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Delft University of Technology 1 Saturday, November 6, 2010 1 TU Delft iGEM 2010 2 Saturday, November 6, 2010 2 TU Delft iGEM 2010 2 Saturday, November 6, 2010 2 From Problem to Project Oil sands tailings ponds 3 Saturday, November 6,

  1. Delft University of Technology 1 Saturday, November 6, 2010 1

  2. TU Delft iGEM 2010 2 Saturday, November 6, 2010 2

  3. TU Delft iGEM 2010 2 Saturday, November 6, 2010 2

  4. From Problem to Project Oil sands tailings ponds 3 Saturday, November 6, 2010 3

  5. Alkanivore A Toolkit for Enabling Hydrocarbon Conversion in Aqueous Environments 4 Saturday, November 6, 2010 4

  6. Alkanivore Enabling Hydrocarbon Conversion in Aqueous Environments Conversion 5 Saturday, November 6, 2010 5

  7. Alkanivore Enabling Hydrocarbon Conversion in Aqueous Environments Conversion Regulation Survival 5 Saturday, November 6, 2010 5

  8. Alkanivore Enabling Hydrocarbon Conversion in Aqueous Environments Conversion Regulation Survival Solubility 5 Saturday, November 6, 2010 5

  9. Alkanivore Enabling Hydrocarbon Conversion in Aqueous Environments Conversion Regulation Survival Solubility 5 Saturday, November 6, 2010 5

  10. Conversion 6 Saturday, November 6, 2010 6

  11. n-alkanes n-alkanol n-alkanal !" !" !" !" n-alkanoic acid β -Oxidation + TCA 6 Saturday, November 6, 2010 6

  12. n-alkanes n-alkanal n-alkanol n-alkanoic acid Enzyme activities LadA AlkB2 ADH ALDH 50 5.0 2.0 20 8-C 16-C 12-C 12-C μ mol min -1 g -1 total protein μ mol min -1 g -1 total protein μ mol min -1 g -1 total protein μ mol min -1 g -1 dry weight 40 16 4.0 1.6 30 3.0 1.2 12 20 2.0 0.8 8 10 4 1.0 0.4 0 0 0 0 E.coli WT E.coli WT E.coli WT E.coli WT AH strain LadA strain ADH Strain ALDH strain 7 Saturday, November 6, 2010 7

  13. RBS characterizations 40% Relative strength (100%=B0034) 32% 24% 16% 8% 0% J61117 J61100 J61127 J61107 J61101 B0032 Anderson Family Member Ref 8 Saturday, November 6, 2010 8

  14. Enabling Hydrocarbon Conversion in Aqueous Environments Conversion Regulation Survival Solubility 9 Saturday, November 6, 2010 9

  15. Regulation 9 Saturday, November 6, 2010 9

  16. Regulation pCaiF pCaiF 10 Saturday, November 6, 2010 10

  17. Regulation pCaiF Glucose phase pCaiF 10 Saturday, November 6, 2010 10

  18. Regulation pCaiF Substrate limited phase pCaiF 10 Saturday, November 6, 2010 10

  19. pCaiF 0.30 300 Glucose phase Limited phase Low cAMP High cAMP 0.25 250 Fluorescence (520nm) Biomass (600nm) 0.20 200 0.15 150 100 0.10 Biomass GFP 50 0.05 Model Biomass Model GFP 10 -0.01 0 3 5 8 10 13 Time (h) 11 Saturday, November 6, 2010 11

  20. Salt Tolerance Enabling Hydrocarbon Conversion in Aqueous Environments Conversion Regulation Survival Solubility 12 Saturday, November 6, 2010 12

  21. Salt Tolerance 2.0 100% E.coli WT Salt tolerance strain Growth rate % Growth rate increase (%) 1.5 75% Growth rate (h -1 ) 1.0 50% 0.5 25% 0 0% 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 NaCl (M) 13 Saturday, November 6, 2010 13

  22. Solvent Tolerance 0.500 100% E.coli WT Solvent Tolerance strain Growth rate % Growth rate increase (%) 0.375 75% Growth rate (h -1 ) 0.250 50% 0.125 25% 0 0% 0% 4% 8% 10% n-hexane (%) 14 Saturday, November 6, 2010 14

  23. Homolog Interaction Mapping Putative Interactions Known Interactions Homologs Target organism Source organism 15 Saturday, November 6, 2010 15

  24. Enabling Hydrocarbon Conversion in Aqueous Environments Conversion Regulation Survival Solubility 16 Saturday, November 6, 2010 16

  25. Solubility AlnA AlnA !"#!$ 17 Saturday, November 6, 2010 17

  26. Solubility AlnA AlnA !"#!$ 17 Saturday, November 6, 2010 17

  27. Solubility 0.20 Absorbance (493nm) 0.15 0.10 0.05 0 E.coli WT AlnA strain 18 Saturday, November 6, 2010 18

  28. Solubility 0.20 Absorbance (493nm) 0.15 0.10 0.05 0 E.coli WT AlnA strain 18 Saturday, November 6, 2010 18

  29. Enabling Hydrocarbon Conversion in Aqueous Environments Conversion Regulation Survival Solubility 19 Saturday, November 6, 2010 19

  30. Scientific Achievements Added 16 BioBricks to the Toolkit Developed methods for characterization Proof of principle for all 4 sub-parts 20 Saturday, November 6, 2010 20

