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

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 BiosphereERASMUS EDUCATION PROGRAMME

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

CONTENTSCONTENTS

• 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 LifeINTRODUCTION: The Origin of Life

• Definition: self-organized, endogenously ordered, spherical collection of

macromolecules proposed as a stepping-stone to the origin of life

nutrient uptake

waste release

primitive metabolismoutside

inside

template

en.wiktionary.org/wiki/protocell

INTRODUCTION: ProtocellsINTRODUCTION: Protocells

INTRODUCTION: Synthetic BiologyINTRODUCTION: Synthetic Biology

INTRODUCTION: Synthesis of a Minimal Cell INTRODUCTION: Synthesis of a Minimal Cell

Solé et al. (2010)

• 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 CellINTRODUCTION: Minimal Cell

Stano Presentation Banyuls 2010

- cell-free/in vitro systems

- liposome technology

INTRODUCTION: Semi-Synthetic Minimal CellINTRODUCTION: Semi-Synthetic Minimal Cell

1. Compartments (vesicles)2. Simple (bio)reactions in

compartments3. Protein expression4. Self-replication of the components 5. Shell (membrane) reproduction6. Core-and-shell reproduction7. … more?

M,N

A+BR

1

2

3 4 5,6

P.L.Luisi – P.Walde – T.Oberholzer (ETH 1995-2003)

INTRODUCTION: A Road Map to the Minimal CellINTRODUCTION: A Road Map to the Minimal Cell

Stano Presentation Banyuls 2010

SCIENTIFIC OBJECTIVESCIENTIFIC OBJECTIVE

EXPRESSION SYSTEM

hv

H+H+

H+H+

H+H+

H+H+ATP

ADP + Pi

ATP

ADP + Pi

Giant vesicle

Artificial organelle

Pore

RibonucleotidesAmino acids

EXPERIMENTAL SETTINGSEXPERIMENTAL SETTINGS

1. ENCAPSULATION OF A CELL-FREE EXPRESSION SYSTEM (LIKE THE PURESYSTEM®) INTO GIANT VESICLES

2. FORMATION OF PROTEOLIPOSOMES WITH BACTERIORHODOPSIN AND F0F1 - 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

COMPONENTS: The PURESYSTEM®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

COMPONENTS: The PURESYSTEM®COMPONENTS: The PURESYSTEM®

Shimizu et al. (2005)

COMPONENTS: The PURESYSTEM®COMPONENTS: The PURESYSTEM®

Shimizu et al. (2005)

COMPONENTS OF THE PURESYSTEM®

BACTERIORHODOPSIN from Halobacterium salinarum

Goodsell (March 2002) PDB Molecule of the Month

COMPONENTS: ProteinsCOMPONENTS: Proteins

Chromophore: Retinal

COMPONENTS: ProteinsCOMPONENTS: Proteins

F0F1 - 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

Green Fluorescent Protein from Aequorea victoria

Source: PDB

Alpha-hemolysin from Staphylococcus aureous

Source: PDB

COMPONENTS: ProteinsCOMPONENTS: Proteins

THE IMPORTANCE OF BEING A COMPARTMENT

COMPONENTS: The CompartmentCOMPONENTS: The Compartment

Hydrophobic interactions are the main factors for the association of fatty acids in an aqueous solvent

FROM PHOSPHOLIPIDS TO VESICLES

COMPONENTS: The CompartmentCOMPONENTS: The Compartment

GIANT UNILLAMELAR VESICLES (GUVs)

Walde et al. (2010)

COMPONENTS: The CompartmentCOMPONENTS: The Compartment

METHODSMETHODS

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)

METHODSMETHODS

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

METHODSMETHODS

3. INTRODUCING PROTEOLIPOSOMES INTO GIANT VESICLES

Holding micropipette Giant vesicle

(10-50 μm of diameter)

Proteoliposomes(50-200 nm)

Injection micropipette

(0.5 μm of diameter)PURESYSTEM®

RESULTS TESTINGRESULTS 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).

APPLICATIONSAPPLICATIONS

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)

ETHICAL IMPLICATIONSETHICAL IMPLICATIONS

Protocells do not yet exist 5 or 10 years

Existence out of the laboratory 20 years

ProtocellsNowadays: Basic Research

Tomorrow: Private Enterprise

Today the risks linked with protocells research are negligible (Cranor, 2009)

BUTBUT

The perception of the risks is not negligible

ETHICAL IMPLICATIONSETHICAL 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

ETHICAL IMPLICATIONSETHICAL 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

ETHICAL IMPLICATIONSETHICAL IMPLICATIONS

POSSIBLE RISKS

Top DownOnly social risk•Laboratory security•Bioterrorism•Environmental issues

Bottom Up

No special laboratory security

But Protocells arise Protocells arise “From Scratch”“From Scratch”

Protocells: Unpredictability of chemical programmability

Dynamic entitiesDynamic entities

ACKNOWLEDGMENTSACKNOWLEDGMENTS

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

SELECTED LITERATURESELECTED 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: 65-75.

DE LORENZO, V. and DANCHIN, A. (2008). Synthetic biology: discovering new worlds and new words. EMBO. 9: 822-827.

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: 17669-17674.

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: 223-246.

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: 299-304.

SELECTED LITERATURESELECTED 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: 1727-1739.

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: 848-865.

ANNEX

MATERIALS AND METHODSMATERIALS 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)