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Effects of Macromolecular Crowding on Gene Expression Studied In Effects of macromolecular crowding on gene expression studied in protocell models The work described in this thesis was supported by a European Research Council (ERC), Advanced Grant (246812 Intercom) and a VICI grant from the Netherlands Organisation for Scientific Research (NWO) ISBN: 978-94-6295-151-8 Cover design: Denis Arslanov and Ekaterina Sokolova Printed & Lay Out by: Proefschriftmaken.nl || Uitgeverij BOXPress Published by: Uitgeverij BOXPress, ’s-Hertogenbosch Effects of macromolecular crowding on gene expression studied in protocell models Proefschrift ter verkrijging van de graad van doctor aan de Radboud Universiteit Nijmegen op gezag van de rector magnificus prof. dr. Th.L.M. Engelen, volgens besluit van het college van decanen in het openbaar te verdedigen op dinsdag 12 mei 2015 om 14.30 uur precies door Ekaterina Alexandrovna Sokolova geboren op 30 augustus 1987 te Angren (Uzbekistan) Promotor: Prof. dr. W. T. S. Huck Manuscriptcommissie: Prof. dr. ir. J. C. M. van Hest (voorzitter) Prof. dr. H. N. W. Lekkerkerker (Universiteit Utrecht) Dr. V. Noireaux (University of Minnesota, VS) Effects of macromolecular crowding on gene expression studied in protocell models Doctoral Thesis to obtain the degree of doctor from Radboud University Nijmegen on the authority of the Rector Magnificus prof. dr. Th.L.M. Engelen, according to the decision of the Council of Deans to be defended in public on Tuesday, May 12, 2015 at 14.30 hours by Ekaterina Alexandrovna Sokolova Born on August 30, 1987 in Angren (Uzbekistan) Supervisor: Prof. dr. W. T. S. Huck Doctoral Thesis Committee: Prof. dr. ir. J. C. M. van Hest (chairman) Prof. dr. H. N. W. Lekkerkerker (University of Utrecht) Dr. V. Noireaux (University of Minnesota, USA) To my parents - Tatyana and Aleksandr Contents 1 Macromolecular crowding and its influence on the chemistry of the cell 1 1.1 What is macromolecular crowding? . .1 1.2 Theory of the effects of crowding . .3 1.2.1 Thermodynamical framework . .4 1.2.2 Molecular interpretations (hard spheres approximation and de- pletion forces) . .6 1.2.2.1 BMCSL (Boublik-Mansoori-Carnahan-Starling-Leland) model . .7 1.2.2.2 Scaled-particle theory (SPT) . 10 1.2.2.3 Depletion interactions . 12 1.2.3 Dynamics effects of crowding . 14 1.3 Experimental investigations of crowding . 16 1.3.1 Approaches to mimic crowded media in vitro . 16 1.3.2 Type of crowders: advantages & disadvatages . 16 1.3.3 Reported effects of macromolecular crowding . 17 1.4 Biochemical model reactions . 18 1.4.1 Association equilibria . 18 1.4.2 Enzymatic activity . 18 1.4.3 Conformational changes . 19 1.4.4 Protein folding . 20 1.4.5 Other effects . 20 1.4.6 Attractive interactions . 21 1.5 Aim of the research presented in this thesis . 21 v vi Contents 2 A microfluidics platform for quantitative characterization of IVTT reaction in droplets 27 2.1 Introduction . 28 2.1.1 The course of gene expression in the cell . 28 2.1.2 Transcription and translation . 29 2.1.3 Cell-free gene expression . 29 2.1.4 Cell free expression in vesicles . 31 2.1.5 Cell free expression in microdroplets . 33 2.1.5.1 Overview of microfluidics . 34 2.1.5.2 In vitro gene expression in droplets . 35 2.2 Experimental protocols . 36 2.2.1 Materials . 36 2.2.2 Laser-induced fluorescence (LIF) and microfluidic setup operation 37 2.2.3 Device fabrication . 38 2.2.4 Operation of chamber and bilayer devices . 40 2.3 Results and Discussion . 41 2.3.1 In vitro gene expression from a single copy of DNA in droplets 42 2.3.2 Kinetic measurements of IVTT in droplets . 44 2.3.3 Control over water contents of the droplets . 45 2.4 Conclusion: From “bulk” to “cell-like” . 45 3 Cell lysate coacervates - possible models of crowding in cells 49 3.1 Introduction . 49 3.1.1 Spatial organization of the cell . 49 3.1.2 Compartmentalization induced by phase separation . 50 3.1.3 Coacervation as a route to crowding . 51 3.2 Results and Discussion . 52 3.2.1 Physical properties of cell lysate coacervates . 53 3.3 Materials and methods . 60 3.3.1 Phase separation of cell-free expression kit containing fluores- cent PEG and lysate . 61 3.3.2 Phase separation of PEG in the absence of cell-free expression kit 61 3.3.3 Calculation of the distribution of PEG over the two phases . 61 3.3.4 Inductively-coupled plasma optical emission spectrometry (ICP- OES) . 62 3.3.5 Covalent labeling of the plasmid . 62 3.3.6 Data Acquisition and Analysis . 63 3.3.7 Calculation of partition coefficients of PEG and fluorescently labelled lysate . 63 3.3.8 Fluorescence recovery after photobleaching experiments . 63 Contents vii 3.4 Conclusion . 