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Towards a semi-synthetical cell GROUP 1 Catherine Acquah Jose Aguilar-Rodríguez Susanna Bisogni Quentin Defenouillere Natalie Jayne Haywood Banyuls-sur-Mer,

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Presentation on theme: "Towards a semi-synthetical cell GROUP 1 Catherine Acquah Jose Aguilar-Rodríguez Susanna Bisogni Quentin Defenouillere Natalie Jayne Haywood Banyuls-sur-Mer,"— Presentation transcript:

1 Towards a semi-synthetical cell GROUP 1 Catherine Acquah Jose Aguilar-Rodríguez Susanna Bisogni Quentin Defenouillere Natalie Jayne Haywood Banyuls-sur-Mer, 3 September 2010 Evolution of the Biosphere ERASMUS EDUCATION PROGRAMME

2  INTRODUCTION: SCIENTIFIC BACKGROUND  SCIENTIFIC OBJECTIVE AND EXPERIMENTAL SETTINGS  COMPONENTS OF OUR SYSTEM: THE PURESYSTEM®, PROTEINS AND THE COMPARTMENT  METHODS  RESULTS TESTING AND APPLICATIONS  ETHICAL IMPLICATIONS CONTENTS

3 Chemical building blocks (simple molecules) Biochemical building blocks (bio-organic molecules) Functional oligomers and polymers Cellular organisms Multi cellular organisms complexity organization Luisi (2006) INTRODUCTION: The Origin of Life

4 Definition: self-organized, endogenously ordered, spherical collection of macromolecules proposed as a stepping-stone to the origin of life nutrient uptake waste release primitive metabolism outside inside template en.wiktionary.org/wiki/protocell INTRODUCTION: Protocells

5 INTRODUCTION: Synthetic Biology

6 INTRODUCTION: Synthesis of a Minimal Cell Solé et al. (2010)

7 Cell-like compartment containing the minimal and sufficient number of components in order to be alive Chemical system capable of replication and evolution, fed only by small molecule nutrients It would define the components sufficient for each subsystem, allowing detailed kinetic analyses Luisi (2006) INTRODUCTION: Minimal Cell

8 Stano Presentation Banyuls cell-free/in vitro systems - liposome technology INTRODUCTION: Semi-Synthetic Minimal Cell

9 1.Compartments (vesicles) 2.Simple (bio)reactions in compartments 3.Protein expression 4.Self-replication of the components 5.Shell (membrane) reproduction 6.Core-and-shell reproduction 7.… more? M,N A+B  R ,6 P.L.Luisi – P.Walde – T.Oberholzer (ETH ) INTRODUCTION: A Road Map to the Minimal Cell Stano Presentation Banyuls 2010

10 SCIENTIFIC OBJECTIVE EXPRESSION SYSTEM hv H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ ATP ADP + P i ATP ADP + P i Giant vesicle Artificial organelle Pore Ribonucleotides Amino acids

11 EXPERIMENTAL SETTINGS 1.ENCAPSULATION OF A CELL-FREE EXPRESSION SYSTEM (LIKE THE PURESYSTEM®) INTO GIANT VESICLES 2.FORMATION OF PROTEOLIPOSOMES WITH BACTERIORHODOPSIN AND F 0 F 1 - ATP SYNTHASE 3.INTRODUCTION OF THE PROTEOLIPOSIMES INTO THE GIANT VESICLES BY MICROINJECTION 4. EXPRESSION BY OUR SYSTEM OF AN HETEROLOGOUS PROTEIN, PRODUCT OF THE FUSION OF THE GREEN FLUORESCENT PROTEIN (REPORTER) WITH THE α-HEMOLYSIN (PROTEIN PORE) 5. OBSERVATION OF SYNTHESIZED ENTITIES BY ELECTRONIC AND FLUORESCENT MICROSCOPY 6. COMPARAISON OF THE SYSTEM LIFESPAN WITH SEVERAL NEGATIVE CONTROLS

12 COMPONENTS: The PURESYSTEM® Protein synthesis is one of the most complicated biological processes. Shimizu et al. (2001) have shown that protein synthesis can be recreated from its purified parts. Called the "protein synthesis using recombinant elements" (PURE). The PURE system is now commercially available from Post Genome Institute Co., Ltd (Japan) as PURESYSTEM® kits (Shimizu et al., 2005). A molecular kit of 36 purified enzymes, ribosomes, t-RNAs, and low molecular weight compounds, which synthesize proteins starting from the corresponding DNA

13 COMPONENTS: The PURESYSTEM® Shimizu et al. (2005)

14 COMPONENTS: The PURESYSTEM® Shimizu et al. (2005) COMPONENTS OF THE PURESYSTEM®

15 BACTERIORHODOPSIN from Halobacterium salinarum Goodsell (March 2002) PDB Molecule of the Month COMPONENTS: Proteins Chromophore: Retinal

16 COMPONENTS: Proteins F 0 F 1 - ATP SYNTHASE “All enzymes are beautiful, but ATP synthase is one of the most beautiful as well as one of the most unusual and important.” Paul Boyer “All enzymes are beautiful, but ATP synthase is one of the most beautiful as well as one of the most unusual and important.” Paul Boyer Goodsell (Dec 2005) PDB Molecule of the Month

17 Green Fluorescent Protein from Aequorea victoria Source: PDB Alpha-hemolysin from Staphylococcus aureous Source: PDB COMPONENTS: Proteins

18 THE IMPORTANCE OF BEING A COMPARTMENT COMPONENTS: The Compartment

19 Hydrophobic interactions are the main factors for the association of fatty acids in an aqueous solvent FROM PHOSPHOLIPIDS TO VESICLES COMPONENTS: The Compartment

