Towards the assembly of a minimal oscillator

(1)

T

OWARDS THE

A

SSEMBLY OF A

M

INIMAL

O

SCILLATOR

(2)

(3)

T

OWARDS THE

A

SSEMBLY OF A

M

INIMAL

O

SCILLATOR

G

ENETIC

N

ETWORKS IN

L

IPOSOMES

Proefschrift

ter verkrijging van de graad van doctor aan de Technische Universiteit Delft,

op gezag van de Rector Magnificus prof. ir. K. C. A. M. Luyben, voorzitter van het College voor Promoties,

in het openbaar te verdedigen op maandag 8 juni 2015 om 12:00 uur

door

Zohreh N

OURIAN

natuurkundig ingenieur geboren te Karaj, Iran.

(4)

Dit proefschrift is goedgekeurd door de promotor: Prof. dr. M. Dogterom

Copromotor: Dr. C. J. A. Danelon Samenstelling promotiecommissie:

Rector Magnificus, voorzitter

Prof. dr. M. Dogterom, Technische Universiteit Delft, promotor Dr. C. J. A. Danelon, Technische Universiteit Delft, copromotor Prof. dr. A. Forster, Uppsala University

Prof. dr. D. Stamou, University of Copenhagen

Prof. dr. G. Rivas Caballero, Centro de Investigaciones Biologicas Dr. E. Mastrobattista, Universiteit Utrecht

Dr. C. Joo, Technische Universiteit Delft

Keywords: Minimal cell, Cell-Free System, Minimal Oscillator, Gene Expression, PURESystem, PUREFrex, Liposome, Genetic Network

Printed by: Gildeprint

Front & Back: Alex de Mulder

Casimir PhD Series, Delft-Leiden 2015-14 ISBN 978-90-8593-223-9

An electronic version of this dissertation is available at http://repository.tudelft.nl/.

(5)

Chapter 1 Introduction

Given the complexity of living organisms, one can question if such complexity is really essential for life, or whether instead cellular life might be possible with a much smaller number of components. The simplest representation of a living cell can be envisioned as a reconstituted synthesis machinery carrying out the expression of a minimal genome inside a synthetic lipid vesicle. As a new constructive paradigm, we hypothesize that even an elementary cell should be able to harness the synthesis apparatus to control in time the levels of messengers and proteins, an essential step toward the orchestration of more elaborate functions, such as growth, division and genome replication.

(10)

1

2 1.INTRODUCTION

1.1 Building a minimal cell

H

OW simple is the simplest cell? Given the extraordinary complexity of even the simplest existing organism, one can question if such a complexity is really essential for life, or whether instead cellular life can be supported with far less components than we see in nature. This question can be addressed using two complementary research directions. In the top-down approach the extant genome of simple modern organisms is reduced in order to infer a minimal genome size or to define the vital cellular functions [1]. In the alternative bottom-up direction -or constructive approach-essential cellular functions are assembled using a minimal set of purified constituents under well-controlled conditions [2–5]. Ultimately, both in vivo reduction and in vitro construction approaches to develop an elementary (or minimal) irreducible cell will ar-guably unveil new insights about the design principles of life as well as about the nature of the last universal common ancestor (LUCA). The term LUCA originally referred to an antique organism from which all living systems that ever existed on Earth descended [6]. The picture of such a rudimentary living entity that ultimately developed into the three primary branches of the universal phylogenetic tree, namely Bacteria, Archaea and Eukarya, is, however, arguably questionable. An attractive hypothesis has been formulated, in which a universal ancestor should rather be apprehended as a diversified population of primitive cells [7].

Possible roadmaps for the realization of a minimal cell have been suggested and will not be reviewed here [2, 8–10]. A distinction has to be made between the bottom-up synthetic biology approach to a minimal cell and the truly synthetic approach to protocell models [11–14]. In the former, extant molecules provided to us by evolution, such as proteins and DNA, are used as building blocks. Hence, the produced minimal cell should formally be referred as semi-synthetic. In the latter, researchers attempt to create primitive cells from scratch by using merely organic and inorganic materials and natural processes presumably available on the early Earth. Note also the difference with the semi-synthetic cell of Craig Venter’s group, obtained by transplantation of a synthetic genome into a recipient living bacterium [15].

Though the definition of life itself is controversial, one can reasonably say that a molecular assembly should be considered alive if it is capable of self-maintenance, self-replication and evolvability. An essential trait of a living cellular system, even in its simplest representation, is the compartment: a continuous membrane that acts as a functional interface to regulate the ionic and molecular exchanges with the environ-ment. Self-reproduction of a cell implies that both the compartment and the genetic material supporting life self-replicate in a coordinated manner.

(11)

1.2.CELL-FREE GENE EXPRESSION INSIDE LIPOSOMES AS A PLATFORM FOR THE CONSTRUCTION OF A MINIMAL CELL

1

3

1.2 Cell-free gene expression inside liposomes as a

plat-form for the construction of a minimal cell

The expression of genes into proteins is a fundamental and ubiquitous cellular process. The transfer of the DNA sequence information to proteins occurs in two steps form-ing the central dogma in molecular biology: the transcription of the gene in RNA and the subsequent translation of that RNA into a protein. During transcription the double stranded DNA is copied by an RNA polymerase protein into a single stranded messenger RNA (mRNA). Translation is a more complex process involving many different compo-nents, with the ribosome as a key player. The synthesis of proteins from DNA templates inside lipid vesicles offers an elegant and unavoidable alternative to the exclusive use of purified constituents. By regulating the timely synthesis of the multiple proteins, the gene expression machinery is arguably at the heart of a programmable, genetically con-trolled, artificial cell.

Figure 1.1: PURE system-based representation of a minimal cell model. A programmable, genetic controlled minimal cell can be represented as a gene expression apparatus, the PURE system, encapsulated inside a semi-permeable liposome. (A) Three essential requirements for building a minimal cell are: growth, replication and division. (B) The PURE system can be broken down in four functional modules: transcription, translation, aminoacylation and energy regeneration. The numbers of purified proteins involved to execute the specific reactions are indicated in parentheses. Abbreviations: AAs, amino acids; NMPs, nucleotide monophosphates; NDPs, nucleotide diphosphates; NTPs, nucleotide triphosphates.

Cell-free protein synthesis inside liposomes has first been performed using cellular extracts derived from cytoplasmic parts of E. Coli bacteria [16, 17]. Despite many advantages, including reduced costs [18], high yield of protein expression [19] and the possibility to use the endogenous bacterial RNA polymerases [20], crude extracts are poorly characterized mixtures containing plenty of undesired components that

(12)

1

4 1.INTRODUCTION

can interfere with the intended reactions. Alternatively, the PURE (Protein synthesis Using Recombinant Elements) system developed by the group of Takuya Ueda (Tokyo University) [21, 22], is a minimal gene expression system reconstituted solely from pu-rified enzymes and cofactors (36 different proteins) for transcription, translation, tRNA aminoacylation, energy regeneration and pyrophosphate hydrolysis (Fig. 1.1 B). Unless otherwise indicated, gene expression was conducted in the PUREfrex (GeneFrontier, Japan). The constituting enzymes in the PUREfrex are similar to those in the original expression system [21], with the exception that they are devoid of a histidine tag [23]. All components are from E. Coli with the exception of the RNA polymerase that is from the T7 bacteriophage. Since the PURE system is made of well-defined elements with known concentrations and intended functions, it is the preferred core synthesis apparatus of a minimal cell. Moreover, it provides a unique platform for reliable modeling and quan-titative understanding of gene expression dynamics, which is essential for high-level control of protein production. Hence, the simplest representation of a living cell can be envisioned as a reconstituted biosynthesis machinery carrying out the expression of a minimal genome inside a self-assembled lipid vesicle (Fig. 1.1 A).