65 4 Gene expression in crowded membrane-free protocells 69 4.1 Introduction . 69 4.1.1 Overview of the protocell models . 69 4.2 Results and Discussion . 73 4.2.1 Modeling of transcription and translation in cell lysate with de- terministic rate equations . 73 4.2.2 Transcription and translation in membrane-free protocells formed by coacervation of cell lysate . 76 4.3 Materials and Methods . 81 4.3.1 Materials . 81 4.3.2 Home made in vitro transcription translation system . 82 4.3.3 Molecular beacons for mRNA labeling . 83 4.3.4 Data acquisition and analysis . 84 4.3.5 Method for mRNA production experiments in cell lysate . 84 4.3.6 Method for GFP production experiments in cell lysate . 84 4.3.7 Method for transcription in bulk at various PEG concentrations . 85 4.3.8 Method for translation in bulk at various PEG concentrations . 86 4.4 Conclusion . 86 5 Understanding the effect of macromolecular crowding on genetic networks in synthetic cellular systems 89 5.1 Introduction . 90 5.2 Results and Discussion . 91 5.3 Conclusion . 97 6 Stochastic gene expression in a crowded environment 101 6.1 Introduction . 102 6.1.1 Stochastic gene expression in vivo . 102 6.1.2 In vitro system for cell mimics . 104 6.2 Materials & Methods . 105 6.2.1 IVTT system . 105 6.2.2 Data acquisition and analysis . 106 6.3 Results and Discussion . 108 6.3.1 Aim of this study and limitations of approach taken . 108 6.3.2 Influence of different molecular weights dextran on expression of b-glucuronidase . 108 6.3.3 Stochastic b-glucuronidase expression in droplets . 109 6.3.3.1 Time of arrivals and kinetics distributions . 110 viii Contents 6.3.3.2 Individual droplet analysis . 114 6.4 Conclusions and future perspectives . 116 6.4.1 Microfluidics and IVTT system . 116 6.4.2 Implications of noise and crowding . 117 Summary 119 Samenvatting 123 Dankwoord 127 Chapter 1 Macromolecular crowding and its influence on the chemistry of the cell 1.1 What is macromolecular crowding? A cell is the common structural unit shared by all living organisms. From a physico- chemical point of view cells are extremely complex systems, characterized by small vol- umes, highly concentrated in large molecules and complexes, but low copy numbers of each individual component, all together surrounded and subdivided into compartments by ubiquitous interfaces. The typical concentration of macromolecules (for example en- zymes, filaments, nucleic acids, glycoproteins, lipids, etc.), in the cell is 300-400 g/L [1]. Despite significant progress being made it is still unclear how high abundance of macromolecules in the cell can influence the outcome of biochemical reactions. An artistic impression of the complex environment inside a eukaryotic cell can be seen in Fig. 1.1. The outside surface area of a typical 20 µm human tissue cell is approx- imately 2400 µm2, but the total area covered by internal membranes (rough and smooth endoplasmic reticulum, Golgi complex, etc.) is around 50 times larger (>120000 µm2) [1]. As a result, the average separation between interfaces (i.e., membranes) within the cell is only about 50 nm, which, coupled to the high concentration of proteins inside a cell leads to a dense and highly confined environment. Cryo-electron tomography gives direct evidence of the crowded interior of the cells. In this technique cells are rapidly frozen and studied under electron microscope with a spatial resolution of 5-6 nm. The 1 2 Chapter 1: Macromolecular crowding and its influence on the chemistry of the cell Figure 1.1: Cartoon of eukaryotic cytoplasm magnified ×106 (reproduced from [2]), modi- fied [3] high density of actin filaments and ribosomes seen in reconstructed images (Fig. 1.2) of slime mould Dictyostelium indicates that cytoplasm is filled with large amounts of macromolecules assembled into complexes, rather than with freely diffusing and collid- ing macromolecules [4]. Figure 1.2 clearly shows that due to the high concentration of macromolecules, there is very little‘free space’ in the cell. It is important to mention that it is not the high concentration of a single macromolecule that causes the cell to be ’fully packed’, but the total concentration of macromolecules. Those macromolecules are commonly called ‘crowders’ if they display no specific interactions. This phenomenon was termed ‘macromolecular crowding’ by Minton in 1981 [5]. The number and type of molecules in the cell depend on the cell type and probably on the cell cycle stage [6]. The total protein content of the cell is estimated to be 50-400 g/L corresponding roughly to 5- 40 % of the total cell volume [2, 3]. Zimmerman and Trach estimated the protein content of E. coli to be around 10-40 % in units of weight/volume [7]. Similarly, Lanni et.
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