Transcript chassis
Preparing a cyanobacterial chassis for H2 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,30th July 2009 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 Standardized parts Assembly of modules and circuits Incorporation in a chassis BioBrick™ parts assembly 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 http://partsregistry.org/ Chassis “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” Escherichia coli Yeast Bacillus subtilis Jan.07- Jan.10 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 BioModularH2 Design In silico analysis Computational design of parts and modules Construction Synthesis of parts Assembly of modules Preparation of the chassis Caracterization Caracterization of parts/modules Incorporation in the chassis Evaluation of the final product Final goal A cyanobacterial chassis that together with the designed devices will harvest solar energy for H2 production. The synthetic parts and modules will be available for other biotechnological applications Photoautotrophic chassis - Synechocystis sp. PCC 6803 the most studied cyanobacteria unicellular and non-N2-fixing simple nutritional requirements naturally transformable molecular tools for manipulation available small genome comprising a 3.6 Mb genome and 7 plasmids (1st cyano genome sequenced) Preparation of the chassis Tuning respiration Oxygen sensing Oxygen consumption Native hydrogenase (s) etc… Nuclease(s) Highly-efficient O2-tolerant hydrogenase • Reduce constraints, e.g. enhance transformation efficiency • Remove redundant genes / parts • Minimize O2 production / maximize O2 consumption H2ases are very sensitive to O2 Deletion of redundant parts – generation of a hydrogenase deficient mutant hoxY hoxH Possesses hoxYH Sensitive to kanamycin Resistant to sucrose Deletion of hoxYH Resistant to kanamycin Sensitive to sucrose Lacks hoxYH Sensitive to kanamycin Resistant to sucrose Hydrogen Producing Device (HPD) HydA1_Fd Hydrogenase module Fe-only hydrogenase fused to ferredoxin – Chlamydomonas reinhardtii Maturation module HydEF + HydG – Chlamydomonas reinhardtii Homology models based on Chang et al. 2007 (Biophys J, 93:3034-45) 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 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. Design and characterization of parts for H2 production Oxygen Consuming Device (OCD) SINGLE-protein modules O2 H 2 O - A-type flavoprotein (ATF) – Synechocystis sp. PCC 6803 - Laccase – Escherichia coli TWO-protein module O2 H2O2 ½ O2 + H2O - Glucose oxidase – Penicillium amagasakiense + Catalase – Synechocystis sp. PCC 6803 Testing the expression of A-type flavoprotein (ATF) module in Escherichia coli Constructions used in the test: Wild-type MW - + T9002 * - + Promoterless LuxR controled ATF ** ATF *** + - + - AHL 100 kDa T9002 * 75 kDa PtetR luxR PluxR gfp ATF 50 kDa (63 kDa) Promoterless ATF ** PtetR luxR 37 kDa gfp ATF LuxR controled ATF *** PtetR luxR PluxR ATF gfp 25 kDa GFP (27 kDa) - A synthetic module that can be used for the controlled expression of the ATF was obtained. The Cellular and Applied Microbiolgy group Thank you for your attention