1. The 2007 Virginia Genetically Engineered Machine Team 2007 iGEM Jamboree 3 Nov. 2007 http://www.etcgroup.org/en/issues/s ynthetic_biology.html

2. The 2007 VGEM Team University-wide, interdisciplinary science and engineering collaboration among students and faculty Amy, George, Kevin, Ranjan, and Emre

3. Advisors Ron Bauerle Jason Papin Brianne Ray Kay Christopher Erik Fernandez

4. Birth of the Team First UVA iGEM team Founded, organized and run by us 16 undergraduates applied We raised $50,000 and secured lab space No prior synthetic biology research at UVA We taught ourselves basic synthetic biology

5. Project Brainstorming Bacterial melanogenesis Ethylene biosensing Autonomous drug delivery Cellular photosignalling Directed angiogenesis Nature. 2000 Jan 20;403(6767):335-8. http://www.calvin.edu/academic/chemi stry/faculty/muyskensmark http://news.bbc.co.uk/cbbcnews/hi/ani mals/newsid_3233000/3233962.stm

6. Butanol Biosynthesis: Background & Motivation 90 years ago- Butanol first produced in a lab setting via fermentation 1950s- Butanol produced petrochemically due to lowered cost Today- Need for alternate energy source BP DuPont Biofuels. www2.dupont.com/Biofuels/en_US/index.

7. Butanol Biosynthesis: Background & Motivation Advantages of Butanol over Ethanol  Less corrosive  Lower latent heat of vaporization  Higher energy density  Less hygroscopic Fits within current infrastructure  Liquid at atmospheric temperature and pressure  Easily blends with other fuels  Can be used in existing internal combustion engines http://www.bioenergywiki.net /index.php/Biobutanol

8. Butanol Biosynthesis: Background & Motivation Butanol is renewable! However, production is not currently economically feasible More research is necessary  High cost of substrate  Toxicity to the fermenting organisms

9. Butanol Biosynthesis: Objectives Design, model, and modularly construct a metabolic pathway in E. coli that includes the following:  A butanol-producing metabolic pathway From Clostridium acetobutylicum  A cellulase system (cheap carbon source) Select enzymes from Saccharophagus degradans  Proteorhodopsin, a light metabolism system (free energy) Discovered via marine metagenomic analysis Increase E.coli butanol tolerance via engineering and/or evolution

11. BioBrick construction plan

12. Proteorhodopsin Requires retinal Added to media, not biosynthesized Martinez, A., A. Bradley, J. Waldbauer, R. Summons and E. F. DeLong. 2007. Proteorhodopsin photosystem gene expression enables photophosphorylation in a heterologous host. PNAS.

13. Proteorhodopsin Results

14. Results: Butanol Tolerance Typical Tolerance: 0.8-1.2% (vol/vol) Unable to get consistent results At higher butanol concentrations (>1.2%), occasionally cells would survive These cells were unable to keep butanol tolerance and would be killed if transferred to a new broth

15. Mathematical Model Model → experiment → model → etc. Metabolic pathway → stoichiometric matrix Flux balance analysis  Fermentation products  Pathway bottlenecks  Knockout simulations

16. Mathematical Model

17. Conclusions Cellulases and butanol-producing enzymes may be toxic to E. coli Simple directed evolution of E. coli may not be an effective way of increasing butanol tolerance Proteorhodopsin is a potential energy- provider for chemical synthesis in E. coli under anaerobic conditions

18. Ongoing Work Determine what went wrong with our BioBrick construction and whether or not they are lethal to E. coli If possible, assemble BioBricks into composite systems Incorporate better or more membrane-associated solvent efflux pumps to increase tolerance Incorporate the retinal pathway Incorporate more diverse cellulases Complete the central butanol biosynthesis pathway Design and optimize complete bioprocess

19. Improvements Expansion of the team to include more departments at UVA (materials science, electrical engineering, chemistry) Future funding by corporate sponsorship Potential intro synthetic biology course to educate new researchers and spread the word. Better communication with iGEM personnel and other teams (more community-oriented) to solve common problems

20. Acknowledgements Our advisors: Erik Fernandez, Jason Papin, Ron Bauerle, Brianne Ray, and Kay Christopher Our academic sponsors: Office of the VP for Research, School of Engineering, U.Va. Engineering Foundation, School of Medicine, and the departments of Biomedical Engineering, Chemical Engineering, Biology, and Microbiology Our corporate sponsor: DNA2.0

21. References [1] Andrykovitch, G., & Marx, I. (1988). Isolation of a new polysaccharide-digesting bacterium from a salt marsh. Applied and Environmental Microbiology, 54(4), 1061-1062. [2] Beja, O., Aravind, L., Koonin, E., Suzuki, M., Hadd, A., Nguyen, L., et al. (2000). Bacterial rhodopsin: Evidence for a new type of phototrophy in the sea. Science, 289(5486), 1902-1906. [3] Borden, J., & Papoutsakis, E. (2007). Dynamics of genomic-library enrichment and identification of solvent tolerance genes for clostridium acetobutylicum. Applied and Environmental Microbiology, 73(9), 3061-3068. [4] Martinez, A., Bradley, A. S., Waldbauer, J. R., Summons, R. E., & DeLong, E. F. (2007). Proteorhodopsin photosystem gene expression enables photophosphorylation in a heterologous host. Proceedings of the National Academy of Sciences, 104(13), 5590- 5595. [5] Nolling, J., Breton, G., Omelchenko, M., Makarova, K., Zeng, Q., Gibson, R., et al. (2001). Genome sequence and comparative analysis of the solvent-producing bacterium clostridium acetobutylicum. The Journal of Bacteriology, 183(16), 4823-4838. [6] Taylor, L.,II, Henrissat, B., Coutinho, P., Ekborg, N., Hutcheson, S., & Weiner, R. (2006). Complete cellulase system in the marine bacterium saccharophagus degradans strain 2-40T. The Journal of Bacteriology, 188(11), 3849-3861.

22. Thanks for your time! Questions?

23. Supplemental Materials

24. Experimental Design

25. Experiment 1: Testing Alcohol Dehydrogenase aad, aad2 + alcohol dehydrogenase? aad, aad2

26. Experiment 2: Butanol biosynthesis from butyric acid atoA atoD

27. Experimental Design Summary: Butyrate to Butanol

28. Experimental Design Summary: Central Pathway

29. Experiment 3: Cellulose as an Alternate Energy Source cellobiohydrolase, cellobiase Native

30. Experiment 4: Proteorhodopsin as Alternate Energy Source

31. BioBrick Components BioBrick BBa_I711040 BBa_I711035 BBa_I711037 BBa_I711036 BBa_I711018 BBa_I711034 BBa_I711038 BBa_I711039 BBa_I711021 BBa_I711028 Name Proteorhodopsin Cel5F Cel6A Bgl3C CAP0078 ato aad/adhE aad2/adhE1 alde CAC1869 Description Light-activated proton pump Endo-1,4-β-glucanase Cellobuihydrolase Cellobiase Thiolase Acetoacetyl-CoA transferase Butyraldehyde dehydrogenase Butanol dehydrogenase Regulatory factor (↑tolerance?) Source Marine metagenomic analysis Saccharophagus degradans Clostridium acetobutylicum Escherichia coli Clostridium acetobutylicum Ref. [2], [4] [1], [6] [5] [?] [5] [3]