2. Construction of a chassis and synthetic modules for H 2 production (BioModularH 2 ) Catarina Pacheco Cellular and Applied Microbiology Group IBMC, INEB S12. Biologia de Sistemas Encontro com a Ciência e Tecnologia em Portugal Centro de Congressos de Lisboa,7 th July 2010

3. Synthetic Biology is...... the design and construction of new biological parts, devices and systems and the re-design of existing, natural biological systems for useful purposes. Synthetic Biology is the application of engineering concepts to biology

4. Standardizedparts Assembly of modules and circuits Incorporation in a chassis

5. Consortium members: Instituto de Biologia Molecular e Celular (Portugal) École Polytechnique (France) Universidad Politécnica de Valencia (Spain) Uppsala Universitet (Sweden) University of Sheffield (UK) Weizmann Institute of Science (Israel) FP6-2005-NEST-PATH Contract no.: 043340 Jan.07- Jul.10

6. In silico analysis Computational design of parts and modules Synthesis of parts Assembly of modules Preparation of the chassis Characterization of parts/modules Incorporation in the chassis Evaluation of the final product BioModularH 2 Design Construction Characterization

7. Final goal A cyanobacterial chassis that together with the designed devices will harvest solar energy for H 2 production. The synthetic parts and modules will be available for other biotechnological applications

8. Photoautotrophic chassis - Photoautotrophic chassis - Synechocystis sp. PCC 6803  the most studied cyanobacteria  unicellular and non-N 2 -fixing  simple nutritional requirements  naturally transformable  molecular tools for manipulation available  small genome comprising a 3.6 Mb genome and 7 plasmids (1 st cyano genome sequenced)

9. Preparation of the chassis Tuning respiration Native hydrogenase (s) Nuclease(s) etc… Oxygen consumption Oxygen sensing Highly-efficient O 2 -tolerant hydrogenase Reduce constraints, e.g. enhance transformation efficiency Remove redundant genes / parts Minimize O 2 production / maximize O 2 consumption  H 2 ases are very sensitive to O 2

10. Deletion of redundant parts – generation of a hydrogenase deficient mutant Possesses hoxYH Sensitive to kanamycin Resistant to sucrose hoxYhoxH Deletion of hoxYH Resistant to kanamycin Sensitive to sucrose Lacks hoxYH Sensitive to kanamycin Resistant to sucrose

11. Identification of neutral sites for the insertion of synthetic modules Genes encoding proteins: - unknown or hypothetical - with maximum length of 300 a.a. - without predicted transmembrane domains (TMHMM Server v. 2.0) - primary or secondary structure without relevant homologues - that do not interact with other proteins in two-hybrid system (CyanoBase data) 16 potential neutral sites identified

12. - RT-PCRs were performed using total RNA collected from Synechocystis sp. PCC 6803 cultures in the exponential phase (OD 730 = 0.8). 6 neutral sites selected for the generation of mts Transcription assessment by RT-PCR

13. HydA1_Fd Construction of synthetic devices - Hydrogen Producing Device (HPD) Homology models based on Chang et al. 2007 (Biophys J, 93:3034-45) Hydrogenase module Fe-only hydrogenase fused to ferredoxin – Chlamydomonas reinhardtii Maturation module HydEF + HydG – Chlamydomonas reinhardtii

14. The OCD will create the intracellular microaerobic environment necessary for optimal hydrogenase activity. Construction of synthetic devices – Oxygen Consuming Device (OCD) Hydrogenase OCD High O 2 pressure Low O 2 pressure O 2 Sensor H2H2 H2H2 H2H2 H2H2 H2H2 H2H2

15. - A-type flavoprotein (ATF) – Synechocystis sp. PCC 6803 - Laccase – Escherichia coli SINGLE-protein modules O 2  H 2 O TWO-protein module O 2  H 2 O 2  ½ O 2 + H 2 O - Glucose oxidase – Penicillium amagasakiense + Catalase – Synechocystis sp. PCC 6803 Construction of synthetic devices – Oxygen Consuming Device (OCD)

16. Characterization of synthetic devices – Laccase device characterization (E. coli) M9 Glycerol medium vs. M9 Glycerol medium Cu supplemented induction: - stationary growth phase cell harvest: - 12/24 hours after induction. Lac (56 kDa) MW 97 kDa 66 kDa 45 kDa 30 kDa 20 kDa - - + - + - + - + AHL 12h 24h12h 24h - - - - - + + + + Cu(II) F2026 + laccase 24h wt SDS-PAGE

17. Spectrophotometric assay ( ABTS oxidation) - Specific laccase activity was expressed as units of activity per µg of protein extract, where 1 activity unit represents 1 µmol of ABTS oxidized per min. Characterization of synthetic devices – Laccase device characterization (E. coli) O 2 consumption (Clark-type O 2 electrode) - Oxygen consumption was expressed as nmol O 2 consumed per min and normalized per µg of protein extract.

18. Ongoing Work: Oxygen Consuming Device Chassis Synechocystis sp. PCC 6803 Oxygen Consuming Device Highly-efficient O 2 -tolerant hydrogenase Chassis with Hydrogen Producing Device (Syn. 6803)

19. Cellular and Applied Microbiolgy group BioModularH 2 team - Paula Tamagnini - Catarina Pacheco - Filipe Pinto - Miguel Lopo - Sérgio Ferreira - Zsófia Büttel - Meritxell Llorens SFRH / BPD / 64095 / 2009 (Catarina Pacheco) SFRH/BD/36378/2007 (Filipe Pinto)