1.3 Thesis Outline

In Chapter 2, we describe a porous matrix-assisted protocol to produce large and giant liposomes (µm size range) encapsulating the PURE system and DNA encoding a fluorescent reporter. The protocol describes immobilization of the liposomes on a glass cover slip and the method to visualize those femto-liter size reactors using a laser scanning confocal microscope. We investigated the kinetics of gene expression in bulk and in liposomes. The gene expression is negatively influenced by the length of the phospholipid carbon chains.

In Chapter 3, the link between genotype and phenotype in gene expressing lipo-somes was established. We showed that there exists no linear correlation between the amount of encapsulated genes and the level of output proteins. Moreover, we developed a dual gene expression assay consisting of the production of two orthogonally detectable fluorescent reporter proteins from two different DNA templates to infer the probabilistic occupancy of transcriptionally active genes in protein synthesizing liposomes. These findings will allow us to operate the expression of multiple genes in a controlled manner and to generate the output proteins with predictable dynamics in liposomes.

In Chapter 4, we investigated how membrane deforming proteins, such as NBAR, FtsZ and FtsA, could be integrated into the construction of a programmable minimal cell relying on gene expression inside liposomes. As a first step towards the de novo synthesis of a divisome, we showed that the N-BAR domain protein produced from its gene could assemble onto the outer surface of liposomes and sculpt the membrane into tubular structures. Besides, we performed preliminary experiments on the in situ synthesis of FtsZ and FtsA from their corresponding gene.

(13)

REFERENCES

1

5 In Chapter 5, we aimed at utilizing our model system as a platform for incorporation of regulatory networks towards achieving spatial and temporal dynamics of protein pro-duction. To be able to control the system’s dynamics, the mRNA and protein lifetime has to be tunable. We achieved controlled degradation by supplementing the PURE system with purified MazF and ClpXP which provides an additional level of control of mRNA and protein amount. With the perspective of incorporating genetic regulatory network to achieve spatial and temporal organizations essential for the minimal cell, we then de-signed and characterized (ongoing effort) genetic regulatory networks in bulk.

References

[1] A. Moya, R. Gil, A. Latorre, J. Peretó, M. P. Garcillán-Barcia, and F. de la Cruz, Toward

minimal bacterial cells: evolution vs. design. FEMS Microbiology Reviews 33, 225

(2008).

[2] P. L. Luisi, F. Ferri, and P. Stano, Approaches to semi-synthetic minimal cells: a review. Die Naturwissenschaften 93, 1 (2006).

[3] P. L. Luisi, Toward the engineering of minimal living cells. The Anatomical record

268, 208 (2002).

[4] E. Karzbrun, J. Shin, R. Bar-Ziv, and V. Noireaux, Coarse-Grained Dynamics of

Pro-tein Synthesis in a Cell-Free System, Physical Review Letters 106, 048104 (2011).

[5] P. Schwille, Bottom-up synthetic biology: engineering in a tinkerer’s world. Science

333, 1252 (2011).

[6] C. Darwin, The origin of species by means of natural selection, or, The preservation of

favored races in the struggle for life. Vol. 2, International Science Library (1859).

[7] C. Woese, The universal ancestor, Proceedings of the National Academy of Sciences of the United States of America 95, 6854 (1998).

[8] G. Murtas, Artificial assembly of a minimal cell. Molecular BioSystems 5, 1292 (2009).

[9] V. Noireaux, Y. T. Maeda, and A. Libchaber, Inaugural Article: Development of an

ar-tificial cell, from self-organization to computation and self-reproduction,

Proceed-ings of the National Academy of Sciences 108, 3473 (2011).

[10] A. C. Forster and G. M. Church, Towards synthesis of a minimal cell, Molecular Sys-tems Biology 2 (2006).

[11] H. J. Morowitz, B. Heinz, and D. W. Deamer, The chemical logic of a minimum

pro-tocell. Origins of Life and Evolution of the Biosphere 18, 281 (1987).

(14)

1

6 REFERENCES

[13] D. Loakes and P. Holliger, Darwinian chemistry: towards the synthesis of a simple

cell. Molecular BioSystems 5, 686 (2009).

[14] P. Walde, Building artificial cells and protocell models: experimental approaches

with lipid vesicles. Bioessays 32, 296 (2010).

[15] D. G. Gibson, J. I. Glass, C. Lartigue, V. N. Noskov, R.-Y. Chuang, M. A. Algire, G. A. Benders, M. G. Montague, L. Ma, M. M. Moodie, C. Merryman, S. Vashee, R. Krish-nakumar, N. Assad-Garcia, C. Andrews-Pfannkoch, E. A. Denisova, L. Young, Z.-Q. Qi, T. H. Segall-Shapiro, C. H. Calvey, P. P. Parmar, C. A. Hutchison, H. O. Smith, and J. C. Venter, Creation of a bacterial cell controlled by a chemically synthesized

genome. Science (New York, N.Y.) 329, 52 (2010).

[16] S.-i. M. Nomura, K. Tsumoto, T. Hamada, K. Akiyoshi, Y. Nakatani, and K. Yoshikawa, Gene Expression within Cell-Sized Lipid Vesicles, ChemBioChem 4, 1172 (2003).

[17] V. Noireaux and A. Libchaber, A vesicle bioreactor as a step toward an artificial cell

assembly, Proceedings of the National Academy of Sciences of the United States of

America 101, 17669 (2004).

[18] M. C. Jewett and A. C. Forster, Update on designing and building minimal cells. Cur-rent Opinion in Biotechnology 21, 697 (2010).

[19] F. Caschera and V. Noireaux, Synthesis of 2.3 mg/ml of protein with an all Escherichia

coli cell-free transcription-translation system. Biochimie 99, 162 (2014).

[20] J. Shin and V. Noireaux, Research Study of messenger RNA inactivation and protein

degradation in an Escherichia coli cell-free expression system, Journal of Biological

Engineering (2010).

[21] Y. Shimizu, A. Inoue, Y. Tomari, T. Suzuki, T. Yokogawa, K. Nishikawa, and T. Ueda,

Cell-free translation reconstituted with purified components. Nature biotechnology

19, 751 (2001).

[22] Y. Shimizu, T. Kanamori, and T. Ueda, Protein synthesis by pure translation systems, Methods 36, 299 (2005).

[23] Y. Shimizu, Y. Kuruma, T. Kanamori, and T. Ueda, The PURE system for protein

(15)

Chapter 2 Triggered Gene Expression in

Fed-Vesicle Microreactors With a

Multifunctional Membrane

We established a method to successfully produce lipid vesicles encapsulating a DNA tem-plate and a minimal gene expression system for mRNA and protein synthesis. Our method is compatible with a variety of natural and functionalized lipids, which allowed us to engineer the vesicle membrane for surface immobilization and for promoting selective ex-change with the environment. We demonstrated that gene expression can be triggered by supplying the nutrients " amino acids, nucleotides, tRNAs " in the outside solution. Protein synthesis can take place in liposomes with a broad range of sizes and the level of expression varies markedly between individual vesicles. We believe that our results are of direct relevance for initiating, regulating and monitoring biochemical reaction networks in general, with far-reaching consequences to the construction of artificial cells and next generation drug delivery systems.

This chapter has been published in Angewandte Chemie, Nourian Z, Roelofsen W, Danelon C 51, 13: 3114 (2012) [1].

