Year: 2024
Type: dissertation
Abstract: Understanding the design principles of living membranes is essential to expanding our ability to manipulate and interact with living systems in a bioengineering capacity. Until now, the complexity of living membranes has made it difficult to understand the principles behind the ways in which membrane remodeling is controlled, the membrane biophysical properties that cells regulate most, and even the question of why life has evolved to use so many lipids in its membranes. This complexity has also stymied attempts to build accurate in vitro and ni silico model systems that can accurately mimic dynamic living membranes. In this thesis, I report the creation of a tunable living model membrane system in Mycoplasma mycoides and JCVI-Syn3A. Iminimized the membrane in these organisms to create viable cells with only two lipid species in their membranes: the simplest living membranes reported to date. I then tuned the membrane size and composition of these organisms by systematically reintroducing phospholipids and acyl chains to their membranes, seeking to investigate a fundamental membrane design question on the importance of lipidome complexity versus composition and finding that acyl chain diversity is more important than head group diversity in these organisms. This model membrane system also allows the creation of non-canonical membranes. By tuning lipid chirality, Iexplored the lipid divide between Archaea and the rest of life and investigated the possibility, and potential consequences, of heterochiral membranes. Identification of membrane proteins is a growing field, and this thesis details how this model membrane is an excellent candidate for ni vivo identification of membrane proteins. Finally, using electron microscopy, Iinvestigated the role between minimal lipid diets and membrane morphology, demonstrating a clear link between lipidome and cell morphology. Overall, this work describes the creation and demonstration of a novel and useful living model membrane system with a simple and tunable membrane, and its use in answering fundamental questions on the design principles of living membranes.:Zusammenfassung 6 Summary 7 Abbreviations 8 1. Introduction 9 1.1 Life as an engineerable material 9 1.2 Lipid Membranes 11 1.3 M. mycoides and JCVI-Syn3A as model organisms 13 1.4 Defined Diets and Lipidome Minimization 15 1.5 Membrane Composition and Cell Properties 16 1.6 Identifying Membrane Proteins 17 1.7 Membrane Composition and Cell Morphology 19 2. Materials 20 2.1 Media, Buffers, and Running Solutions 20 2.2 Lipids 20 2.3 Assays 21 2.4 Organisms 21 2.5 Machines 21 3. Methods 22 3.1 Cell Culture 22 3.2 Growth Rates 22 3.3 Lipid Extraction and Thin Layer Chromatography 23 3.4 Liposome Preparation 23 3.5 Propidium Iodide Osmotic Shock Assay 23 3.6 Fluorescein Diacetate Permeability Assay 24 3.7 C-Laurdan General Polarization Assay 24 3.8 Phosphate Assay 25 3.9 Lipidomic Analysis 25 3.10 Transcriptomics 26 3.11 PCR, Protein Expression, and SDS Page separation 27 3.12 Room temperature TEM 28 3.13 Cryo TEM 29 3.14 Statistical Analysis 30 4. Results 32 4.1 Mycoplasma and Syn3A as minimal model membrane systems 32 Fig. 1 34 4.2 A Defined Lipid Diet for M. mycoides and Syn3A 35 Table 1 36 Fig. 2 38 4.3 Creating a minimal diet 39 Fig. 3 41 Fig. 4 42 4.4 Validation of Growth Assays 43 Fig. 5 44 Fig. 6 45 4.5 Tuning Lipidome Composition 46 Fig. 7 49 Fig. 8 50 4.6 Tuning Lipid Chirality 51 Fig. 9 53 Fig 10 54 Fig 11 55 4.7 Identifying Lipid Transporters 56 Table 2 58 4.8 Cell phenotype and diet 62 Fig. 12 63 Fig. 13 64 Fig. 14 65 5. Discussion 66 6. Final Summary and Outlook 69 7. Citations 70
Author: Justice, Isaac Gabriel
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W7167858797