of potassium permeation across the tBMS. Impedance analysis shows good agreement with the ability to control potassium permeation to that of the free vesicle

Surface characterization of lipid biomimetic systems

Zeta potential and dipole potential measures are direct operational methodologies to determine the adsorption, insertion and penetration of ions, amphipathic and neutral compounds into the membranes of cells and model systems. From these results, the contribution of charged and dipole groups can be deduced. However, although each method may give apparent affinity or binding constants, care should be taken to interpret them in terms of physical meaning because they are not independent properties. On the base of a recent model in which the lipid bilayer is considered as composed by two interphase regions at each side of the hydrocarbon core, this review describes how dipole potential and zeta potential are correlated due to water reorganization. From this analysis, considering that in a cell the interphase region the membrane extends to the cell interior or overlaps with the interphase region of another supramolecular structure, the correlation of dipole and electrostatic forces can be taken as responsible of the propagation of perturbations between membrane and cytoplasm and vice versa. Thus, this picture gives the membrane a responsive character in addition to that of a selective permeability barrier when integrated to a complex system.Fil: Disalvo, Edgardo Anibal. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet Noa Sur. Centro de Investigación en Biofísica Aplicada y Alimentos. - Universidad Nacional de Santiago del Estero. Centro de Investigación en Biofísica Aplicada y Alimentos; ArgentinaFil: Frías, María de los Ángeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet Noa Sur. Centro de Investigación en Biofísica Aplicada y Alimentos. - Universidad Nacional de Santiago del Estero. Centro de Investigación en Biofísica Aplicada y Alimentos; Argentin

DEPOLYMERIZABLE AND DISSIPATIVE CHEMICAL SYSTEMS: ROLE IN MATERIAL SYNTHESIS AND APPLICATIONS

Biomimetic systems which can show a functional response when treated with external stimuli are advantageous in applications such as self-healing, drug delivery, diagnostic and sensing. Depolymerizable and dissipative chemical systems are ideal tools to synthesize biomimetic systems that mimic nature. While there is abundance of synthetic chemistry tools to design depolymerizable systems, there is a lack of synthetic and formulation methodologies for developing useful materials from them. Majority of this thesis is directed towards narrowing that gap in knowledge, by utilizing depolymerizable systems to synthesize useful materials such as hydrogel and polyelectrolyte complexes for applications including self-healing, drug delivery and sensing. Minor part of this thesis involves dissipative chemical systems and it is directed towards achieving artificial systems that mimic several dynamic pathways predominant in nature which are regulated in presence of energy input

Realisation of next generation Lab-on-a-Chip devices - key challenges in fundamental materials science

Novel polymers responsive to external stimuli can undergo dramatic changes in properties. These effects can be used to control the functionality of microfluidic manifolds, and suggest that they could form the basis of next-generation, biomimetic-systems capable of long-term autonomous operation

Towards molecular machine functionalised biological and biomimetic systems

Overall this thesis describes the study of the ability of the tetracationic cyclophane CBPQT4+ to form inclusion complexes with electron-rich moieties such as tetrathiafulvalene (TTF) and dioxynaphthalene. These complexes are strengthened by π-stacking and charge transfer interactions, which give rise to coloured complexes. The complexes are fully reversible and can be decomplexed by the addition of a stimulus that can be chemical, electrochemical, and thermal. In addition we have exploited the ability of ferrocene to form inclusion complexes with cyclodextrins in aqueous media. The host-guest interactions that occur between these molecules were investigated using a number of techniques such as UV-Vis, fluorescence, NMR spectroscopy and cyclic voltammetry. Isothermal titration calorimetery (ITC) was also be used to measure the Ka of the complexes. Chapter two describes the synthesis of naphthalene and ferrocene functionalised dihydroimidazophenanthridines (DIPs). These materials were synthesised in order to create DNA intercalating agents that could undergo further host-guest interactions with either CBPQT4+ or β-cyclodextrin. These interactions were studied using ITC in a number of aqueous buffers with calf thymus DNA and the synthetic Dickerson dodecamer D-DNA. Additionally, the host-guest interactions for the naphthalene functionalised DIP with CBPQT4+ were studied using UV-Vis, fluorescence and NMR spectroscopy. The cytotoxic nature of the functionalised DIPs were investigated using MDCK epithelial cell culture experiments. Chapter three describes the synthesis and analysis of silane and disulfides modified with chosen electron rich substrates for the production of functionalised surfaces where self-assembled monolayers were produced on either glass or gold surfaces. These functionalised surfaces were then utilised in cell adhesion experiments with MDCK cells where the modulation of adhesion was attempted by the formation of pseudorotaxanes with either CBPQT4+ or β-cyclodextrin and by changing the oxidation state of the functional group in the case of ferrocene. Chapter four describes the synthesis of functionalised diacetylenes for the formation of polydiacetylene liposomes in aqueous conditions. The liposomes successfully formed were analysed by DLS. UV-Vis spectroscopy and cyclic voltammetry were used to investigate the dual response chromophoric sensing applications of these materials. Chapter five describes the synthesis of functionalised surfactant compounds for the formation of mixed micelles in aqueous conditions with sodium dodecyl sulfate (SDS). The interactions between the surfactant and CBPQT4+ were measured by ITC, NMR, UV-Vis, and fluorescence spectroscopy. Chapter six describes the modification of the protein BSA with a naphthalene functionalised chloroacetate. The modified protein was analysed by ITC, MALDI TOF, UV-Vis, and fluorescence spectroscopy in order to identify the degree of functionalisation that had occurred and whether complexation was possible with CBPQT4+. Chapter seven describes the synthesis of a naphthalene, and two ferrocene functionalised biotin conjugates with a view to investigate the interactions with avidin proteins. The interactions were measured by ITC and UV-Vis spectroscopy. The effect of changing the chain length on binding to neutravidin and β-cyclodextrin was studied in the ferrocene biotin conjugates where the interactions were assessed using ITC, cyclic voltammetry, and NMR spectroscopy. The interactions between the naphthalene based conjugate and CBPQT4+ was measured by UV-Vis and fluorescence spectroscopy

Buckling of stiff polymer rings in weak spherical confinement

Confinement is a versatile and well-established tool to study the properties of polymers either to understand biological processes or to develop new nanobiomaterials. We investigate the conformations of a semiflexible polymer ring in weak spherical confinement imposed by an impenetrable shell. We develop an analytic argument for the dominating polymer trajectory depending on polymer flexibility considering elastic and entropic contributions. Monte Carlo simulations are performed to assess polymer ring conformations in probability densities and by the shape measures asphericity and nature of asphericity. Comparison of the analytic argument with the mean asphericity and the mean nature of asphericity confirm our reasoning to explain polymer ring conformations in the stiff regime, where elastic response prevails

Preclinical platforms to study therapeutic efficacy of human γδT‐cells for oncology indications

In this commentary, we discuss recent advances in the study of γδT cell-based immunotherapeutics. As an allo-compatible cell therapy chassis without clear functional homologs in mice, γδT cells represent a challenge and an opportunity for preclinical modelling. We discuss some of the techniques and approaches that can be used to demonstrate and characterise γδT cell behaviour in biomimetic systems

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