  31. Future prospects Oil contaminated Clean water water !" # $ !" ! " # !"#$%&&' 21 Saturday, November 6, 2010 21

  32. Human Practice Perception Education Awareness 22 Saturday, November 6, 2010 22

  33. Awareness Wiki Media Collaboration Perception Education SynBio Workshops Interviews Game BioTech Room 23 Saturday, November 6, 2010 23

  34. Synthetic Biology Workshops 24 Saturday, November 6, 2010 24

  35. Awareness Wiki Media Collaboration Perception Education SynBio Workshops Interviews Game BioTech Room 25 Saturday, November 6, 2010 25

  36. 26 Saturday, November 6, 2010 26

  37. 26 Saturday, November 6, 2010 26

  38. Awareness Wiki Media Collaboration Perception Education SynBio Workshops Interviews Game BioTech Room 27 Saturday, November 6, 2010 27

  39. Media Attention Dutch National News 28 Saturday, November 6, 2010 28

  40. Awareness Wiki Media Collaboration Perception Education SynBio Workshops Interviews Game BioTech Room 29 Saturday, November 6, 2010 29

  41. Saturday, November 6, 2010 30

  42. Acknowledgements We would like to thank: - Esengül Yildirim - Alessandro Abate - Aljoscha Wahl - Stefan de Kok and all the others that were so kind to help, advise, and support us. 31 Saturday, November 6, 2010 31

  43. And our Sponsors: 32 Saturday, November 6, 2010 32

  44. 33 Saturday, November 6, 2010 33

  45. References Fujii, T., Narikawa, T., Takeda, K., Kato, J. , Biotransformation of various alkanes using the Escherichia coli expressing an alkane hydroxylase system from ! Gordonia sp. TF6 . ! Bioscience, biotechnology, and biochemistry , 68(10) 2171-2177 ( 2004 ) Liu Li, Xueqian Liu, Wen Yang, Feng Xu, Wei Wang, Lu Feng, Mark Bartlam, Lei Wang and Zihe Rao. ! Crystal Structure of Long-Chain Alkane Monooxygenase (LadA) in Complex with Coenzyme FMN: Unveiling the Long-Chain Alkane Hydroxylase. ! Journal of molecular biology , 376: 453-465 ( 2008 ) Tomohisa Kato, Asuka Miyanaga, Mitsuru Haruki, Tadayuki Imanaka, Masaaki Morikawa & Shigenori Kanaya. ! Gene Cloning of an Alcohol Dehydrogenase from Thermophilic Alkane-Degrading ! Bacillus thermoleovorans B23 . ! Journal of Bioscience and Bioengineering 91(1):100-102 ( 2001 ) Tomohisa Kato, Asuka Miyanaga, Shigenori Kanaya, Masaaki Morikawa. ! Gene cloning and characterization of an aldehyde dehydrogenase from long- chain alkane-degrading ! Geobacillus thermoleovorans B23 . ! Extremophiles ! 14:33-39 ( 2010 ) Sulzenbacher, G., et al. , Crystal structure of E-coli alcohol dehydrogenase YqhD: Evidence of a covalently modified NADP coenzyme. Journal of Molecular Biology ! 342 (2):489-502 ( 2004 )://mbel.kaist.ac.kr/lab/research/protein_en1.html Hoffmann F. and Rinas U . Stress Induced by Recombinant Protein Production in ! Escherichia coli ! Advances in Biochemical Engineering/Biotechnology , Vol. 89, pp. 73-92. ( 2004 ) Canosa, I., J. M. Sanchez-Romero, et al. ! A positive feedback mechanism controls expression of AlkS, the transcriptional regulator of the Pseudomonas oleovorans alkane degradation pathway. ! Molecular Microbiology ! 35(4): 791-799 ( 2000 ) Moreno, R., A. Ruiz-Manzano, et al. ! The Pseudomonas putida Crc global regulator is an RNA binding protein that inhibits translation of the AlkS transcriptional regulator. ! Molecular Microbiology ! 64(3): 665-675 ( 2007 ) van Beilen, J. B., S. Panke, et al. ! Analysis of Pseudomonas putida alkane-degradation gene clusters and flanking insertion sequences: evolution and regulation of the alk genes. ! Microbiology-Sgm ! 147: 1621-1630 ( 2001 ) Rojo, F. ! , Degradation of alkanes by bacteria. ! Environmental Microbiology ! 11: 2477-2490 ( 2009 ) Kotte, O, Zaugg, J., Heinemann, M. ! , ‘Bacterial adaptation through distributed sensing of metabolic fluxes’, ! Molecular Systems Biology , 6:355, doi:10.1038/msb. 2010.10 ( 2010 ) Kremling, A., Bettenbrock, K., Gilles, E.D. , ‘Analysis of global control of Escherichia coli carbohydrate uptake’, ! BMC Systems Biology , 1:42, doi: 10.1186/1752-0509-1-42 ( 2007 ) Lin, H. Y., Mathiszik, B., Xu, B., Enfors, S.-O., Neubauer, P. , ‘Determination of the Maximum Specific Uptake Capacities for Glucose and Oxygen in Glucose- Limited Fed-Batch Cultivations of ! Escherichia coli ’, ! Biotechnology and Bioengineering , 73, 347-357 ( 2001 ) Alon, U. (ed.) , An Introduction to Systems Biology: Design Principles of Biological Circuits, ! CRC Press ! ( 2007 ) 34 Saturday, November 6, 2010 34

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