(16)

2

8

2.TRIGGEREDGENEEXPRESSION INFED-VESICLEMICROREACTORSWITH A

MULTIFUNCTIONALMEMBRANE

2.1 Introduction

W

ITHthe aim of constructing an artificial cell having organizational and functional properties mimicking that of living systems, it is now possible to assemble lipo-somes that enclose biochemical reaction networks as complex as the whole transcrip-tion and translatranscrip-tion machinery capable of synthesizing proteins from a DNA template [2–16]. Further progress towards the development of a minimal cell is critically depen-dent on technological advances to efficiently encapsulate in vitro gene expression sys-tems within cell-sized (>1 µm) liposomes [17–19]. Ideally, the vesicle formation method should be compatible with any buffer and membrane compositions in order to tailor the container properties, e.g. for promoting selective exchange of matter with the environ-ment or to facilitate vesicle handling and characterization. However, current techniques to meet those demands are inadequate and technological challenges remain in order to pave the way to the assembly of a semi-synthetic minimal cell.

2.1.1 Liposome formation methods

A variety of liposome preparation methods have been employed for compartmentalized gene expression: lipid film swelling [2], mechanical lipid film resuspension [3], freeze-dried empty liposome (FDEL) [5–11], ethanol injection [12], water-in-oil emulsion trans-fer, and microfluidic-based devices [13–15]. Commonly used methods for liposome-based compartmentalization of gene expression include the water-in-oil emulsion and the freeze-dried empty liposome (FDEL) methods. With methods using lipids dissolved in oil as precursors to generate liposomes, solvent remains trapped in the bilayer, which affects membrane permeability. Moreover, the yield of vesicle formation is poor with some lipid compositions or unbalanced osmolarity between the inside and outside of the vesicles [13–15]. As for the FDEL technique, it leads to small vesicles that are diffi-cult to observe by fluorescence microscopy.The lipid film swelling method offers unique advantages to create large and giant (>10 µm) liposome-based bioreactors with almost unrestricted choice in membrane components making it a popular approach to produce vesicles with various membrane functionalities, such as surface positioning[20], target-ing and selectively delivertarget-ing cargo to live cells[21], and temperature-responsive perme-ability [22]. However, this method suffers from several deficiencies including i) a low yield of formed liposomes in physiological buffers, ii) lack of compatibility with micro-liter volume handling during lipid film rehydration and iii) low encapsulation efficiency of macromolecules compared to the emulsion-based or FDEL methods. Consequently, the benefits provided by the lipid film swelling method remain largely unexploited in the context of artificial cell assembly.

To date, in all studies of protein biosynthesis inside liposomes, phospholipids of 16 or 18 carbons were used [2–16, 23, 24], rendering bilayer permeability very low in non-stressed conditions. Two approaches have been presented to create an active interface with the environment: i) to generate membrane defects under osmotic pressure, and ii) to in-corporate transmembrane ion channels in the liposome membrane [13]. Both meth-ods enabled the exchange of low-molecular weight solutes with the surrounding feed-ing environment and thus increased the production time of a cell extract-based expres-sion system as compared to that in sealed vesicles. Although the influence of charged

(17)

2.2.RESULTS ANDDISCUSSIONS

2

9 and PEG-bearing lipids on the yield of protein synthesis has been investigated [9, 11], the role of the length and saturation level of the lipid carbon chain–with the associated temperature- and pressure-dependent bilayer permeability–to regulate molecular diffu-sion across the vesicle boundary remains unexplored.

2.2 Results and Discussions

H

EREIN, we describe the use of a porous matrix-assisted method to produce by lipid

film swelling large and giant liposomes encapsulating a minimal gene expression system. We reasoned that the increased active surface area provided by submillimeter glass beads may lead to higher concentration of liposomes generated within inter-bead cavities (Figure 2.1), while enhancing encapsulation of the multiple components (about 40 different proteins) by promoting membrane reactivity. A similar technique has proven increased encapsulation efficiency of small vesicles into larger ones [25].

!

Figure 2.1: Phase contrast micrographs of (left) lipid film swelling from glass beads and (right) liposomes formed within bead cavities. The lipid film swells from the bead surface forming long cylindrical membranes. Most of the tethered liposomes remain attached to the beads until the mixture is gently agitated.

Moreover, we report for the first time on the use of short dimyristoylated, 14 carbon-acyl chain phospholipids, to create semi-permeable membranes making possible the uptake of nutrients from the outside feeding environment, while keeping the macro-molecules entrapped inside the liposome. We exploited the capability of the lipid film swelling method to accommodate a variety of membrane constituents for integrat-ing multiple functionalities: biotin-PEG-lipids (PEG = poly(ethylene glycol)) for lipo-some immobilization on neutravidin-coated surfaces and vesicle stabilization through the PEG spacer, negatively charged lipids to improve swelling, TRITC-conjugated lipids (TRITC = N-(6-tetramethylrhodaminethiocarbamoyl)) for membrane localization using fluorescence imaging, and lipids of different phase transition temperatures (Tm) for tun-ing membrane permeability as a response to temperature and osmotic pressure. For the in vitro gene expression system, we used the PURE system reconstituted solely from purified enzymes and cofactors including E. Coli ribosomes for translation, the T7 RNA polymerase for transcription, and others for aminoacylation and energy regenera-tion [26]. In addiregenera-tion to this enzymatic mix the PURE system comprises a feeding solu-tion containing the nutrients – mainly nucleotides and amino acids – as well as tRNAs.

(18)

2

10

As compared to cell extracts, the PURE system is made of well-defined elements and it is therefore preferred as a constructive paradigm of a minimal cell.

!

Figure 2.2: Preparation of surface-immobilized liposome reactors using a bead-based lipid film swelling strat-egy. a) 200-µm glass beads are mixed with lipid-containing organic solvent in a round-bottom flask. A lipid film is formed onto the glass beads by rotary evaporation of solvent. b) Lipid-coated glass beads are trans-ferred in a reaction tube, where the lipid film is rehydrated in the presence of a DNA template and the enzyme mix of the PURE system. Then, the formed liposomes are subjected to a few freeze-thaw cycles in situ. c) The liposome-containing solution is transferred in a PDMS chamber for surface immobilization on a microscope coverslip using biotneutravidin recognition sites. Transcription and translation reactions are initiated in-side the liposomes by diluting the bulk phase with the feeding solution. Two-colour fluorescence imaging measurements are carried out to characterize vesicle sizes and structures, and protein synthesis.

Figure 2.2 illustrates the main experimental steps to produce surface-tethered protein-synthesizing liposome microreactors. Briefly, lipid-coated beads are prepared by organic solvent evaporation under continuous rotation in a round-bottom glass flask and can be stored under nitrogen gas at -20±_{C for several weeks. The equivalent of about} 10 µL of lipid-coated beads are transferred in a reaction tube, gently packed, and the lipid film is rehydrated for 1 to 2 hours with 2 to 10 µL of a solution containing the enzyme mix and a linear DNA template encoding for an autofluorescent protein. As repetitive li-posome freezing and thawing is recognized to break lipidic multilamellar structures and enhance encapsulation efficiency [27], the reaction tube is then subjected to four freeze-thaw cycles (see below). Next, the vial is tilted and gently rotated to unpack the glass beads and release the liposomes. Then, 1 to 2 µL of the vesicle-containing solution is transferred into a poly(dimethylsiloxane) (PDMS) chamber mounted on a microscope coverslip pre-coated with neutravidin for liposome immobilization. At this stage, no ex-pression is possible as the nutrients and tRNAs have not been supplemented yet. Finally, the surface-tethered vesicles are diluted in – or the external solution is exchanged with – the feeding solution (supplemented with DNase proteins, when indicated) such that outside protein synthesis is inhibited, and incubated at 37±_C.

