2. Preparing a cyanobacterial chassis for H 2 production: a synthetic biology approach Catarina Pacheco Cell and Applied Microbiology Group IBMC, INEB E4. Genómica funcional e biologia sintética Encontro Nacional de Ciência - Ciência 2009 Fundação Calouste Gulbenkian,30 th July 2009

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. BioBrick™ parts assembly BioBrick BioBrick ™ Standardized DNA fragment designed for a specific purpose and that can be easily assembled with other bricks to generate modules and devices. e.g. promoter sensor modified gene

6. http://partsregistry.org/

7. “The candidates for chassis should be well studied organisms with high throughput genomic and proteomic data available, minimalist in terms of the subset of genes that will allow retaining viability, and easy to engineer with the available molecular tools, becoming a versatile platform for multiple purpose applications” Chassis Escherichia coli Bacillus subtilis Yeast

8. 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- Jan.10

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

10. 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

11. 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)

12. 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

13. 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

14. HydA1_Fd 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

15. 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

16. Analysis of gene expression by RT-PCR Generation of deletion mutants in the ORFs corresponding to the neutral sites N5, N7, N8, N10, N15 and N16. Mutant analysis will reveal the true neutral sites that can be used for the integration of synthetic modules and devices.

17. Design and characterization of parts for H 2 production Oxygen Consuming Device (OCD) - 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

18. Testing the expression of A-type flavoprotein (ATF) module in Escherichia coli - A synthetic module that can be used for the controlled expression of the ATF was obtained. Constructions used in the test:

19. Thank you for your attention The Cellular and Applied Microbiolgy group