20 GIANT UNILLAMELAR VESICLES (GUVs) Walde et al. (2010) COMPONENTS: The Compartment

21 METHODS 1. ENCAPSULATING THE PURESYSTEM® INTO GIANT VESICLES 1.A small volume of the aqueous solution containing the Puresystem® is added to mineral oil with disolved phospholipids. 2.Agitate to create an aqueous-oil emulsion. The microdroplets generated are stabilized by a monolayer of phospholipids. 3.The emulsion is placed on top of the feeding solution. 4.The giant vesicles (>10 μl) are created when the droplets pass through the monolayer at the interface of the biphasic solution. Noireaux and Libchaber (2004) Ribonucleotides, amino acids, buffer (pH 7.4-8)

22 METHODS 2. PREPARATION OF PROTEOLIPOSOMES (detergent-mediated reconstitution) CELLULAR MEMBRANE + Detergent PROTEOLIPOSOMES MICELLES Adapted from Rigaud et al. (1995) Detergent removal by dialysis Overexpression of the desired protein

23 METHODS 3. INTRODUCING PROTEOLIPOSOMES INTO GIANT VESICLES Holding micropipette Giant vesicle (10-50 μm of diameter) Proteoliposomes ( nm) Injection micropipette (0.5 μm of diameter) PURESYSTEM ®

24 RESULTS TESTING Internal structure of synthesized entities can be checked by electronic microscopy Location of GFP::α-hemolysine at the membrane can be verified by fluorescent microscopy Comparaison of the lifespan of biological entities and the fusion protein production rate with several negative controls : Same system with a GFP::Albumine fusion protein instead, PureSystem in giant unilamellar vesicles without liposomes, Puresystem in vitro without compartment. This data can be measured by following the fluorescence intensity of GFP over a period of time (the GFP intensity rate is a direct witness of the biological entities living rate).

25 APPLICATIONS 1.THE FIRST STEP TOWARDS THE ASSEMBLY OF A SYNTHETIC MINIMAL CELL 2.TEST CHAMBER TO DEVELOP AND TEST SYNTHETIC GENOMES 3.CREATION OF A CELL-LIKE BIOREACTOR Adventures in Synthetic Biology Synthetic M. mycoides genome (Gibson et al., 2010)

26 ETHICAL IMPLICATIONS Protocells do not yet exist 5 or 10 years Existence out of the laboratory20 years Protocells Nowadays: Basic Research Tomorrow: Private Enterprise Today the risks linked with protocells research are negligible (Cranor, 2009) BUT The perception of the risks is not negligible

27 ETHICAL IMPLICATIONS Protocell: Self assembling and self reproducing chemical system, having the following properties (Rasmussen et al., 2009): Comparmentalization Metabolism Genetical Information Protocell can reproduce themselves Genetic information can mutate Population can adapt and evolve Protocell can undergo natural (or artificial) selection

28 ETHICAL IMPLICATIONS POSSIBLE PROBLEMS Social, cultural and religious Conflict with religious principles Violating nature (Fukuyama, 2002) Playing God (Bedau and Parke, 2009) Potential use as weapons Biocompatibility Fixed percentage of the funds for (Gaisser et al. 2008): ethical and legal implications Public education Public communication of each reached result Intellectual property regulations RESPONSIBLE STEPS TO UNDERTAKE

29 ETHICAL IMPLICATIONS POSSIBLE RISKS Top Down Only social risk Laboratory security Bioterrorism Environmental issues Bottom Up No special laboratory security Protocells arise “From Scratch” But Protocells arise “From Scratch” Protocells: Unpredictability of chemical programmability Dynamic entities

30 ACKNOWLEDGMENTS We thank Pasquale Stano from the RomaTre University in Rome for his advise and technical help.

31 SELECTED LITERATURE BEDAU, M.A., PARKE, E.C., TANGEN, U., HANTSCHE-TANGEN, B. (2009). Social and ethical checkpoints for bottom-up synthetic biology, or protocellS. Systems and Synthetic Biology. 3: DE LORENZO, V. and DANCHIN, A. (2008). Synthetic biology: discovering new worlds and new words. EMBO. 9: LUISI, P.L. (2006). The Emergence of Life - From Chemical Origins to Synthetic Biology. Cambridge University Press. NOIREAUX, V. and LIBCHABER, A. (2004). A vesicle bioreactor as a step toward an artificial cell assembly. Proceedings of the National Academy of Sciences. 101: RIGAUD, J-L., PITARD, B. and LEVY, D. (1995). Reconstitution of membrane proteins into liposomes: application to energy-transducing membrane proteins. Biochimica et Biophysica Acta. 1231: SHIMIZU, Y., INOUE, A., TOMARI, Y., SUZUKI, T., YOKOGAWA, T., NISHIKAWA, K., UEDA, T. (2001) Cell-free translation reconstituted with purified components. Nature Biotechnology 19,: 751 – 755. SHIMIZU, Y., KANAMORI, T., UEDA, T. (2005). Protein synthesis by pure translation systems. Methods. 36:

32 SELECTED LITERATURE SOLÉ, R.V., MUNTEANU, A., RODRIGUEZ-CASO, C. and MACIA, J. (2007). Synthetic protocell biology: from reproduction to computation. Philosophical Transactions of the Royal Society-Biological Sciences. 362: SWARTZ, J. (2001) A PURE approach to constructive biology. Nature Biotechnology 19: 732 – 733. WALDE, P., COSENTINO, K., ENGEL, H. and STANO, P. (2010). Giant vesicles: preparations and applications. ChemBioChem. 11:

33 ANNEX

34 MATERIALS AND METHODS 1. PREPARATION OF GIANT VESICLES (lipid film hydration method; Reeves and Dowben, 1969) Electroformation: hydration in the presence of an electric field (Angelova and Dimitrov, 1988)


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