In a first series of batch reactor measurements, we mimicked the conditions applied during vesicle preparation and examined the influence of temperature and freeze-thaw cycles, two potentially denaturating parameters, on PURE system activity. Figure 2.4a shows the time courses of emGFP synthesis at different temperatures applied for

(19)

lipo-2.2.RESULTS ANDDISCUSSIONS

2

11 some formation or protein synthesis. All kinetic curves exhibit three distinct regimes: a lag phase, a linear increase and a slow (almost saturating) regime. It can be observed that increasing temperature accelerates the rate of emGFP production and reduces the expression period. The total amount of synthesized proteins is not monotonic in tem-perature as at 45±_{C the maximal fluorescence intensity is substantially decreased. To} in-vestigate the effect of freeze-thaw, reaction tubes containing the enzyme mix and DNA templates underwent different numbers of freeze-thaw cycles prior incubation in the presence of the feeding solution, and the yield of emGFP production was evaluated by performing end point fluorescence measurements (Figure 2.4b). The PURE system re-tains about 60% of its activity after applying four cycles; we found that this number offers the best compromise between the amount of synthesized proteins and the yield of im-mobilized unilamellar liposomes.

Using the protocol described in Figure 2.2, we prepared liposomes with different lipid compositions, all comprising 0.5% mole of DSPE-PEG-biotin for vesicle stabilization and surface immobilization, and 0.5% mole of DHPE-TRITC for liposome localization: DMPC/DMPG = 4:1 molar ratio (termed DM liposomes; phase transition temperature, Tm º 23±C), DPPC/DPPG = 4:1 molar ratio (DP liposomes; Tm º 41±C) and DOPC (DO liposomes; Tm º -20±_{C). To determine if the presence of DH- and DS-containing lipids} affects the Tm value, we performed calcein efflux measurements with DM and DP lipo-somes and found a slight shift of about +1±_{C from that of pure DMPC or DPPC vesicles} (Figure 2.3).

!

Figure 2.3: Carboxyfluorescein efflux assays with (left) DM liposomes and (right) DP liposomes.

Liposomes were generated by lipid film swelling above the Tm, at 30±_{C for DM} (un-less differently indicated) and DO liposomes, and at 45±_{C for DP liposomes. As shown} in Figures 2.4 and 2.6, unilamellar and multilamellar vesicles with sizes ranging from >1

µm to about 20 µm could be formed and immobilized at high density with all liposome compositions tested. While large and giant unilamellar vesicles are also observed, most liposomes have internal bilayer structures that form sub-compartments.

(20)

2

12

2.2.1 Effects of liposome size on internal gene expression

Heterogeneity in the expression levels is high between individual vesicles for all time points analyzed and no obvious size dependence could be observed. As exemplified in Figure 2.4c, e with DM vesicles, sub-compartments with different expression levels are frequently observed and compartment-specific fluorescence intensity can be quantified (Figure 2.4d).

As compared to batch reactor experiments, liposomal compartmentalization of the tran-scription/translation apparatus re-establishes the stochastic nature of gene expression observed in cells, which leads to a variability of protein expression levels in an isogenic population of cells exposed to the same environment [30]. In addition to the random partitioning of all reactant molecules in the vesicles, the observed variation in the levels of gene expression between single liposomes/sub-compartments might be attributed to a combination of several effects, such as the efficacy of matter exchange with the exter-nal solution, surface effects, confinement-enhanced phenomena [2].

Confined protein synthesis relies on two main parameters: the presence of all necessary components for transcription and translation, and the efficient exchange of matter (up-take of nutrients and efflux of waste products) between the interior and the exterior of the liposome. The former is probabilistic and may vary between vesicles of equal sizes, while the latter depends on the surface to volume ratio and membrane composition. Interestingly liposome reactors with >1 µm diameter (lower probability to enclose all necessary reactants and higher exchange capability) or º 10 µm diameter (higher prob-ability to enclose all necessary reactants and lower exchange capprob-ability) exhibit fluores-cence (Figures 2.4, 2.6). We rarely observed liposomes with diameters larger than 10 µm expressing emGFP or CFP. To explain protein synthesis in submicrometer liposomes, a molecular crowding effects occurring during liposome formation can be hypothesized, as previously shown using the ethanol injection method for vesicle production [12].

2.2.2 Liposome immobilization and shape

We observed that after immobilization at 37±_{C liposomes undergo considerable} rear-rangements. After the first two hours of incubation, growth of population of smaller, more spherical and unilamellar liposomes is obvious.

2.2.3 Possible mechanisms for membrane permeability

We first investigated gene expression in surface-tethered DM liposomes. mRNA and pro-tein syntheses can be triggered inside individual vesicles by external supply of nutrients and tRNAs as shown in Figure 2.4c, e with two different fluorescent proteins. How does internal resource allocation take place? Two distinct effects are likely to influence mem-brane permeability and enable liposome feeding. First, diluting enzyme-loaded vesicles with the feeding solution leads to an osmotic pressure due to the difference of osmo-larity between the inside and outside of the liposomes, which generates membrane de-fects. Second, when temperature is close to the lipid bilayer Tm, molecular diffusion,

(21)

2

13

!

Figure 2.4: a) Time course of emGFP expression in batch reaction at different temperatures. b) End-point flu-orescence measurements of emGFP synthesis in batch reaction after different numbers of freeze-thaw cycles. Data points from two independent experiments are shown. c) Typical fluorescence confocal micrographs of

surface-tethered DM liposomes (red) expressing emGFP (green). Swelling was performed at 30±_{C for 2 hours}

and liposomes were imaged after 23 or 5 hours (inset) incubation at 37±_{C. Arrowheads indicate}

submicrome-ter liposomes expressing emGFP. Scale bars 5 µm. d) Intensity plots of TRITC (red) and emGFP (green) along the dotted line in fig. 2.4c) inset. e) Dual gene expression assay in surface-immobilized DM liposomes. TRITC (red), CFP (cyan) and emGFP (green). 250 ng of CFF- and emGFP-encoding DNAs were co-encapsulated and

liposomes were imaged after 2 hours incubation at 37±C. Scale bars 2 µm.

including that of nucleotide triphosphates [28, 29], across the vesicle membrane is en-hanced. For the reactions to occur, liposomes are incubated at 37±_{C, that is, far above} the Tm. Therefore temperature-assisted permeation across DM membrane is unlikely at these conditions. It is remarkable that the initial osmotic stress is not too large for bursting the liposomes but large enough to generate membrane defects that can accom-modate the diffusion of nutrients and tRNAs, while maintaining the DNA template, the enzymes/cofactors and synthesized proteins trapped.

(22)

2

14

2.2.4 Nutrient and tRNA uptake

To initiate gene expression in surface-immobilized liposomes, the nutrients (mainly amino acids and nucleotide triphosphates), as well as a tRNA mixture, were supplied in the outside solution. Unexpectedly, tRNAs (molecular mass about 20 kDa) are capable to diffuse across DM and DO bilayers and enter into the liposomes, where they are aminoa-cylated and used for translation. It was previously reported that low molecular weight nutrients, under osmotic pressure conditions or through Æ-hemolysine pores, could penetrate inside liposomes and be consumed for gene expression using an entrapped bacterial cytoplasmic extract that already contains tRNAs [13]. To our knowledge we provide the first evidence of the uptake inside liposomes of tRNAs initially present in the external feeding solution.

Passive diffusion through fatty acid or phospholipid membranes has been shown to be a plausible mechanism for the selective uptake of uncharged nutrients by primitive cells [31, 32]. However, phospholipid bilayers are known to be an efficient barrier against ionic nutrients, such as amino acids and nucleotides [32]. Figure 2.5 shows the relative dimensions of tRNA, GFP and DMPC bilayer. Obviously, large membrane rearrange-ments have to occur in order to enable the translocation of tRNA and GFP molecules. Hence, the uptake of tRNAs through a passive diffusion mechanism can be ruled out. Increased membrane permeability to ionic nutrients near the lipid melting transition is well documented [28, 29], and its microscopic nature has been quantitatively stud-ied [33]. To determine whether DM and DP liposome membranes could be made more permeable thus providing a more efficient exchange platform with the feeding environ-ment, we applied temperature cycles to cross their respective Tm. The fact that no fur-ther gene expression could be observed when crossing the lipid phase transition temper-ature multiple times (Figure 2.6d and Figure 2.8) indicates that tRNAs, and to a less ex-tent nutrients, can not efficiently penetrate inside the liposomes. Therefore, membrane defects generated near lipid melting transition is not the dominant cause of membrane permeability to resources.

Although a thorough understanding of the mechanism underlying tRNA permeation across DM and DO membranes would require further investigation, we propose two ef-fects that could explain tRNA uptake by liposomes:

(1) Osmotic pressure-induced membrane defects. It has long been reported that os-motic pressure (even small), by generating defects or pores in the membrane of lipo-somes made of phospholipids, causes an increase in flip-flop rate and of permeability to charged substances [34]. This osmotic stress-induced membrane permeability depends on the lipid composition and occurs through transient membrane rupture followed by resealing [35–37]. For instance, DOPC liposomes subjected to osmotic gradients of more than about 400 mOsm become leaky to charged molecules and their rupture (or max-imal sustainable) membrane tension is about 10 dyn/cm [38]. In a recent study, the formation of large hydrated pores induced by a difference of osmolarity between the in-side and the outin-side of liposomes, and their subsequent relaxation could be measured in real-time [39]. The large morphological changes following the osmotic shock suggest that molecules as large as tRNAs could cross DM and DO lipid bilayers in the fluid phase. As the DP liposome membrane is in the gel phase at 37±_{C , it is likely that its mechanical} properties and thickness are not compatible with efficient permeability to nutrients and

(23)

2

15

!

Figure 2.5: Comparison of the dimensions of tRNA (left) and GFP (middle) molecules, and the thickness of a

DMPC bilayer (right). The tRNA and GFP structures were generated with PyMOL (:http://www.pymol.org/)

using the PDB IDs 1TN2 for the tRNA and 1GFL for the GFP. The DMPC bilayer image is from Dr. Pe-ter Tieleman?s laboratory at the University of Calgary Biocomputing Group and is available online from:

:http://moose.bio.ucalgary.ca/index.php?page=Structures_and_Topologies. The chemical structure of DMPC

molecule is also represented.

tRNAs.

(2) tRNA-bilayer interaction. There are several reports on the interaction between tR-NAs and lipid bilayers [40–42]. Adsorption is not only controlled by electrostatic inter-action between the phosphate groups of the tRNAs and the lipid headgroup, but also by hydrophobic interaction between the exposed nucleobases and the hydrophobic part of the bilayer. The interaction is reversible and is stronger in fluid-state bilayers, where lipid packing is less dense. Importantly, interaction with zwitterionic and negatively charged lipids as those used in the present study had little effects on the conformation of tRNA [41, 42]. Of note, in these studies no osmotic pressure was exerted on the lipid bilayer. We suggest that the conjunction of tRNA-bilayer interaction and osmotic pressure-induced membrane defects could lead to the permeation of tRNA across a lipid bilayer and its delivery inside liposomes. Reversible adsorption of tRNAs onto liposome mem-brane increases their local concentration, while large hydrated defects induced by the osmotic stress generated upon resource supply would provide the permeation pathways for tRNAs.

2.2.5 Leakage of intravesicular content

Then, one can question how some translation factors of lower or similar molecular weight as tRNAs remain entrapped within liposomes. Had the liposomes be leaky for these compounds, one would expect gene expression not to occur in the intravesicular space. Continuous cell-free translation devices are based on semipermeable ultrafiltra-tion membranes with pore sizes large enough for the penetraultrafiltra-tion of small proteins (typi-cal molecular mass cutoff is 30 kDa) [43, 44]. It has been suggested that translation com-ponents are continuously involved in interaction with each others and thus are trapped

(24)

2

16

!

Figure 2.6: a-c) Fluorescence confocal micrographs of surface-immobilized liposomes diluted with the NutM

solution and incubated at 37±_{C for emGFP synthesis. a) DP liposomes after 5 hours incubation. No expression}

of emGFP could be detected. The image is a montage obtained on the same sample from two different fields of view under identical acquisition settings. b) DO liposomes after 21 hours incubation. c) DM liposomes

generated at 45±C, immobilized and incubated for 21 hours prior imaging. d) Single liposome quantification

of emGFP intensity at 0, 2, 5 and 21 hours incubation times in the feeding solution. The main lipid in the liposome composition and the swelling temperature are indicated. To cross the Tm in DM liposomes, one

temperature cycle (37±_{C to 4}±_{C and back, thus the Tm is crossed twice per cycle) was applied between each}

time points. Scale bars 10 µm.

in the reaction chamber [43, 44]. Similarly, we suggest that the different reactional com-ponents in vesicles are engaged in functional multiprotein/mRNA complexes, which are incapable of passing through the bilayer defects.

One may also ask why internally synthesized fluorescent proteins (molecular mass of about 27 kDa) do not completely leak out of the vesicles even after more than 20 hours incubation (Figure 2.6d).

To examine the permeability of DM liposome membrane to fluorescent proteins, we produced CFP-expressing vesicles with pre-synthesized emGFP present in the outside solution (see Materials and Methods). Figure 2.7 shows that two hours after supplying the emGFP-containing feeding solution, a significant number of liposomes expressing CFP exhibit also emGFP fluorescence. This result indicates that fluorescent proteins can

(25)

2

17 diffuse across DM liposome bilayer when supplemented together with the feeding solu-tion. It has to be noted that, in most liposomes, internal emGFP fluorescence is markedly lower than that of the outside solution demonstrating that DM membrane, though not impermeable, significantly restricts the permeation of emGFP.

!

Figure 2.7: Permeability assay of DMPC membrane to pre-synthesized emGFP. a-c) Fluorescence confocal im-ages of CFP (blue)-producing DM liposomes (red) with pre-synthesized emGFP proteins (green) supplemented in the outside feeding solution. Scale bars are 5 µm. d) Intensity plots along the dotted line in a) with similar color coding. The green dashed line corresponds to background fluorescence level measured in the emGFP channel for emGFP-free liposomes.

We envision two possible scenarios to explain the long-term presence of fluorescent proteins inside liposomes. First, we expect the osmotic stress to occur mainly when supplying the feeding solution. Then, membrane tension decreases and less defects are generated. Since emGFP/CFP molecules are synthesized with a time lag of several min-utes following the addition of resources, we hypothesize that the fluorescent proteins are produced in more "relaxed", less leaky, liposomes. Second, prolonged synthesis of flu-orescent proteins in semipermeable vesicles could compensate for their partial efflux. In that case, one assumes that membrane is permeable to nutrients and tRNAs for sev-eral hours to sustain liposome fuelling. However, we could not measure any significant increase of fluorescence in the external solution over time, which, despite the dilution effect upon diffusion of the fluorescent proteins outside the liposomes, suggests that li-posome membranes become poorly leaky to fluorescent proteins.

2.2.6 Possible mechanisms for prolonged expression in liposomes

We observed that gene expression could be re-established in DMPC liposomes swelled at 45±_{C and fuelled with fresh resources (Figure 2.6c, d). This finding suggests that toxic}

(26)

2

18

reactional side products reducing the transcription/translation lifetime can efficiently be cleared out of the vesicle reactor and/or nutrient shortage is delayed due to the in-flux of external resources. Hence, DM liposomes, by providing femtoliter dialysis reac-tion vessels, can be seen as ultraminiaturized semi-continuous gene expression systems [43, 44]. Another possible explanation relies on the conformational stability of tRNA with respect to temperature. The tRNA has a functional clover-leaf (or L-shaped) sec-ondary structure that is stabilized by magnesium ions under heat stress [41]. The melt-ing temperature of tRNA is about 48±_{C in the absence of magnesium and 60}±_{C in the} presence of magnesium [41]. Although the PURE system solution contains magnesium, it could be that gene expression at 45±_{C is less efficient (Figure 2.4a) as more tRNAs adopt} non-functional conformations. Note also that the presence of POPC liposomes (similar headgroup as the lipids used in this study) had minor effect on the tRNA melting temper-ature [41, 42]. Since nutrients and tRNAs are supplied to liposomes at 37±_{C , we expect} temperature-dependent tRNA denaturation to be minimal, which would lead to longer protein synthesis inside vesicles (Figure 2.6c) than in batch mode reactions performed at 45±_{C (Figure 2.4a).}

2.2.7 DNA concentration is not a limiting factor

We then sought to identify whether DNA concentration was limiting the yield of lipo-somes expressing the fluorescent proteins. We performed a dual gene expression assay consisting of co-encapsulating two different DNA molecules, each encoding for a spe-cific coloured fluorescent protein. Figure 2.4e shows three representative liposomes ex-pressing either both or none of the cyan and green fluorescent proteins, suggesting that, at least for >1-µm vesicles/sub-compartments, other components than DNA are not en-closed at sufficient concentration to enable gene expression. On the contrary, synthesis of either CFP or GFP would have indicated that DNA concentration is a limiting factor for the whole reaction.

2.2.8 Effect of lipid composition on internal gene expression

To better understand and further extend the microreactor properties, several lipid com-positions and incubation conditions were tested, and gene expression was quantified at four different time points after addition of the feeding solution. DP vesicles mainly com-posed of 16 carbon-acyl chain phospholipids are in the liquid-ordered phase at 37±_{C and} fail to express fluorescent proteins (Figure 2.6a). As shown in Figure 2.4a, gene expres-sion in a batch reactor ceases after about 30 min and leads to low production at 45±_{C .} To discriminate between a deleterious effect on the PURE system activity when forming DP liposomes at such a high temperature and a reduced membrane permeability due to a thicker bilayer, we also swelled DM liposomes at 45±_{C . Not only can GFP-synthesizing} liposomes be observed (Figure 2.6c), the expression significantly increases for more than 5 hours (Figure 2.6d). These results indicate that the DP membrane acts as a barrier for nutrient uptake. In addition, enzymatic activity in semipermeable DM liposomes can partly be “rescued” and gene expression prolonged for longer time periods than that in batch mode reactions. DO liposomes made of unsaturated 18 carbon-chain phospho-lipids are in the liquid-disordered phase at 37±_{C and successfully express GFP (Figure}

(27)

2.3.CONCLUSION

2

19 2.6b). Note that the average size of immobilized liposomes is smaller than that of DM and DP vesicles. The highly heterogeneous expression level observed between individ-ual liposomes for all conditions tested is featured in Figure 2.6d by the large standard deviation. No further increase of protein synthesis could be observed when crossing the Tm in DM and DP liposomes (Figure 2.6d and Figure 2.8).

!

Figure 2.8: Fluorescence confocal image of DM liposomes (red) expressing emGFP (green). Gene expression was initiated by supplying the PURE system solution A in the outside medium and the image was taken 5 hours

later. The sample was incubated at 37±C and subjected twice to a temperature cycle (37±C to 4±C and back),

one at about 1 hour and the other at 3 hours after the start of the incubation. The image is representative of the liposomes analyzed in Figure 2.6d at 5-hour time point (red bar). Scale bar 10 µm. Crossing the Tm of DP

liposomes (room temperature to 50±_{C and back to 37}±_{C ) failed to result in detectable amounts of emGFP}

(data not shown).

2.3 Conclusion

W

Eestablished a simple methodology to produce highly concentrated lipid vesicles loaded with a minimal transcription and translation machinery. Additionally, it raises the possibility to engineer an active interfacial membrane platform for surface immobilization, fluorescence localization of liposomes and internal membranous struc-tures, and controlling molecular exchange through pressure- and temperature-sensitive bilayer permeability. To eliminate external synthesis and trigger gene expression inside surface-positioned liposome microreactors, we performed in situ buffer exchange with an osmolarity mismatched feeding medium. We found that short 14-carbon chain phos-pholipids are particularly suited for compartmentalized gene expression with a semi-permeable boundary.

Our system offers a unique possibility to analyze compartmentalized gene expression over long time periods in ensemble vesicle array [28, 29], down to the single molecule level. Protein-synthesizing vesicles could be integrated in microfluidic devices with

(28)

po-2

20

tential for further miniaturization and interesting bioanalytical applications for phar-maceutical and medical screening. The field of applications can be extended beyond cell-free gene expression in confined space. Our approach is ideally suited for investigat-ing thermodynamics and kinetics of other complex multiprotein reaction networks in a cell-sized reaction chamber with triggered membrane permeability. Thus, our method-ology has the potential to transcend the applicability of liposome research to the de-velopment of vesicle-based systems with high degree of complexity necessary to create semi-synthetic minimal cells.

2.4 Experimental Section

2.4.1 Materials and Methods

Linear DNA Constructs Linear 1078-bp-long DNA templates encoding for the CFP

or emGFP protein (Scheme S1) were prepared from the pRSET/CFP and pRSET/emGFP vectors (Invitrogen), respectively, by polymerase chain reaction (PCR). The following for-ward and reverse primers were used:

Forward primer: 5’-GCGAAATTAATACGACTCACTATAGGGAGACC-3’ Reverse primer: 5’-TTCGCTATTACGCCAGATCCGGATATAGTTCC-3’

The primers were purchased from Biolegio. For each construct, a PCR with 1

!

Scheme 2.1: Schematic of the DNA templates.

unit of the Phusion enzyme from Finnzymes was performed in the presence of 10 ng of plasmid DNA, 0.2 mM dNTPs, 0.4 muM forward and 0.2 muM reverse primers in a total volume of 50 mul (of which 10 mul was 5x Phusion HF buffer). The generated linear DNA fragment was purified with the PCR Clean-up kit from Promega according to the manufacturer protocol. The purity of the DNA product was checked on a 0.9 % agarose gel by using 50 ng of DNA and ethidium bromide staining. The 1 Kb Plus ladder from Invitrogen was used.

In vitro gene expression in batch reactions The PURE system

The linear DNA templates were expressed in vitro by using the PURExpress kit. This kit, based on the PURE system technology, was purchased from New England Biolabs (NEB # E6800S). The kit consists of two solutions: Solution B contains the T7 RNA polymerase and all essential components for translation and nucleotide triphosphate regeneration. Solution A is the feeding mix and contains all the nutrients needed for the transcription

(29)

2.4.EXPERIMENTALSECTION

2

21 and translation reactions, including tRNAs. Solutions A and B were aliquoted into vials of 10 and 7.5 muL, respectively, and stored at – 80±_{C .}

Spectrofluorometry assays

The effect of temperature on the kinetics of emGFP expression (Figure 2.4a) was stud-ied using the following composition: 7.5 µL of solution B, 10 µL of solution A, 250 ng of DNA template, 0.5 µL of RNase inhibitor (10 or 20 units of Superase or Murine inhibitor, respectively), 20 µL of reaction buffer (50 mM HEPES, 100 mM potassium glutamate) and nuclease-free water to reach a total volume of 53 µL. The solution was kept on ice, transferred into a 40 µL cuvette (Varian) and covered with 20 µL of mineral oil (Sigma-Aldrich). The cuvette was mounted in the temperature-controlled holder of a fluores-cence spectrophotometer (Cary Eclipse from Varian) and fluoresfluores-cence was measured every 2 minutes (excitation 490 nm, emission 510 nm).

To quantify the effect of freeze-thaw cycles on the yield of synthesized emGFP (Fig-ure 2.4b) a solution containing 7.5 µL of solution B, 0.5 µL of DNA (150 ng) and 7 µL of nuclease-free water was split into six aliquots of 2.5 µL. Each solution was subjected to a different number of freeze-thaw cycles (liquid nitrogen – heating block at 30±_{C ). Then,} 1.6 µL of solution A was added to each sample prior incubation at 37±_{C for 2 hours.} Finally, the samples are diluted with 40 µL of reaction buffer (50 mM HEPES, 100 mM potassium glutamate) into a 40 µL cuvette (Varian) and the fluorescence signal was mea-sured with a Cary Eclipse spectrophotometer (Varian) (excitation 490 nm, emission 510 nm).

Gene expression in liposomes Lipids

1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC, 14:0), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, 16:0), 1,2-dimyristoyl-sn-glycero-3-phospho-(1’-rac-glycerol) (DMPG, 14:0), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1’-rac-1,2-dimyristoyl-sn-glycero-3-phospho-(1’-rac-glycerol) (DPPG, 16:0) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000] (DSPE-PEG-biotin, 18:0) were all purchased from Avanti Polar Lipids. The N-(6-tetramethylrhodaminethiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (TRITC-DHPE, 16:0) was purchased from Invitrogen.

Three different lipid compositions were used:

(1) DMPC:DMPG:TRITC-DHPE:DSPE-PEG-biotin = 4:1:0.05:0.025 molar ratio (2) DPPC:DPPG:TRITC-DHPE:DSPE-PEG-biotin = 4:1:0.05:0.025 molar ratio (3) DOPC:TRITC-DHPE:DSPE-PEG-biotin = 1:0.01:0.005 molar ratio

(30)

2

22

Preparation of lipid-coated beads

Glass beads (212-300 µm) were purchased from Sigma-Aldrich. Lipids dissolved in chlo-roform or in chlochlo-roform:methanol (1:1 volume ratio) were mixed to obtain the desired molar ratio and added to the beads in a round bottom glass flask at a ratio of 2 mg of lipids per gram of beads. The solvent was then slowly evaporated in a rotavapor and the beads were further dried in a dessicator for 2 hours at 12 mbar. The lipid-coated beads could be stored under N2 at -20±_{C for several weeks.}

Liposome formation

Liposomes were produced as described in Figure 2.2. The equivalent of about 20 µL of lipid-coated beads were poured in a reaction tube and the swelling solution was added such that the beads were completely immersed. The swelling mixture consists of 7.5 µL of the PURE system solution B, 2.5 µL of nuclease-free water, 0.5 µL of Superase inhibitor (10 units final) and 0.6 µL of DNA template (250 ng). The volume of the swelling solution could be reduced to a few microliters when less beads were used. The lipid film was swelled above the lipid phase transition temperature for 1 to 2 hours. Then, the solution was snap frozen in liquid nitrogen and thawed in a heating block at 30±_{C four times.} The tube was gently rotated to unpack the beads and release the liposomes that were carefully collected and immobilized on a microscope coverslip.

Liposome immobilization and reaction initiation

Microscope coverslips were sonicated for 15 minutes in ethanol. Dried coverslips were incubated with 1 mg/mL BSA:BSA-Biotin (1:1) (BSA was from Sigma-Aldrich, BSA-Biotin was from Thermo Fisher Scientific) for 10 minutes, washed twice with reaction buffer (50 mM HEPES, 100 mM potassium glutamate), incubated for 10 minutes with 1 mg/mL neutravidin (Sigma-Aldrich) and washed twice with reaction buffer. Finally, 1 µL of the liposome solution was added into a 1.5-2-mm thick PDMS chamber fixed onto the neutravidin-coated microscope coverslip. After 5-10 minutes incubation at 37±_{C , 2 µL of} the PURE system solution A were added and the chamber was sealed with a clean glass coverslip. When indicated, liposomes were diluted with solution A supplemented with the RQ1 DNase (0.25 units final; from Promega). The sample was incubated at 37±_{C .}

Fluorescence imaging and data analysis We used a laser scanning confocal

micro-scope (LSM710, Zeiss) equipped with a ×40 oil immersion objective. The following fluo-rescence settings were used: TRITC (Exc 543 nm, Em 553-797 nm), emGFP (Exc 488, Em 492-523 nm) and CFP (Exc 458 nm, Em 465-483 nm). Fluorescence measurements were performed at room temperature (18±_{C ). To quantify the emGFP fluorescence within} in-dividual liposomes over a broad range of intensity levels, images were acquired using two different detector gains. Fluorescence images were analyzed with the software ImageJ. The fluorescence intensity inside single liposomes/sub-compartments was determined as the averaged intensity subtracted from the background intensity. For several vesi-cles, the intensity was monitored with two detector gains and the corresponding ratio

(31)

2.4.EXPERIMENTALSECTION

2

23 values were calculated. The averaged value of intensity ratios was used as a normaliza-tion coefficient to determine gain-corrected intensity levels enabling direct comparison of emGFP production at different time points and membrane compositions (Figure 3d). Typically, more than 50 liposomes were analyzed for each experimental condition.

Carboxyfluorescein efflux assays Preparation of small unilamellar vesicles

Small unilamellar vesicles (SUVs) loaded with a quenching concentration of carboxyflu-orescein (CF, Sigma-Aldrich) and of compositions (1) and (2) (see section 3.a.) were pre-pared for carrying out CF efflux measurements. A total of 5 mg of lipids dissolved in chloroform or chloroform:methanol (1:1 volume ratio) was poured in a round bottom glass flask. A lipid film was formed by rotary evaporation of the solvent and rehydrated with 1 ml of 25 mM CF in 100 mM HEPES. The flask was heated above the Tm of the lipids and extensively vortexed to resuspend the lipid film. SUVs were produced by the extrusion method (Mini-extruder, Avanti Polar Lipids) using a polycarbonate filter with 200-nm pores (Avanti Polar Lipids). The temperature of the extruder holder was main-tained above the Tm and the liposome solution was passed ten times through the filter. The free CF was separated from the CF-loaded liposomes by size exclusion chromatogra-phy using a G50 Sephadex (Sigma-Aldrich) column equilibrated with an isotonic buffer (100 mM HEPES and 50 mM KCl). The chromatography was performed in a cold room with DM liposomes and at room temperature with DP liposomes. The collected SUVs were then used for CF efflux measurements.

Determination of Tm

The CF-loaded SUV solution was diluted 400 times in isotonic buffer (100 mM HEPES and 50 mM KCl) and transferred in a 40 µL cuvette (Varian) placed into the thermostated chamber of a fluorescence spectrophotometer (Cary Eclipse from Varian). A tempera-ture ramp of 0.5±_{C /min was applied and the CF fluorescence was measured (Exc 490,} Em 510). Near the Tm, the entrapped CF is released due to membrane defects and the fluorescence increases upon CF dilution. The Tm value is determined as the temperature at the inflection point (Figure 2.3).

Permeability assay to pre-synthesized emGFP

emGFP proteins were synthesized in a batch mode reaction by incubating the following mix at 37±C for two hours: 3.75 µL of solution B, 5 µL of solution A, 125 ng of emGFP DNA

template, 0.5 µL of RNase inhibitor (10 units of Superase inhibitor), and 3 µL of nuclease-free water. Surface-immobilized DM liposomes containing the CFP gene were prepared as described above. Instead of adding 2 µL of solution A to initiate gene expression, we diluted the liposomes in a feeding medium containing 1.6 µL of solution A and 1 µL of the emGFP-containing PURE system solution. Hence, an osmotic pressure close to the one generated in regular conditions (no pre-synthesized fluorescent proteins) will be exerted to the liposome membrane.

(32)

2

24 REFERENCES

References

[1] Z. Nourian, W. Roelofsen, and C. Danelon, Triggered gene expression in fed-vesicle

microreactors with a multifunctional membrane. Angewandte Chemie

(Interna-tional ed. in English) 51, 3114 (2012).

[2] S.-i. M. Nomura, K. Tsumoto, T. Hamada, K. Akiyoshi, Y. Nakatani, and K. Yoshikawa, Gene Expression within Cell-Sized Lipid Vesicles, ChemBioChem 4, 1172 (2003).

[3] W. YU, K. Sato, M. WAKABAYASHI, T. NAKAISHI, E. P. KO-MITAMURA, Y. SHIMA, I. Urabe, and T. Yomo, Synthesis of Functional Protein in Liposome, Journal of Bio-science and Bioengineering 92, 590 (2001).

[4] Y. Kuruma, P. Stano, T. Ueda, and P. L. Luisi, A synthetic biology approach to the

construction of membrane proteins in semi-synthetic minimal cells, BBA -

Biomem-branes 1788, 567 (2009).

[5] K. Ishikawa, K. Sato, Y. Shima, and I. Urabe, ScienceDirect - FEBS Letters : Expression

of a cascading genetic network within liposomes, FEBS letters (2004).

[6] T. Sunami, K. Sato, T. Matsuura, K. Tsukada, I. Urabe, and T. Yomo, Femtoliter

com-partment in liposomes for in vitro selection of proteins, Analytical Biochemistry 357,

128 (2006).

[7] H. Kita, T. Matsuura, T. Sunami, K. Hosoda, N. Ichihashi, K. Tsukada, I. Urabe, and T. Yomo, Replication of Genetic Information with Self-Encoded Replicase in

Lipo-somes, ChemBioChem 9, 2403 (2008).

[8] K. Hosoda, T. Sunami, Y. Kazuta, T. Matsuura, H. Suzuki, and T. Yomo,

Quantita-tive study of the structure of multilamellar giant liposomes as a container of protein synthesis reaction. Langmuir 24, 13540 (2008).

[9] T. Sunami, K. Hosoda, H. Suzuki, T. Matsuura, and T. Yomo, Cellular compartment

model for exploring the effect of the lipidic membrane on the kinetics of encapsulated biochemical reactions. Langmuir 26, 8544 (2010).

[10] G. Murtas, Y. Kuruma, P. Bianchini, A. Diaspro, and P. L. Luisi, Protein synthesis

in liposomes with a minimal set of enzymes, Biochemical and biophysical research

communications 363, 12 (2007).

[11] M. Amidi, M. de Raad, H. de Graauw, D. van Ditmarsch, W. E. Hennink, D. J. A. Crommelin, and E. Mastrobattista, Optimization and quantification of protein

syn-thesis inside liposomes. Journal of liposome research 20, 73 (2010).

[12] T. Pereira de Souza, P. Stano, and P. L. Luisi, The minimal size of liposome-based

model cells brings about a remarkably enhanced entrapment and protein synthesis.

(33)

REFERENCES

2

25 [13] V. Noireaux and A. Libchaber, A vesicle bioreactor as a step toward an artificial cell

assembly, Proceedings of the National Academy of Sciences of the United States of

America 101, 17669 (2004).

[14] V. Noireaux, R. Bar-Ziv, J. Godefroy, H. Salman, and A. Libchaber, Toward an

artifi-cial cell based on gene expression in vesicles, Physical Biology 2, P1 (2005).

[15] H. Saito, Y. Kato, M. Le Berre, A. Yamada, T. Inoue, K. Yosikawa, and D. Baigl,

Time-Resolved Tracking of a Minimum Gene Expression System Reconstituted in Giant Li-posomes - Saito - 2009 - ChemBioChem - Wiley Online Library, ChemBioChem 10,

1640 (2009).

[16] S. Ota, S. Yoshizawa, and S. Takeuchi, Microfluidic Formation of Monodisperse,

Cell-Sized, and Unilamellar Vesicles, Angewandte Chemie-International Edition 48,

6533 (2009).

[17] P. L. Luisi, F. Ferri, and P. Stano, Approaches to semi-synthetic minimal cells: a review. Die Naturwissenschaften 93, 1 (2006).

[18] P. Schwille and S. Diez, Synthetic biology of minimal systems. Critical reviews in bio-chemistry and molecular biology 44, 223 (2009).

[19] V. Noireaux, Y. T. Maeda, and A. Libchaber, Inaugural Article: Development of an

ar-tificial cell, from self-organization to computation and self-reproduction,

Proceed-ings of the National Academy of Sciences 108, 3473 (2011).

[20] D. Stamou, C. Duschl, E. Delamarche, and H. Vogel, Self-assembled microarrays of

attoliter molecular vessels. Angewandte Chemie (International ed. in English) 42,

5580 (2003).

[21] G. Gopalakrishnan, C. Danelon, P. Izewska, M. Prummer, P.-Y. Bolinger, I. Geiss-bühler, D. Demurtas, J. Dubochet, and H. Vogel, Multifunctional lipid/quantum

dot hybrid nanocontainers for controlled targeting of live cells. Angewandte Chemie

(International ed. in English) 45, 5478 (2006).

[22] P.-Y. Bolinger, D. Stamou, and H. Vogel, An integrated self-assembled nanofluidic

system for controlled biological chemistries. Angewandte Chemie (International ed.

in English) 47, 5544 (2008).

[23] T. Oberholzer, K. H. Nierhaus, and P. L. Luisi, Protein Expression in Liposomes, Bio-chemical and biophysical research communications 261, 238 (1999).

[24] T. Oberholzer and P. L. Luisi, The use of liposomes for constructing cell models. Jour-nal of biological physics 28, 733 (2002).

[25] K. Yamabe, Y. Kato, H. Onishi, and Y. Machida, In vitro characteristics of liposomes

and double liposomes prepared using a novel glass beads method. Journal of

Towards the assembly of a minimal oscillator

T

OWARDS THE

A

SSEMBLY OF A

M

INIMAL

O

SCILLATOR

T

OWARDS THE

A

SSEMBLY OF A

M

INIMAL

O

SCILLATOR

G

ENETIC

N

ETWORKS IN

L

IPOSOMES

Proefschrift

Zohreh N

OURIAN

Contents

Chapter 1

Introduction

1

1.1 Building a minimal cell

H

1

1.2 Cell-free gene expression inside liposomes as a

plat-form for the construction of a minimal cell

1

1.3 Thesis Outline

1

References

1

Chapter 2

Triggered Gene Expression in

Fed-Vesicle Microreactors With a

Multifunctional Membrane

2

2.1 Introduction

W

2.1.1 Liposome formation methods

2

2.2 Results and Discussions

H

!

!

2

2

!

2

2.2.1 Effects of liposome size on internal gene expression

2.2.2 Liposome immobilization and shape

2.2.3 Possible mechanisms for membrane permeability

2

!

2

2.2.4 Nutrient and tRNA uptake

2

!

2.2.5 Leakage of intravesicular content

2

!

2

!

2.2.6 Possible mechanisms for prolonged expression in liposomes

2

2.2.7 DNA concentration is not a limiting factor

2.2.8 Effect of lipid composition on internal gene expression

2

!

2.3 Conclusion

W

po-2