Delft University of Technology 1 Saturday, November 6, 2010 1 TU - - PowerPoint PPT Presentation

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Delft University of Technology 1 Saturday, November 6, 2010 1 TU - - PowerPoint PPT Presentation

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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,

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Delft University of Technology

1 Saturday, November 6, 2010

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SLIDE 2

TU Delft iGEM 2010

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2 Saturday, November 6, 2010

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SLIDE 3

TU Delft iGEM 2010

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2 Saturday, November 6, 2010

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SLIDE 4

Oil sands tailings ponds

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From Problem to Project

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Alkanivore

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A Toolkit for Enabling Hydrocarbon Conversion in Aqueous Environments

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SLIDE 6

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Enabling Hydrocarbon Conversion in Aqueous Environments

Alkanivore

Conversion

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SLIDE 7

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Enabling Hydrocarbon Conversion

in Aqueous Environments

Alkanivore

Conversion Regulation Survival

5 Saturday, November 6, 2010

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SLIDE 8

5

Enabling Hydrocarbon Conversion

in Aqueous Environments

Alkanivore

Conversion Regulation Survival Solubility

5 Saturday, November 6, 2010

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SLIDE 9

5

Enabling Hydrocarbon Conversion in Aqueous Environments

Alkanivore

Conversion Regulation Survival Solubility

5 Saturday, November 6, 2010

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Conversion

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n-alkanes

n-alkanol n-alkanal n-alkanoic acid β-Oxidation + TCA

!" !" !" !"

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7 10 20 30 40 50

E.coli WT AH strain

1.0 2.0 3.0 4.0 5.0

E.coli WT LadA strain

0.4 0.8 1.2 1.6 2.0

E.coli WT ADH Strain

4 8 12 16 20

E.coli WT ALDH strain

μmol min-1 g-1 dry weight 8-C μmol min-1 g-1 total protein 16-C μmol min-1 g-1 total protein 12-C μmol min-1 g-1 total protein 12-C

n-alkanol n-alkanal n-alkanoic acid

n-alkanes

Enzyme activities

LadA AlkB2 ADH ALDH

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RBS characterizations

0% 8% 16% 24% 32% 40% J61117 J61100 J61127 J61107 J61101 B0032

Relative strength (100%=B0034) Anderson Family Member Ref

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SLIDE 14

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Enabling Hydrocarbon Conversion in Aqueous Environments

Conversion Regulation Survival Solubility

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Regulation

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Regulation pCaiF

pCaiF

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Regulation pCaiF

pCaiF

Glucose phase

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Regulation pCaiF

pCaiF

Substrate limited phase

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Biomass (600nm) Time (h)

pCaiF

  • 0.01

0.05 0.10 0.15 0.20 0.25 0.30 3 5 8 10 13

Biomass GFP Model Biomass Model GFP

Glucose phase Limited phase Low cAMP High cAMP

300 250 200 150 100 50 10

Fluorescence (520nm)

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Enabling Hydrocarbon Conversion in Aqueous Environments

Conversion Regulation Survival Solubility

Salt Tolerance

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Growth rate (h-1) Growth rate increase (%) NaCl (M)

Salt Tolerance

13 0.5 1.0 1.5 2.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 0% 25% 50% 75% 100% E.coli WT Salt tolerance strain Growth rate %

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Solvent Tolerance

Growth rate increase (%) n-hexane (%)

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Growth rate (h-1)

0.125 0.250 0.375 0.500 0% 4% 8% 10% 0% 25% 50% 75% 100% E.coli WT Solvent Tolerance strain Growth rate %

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Homolog Interaction Mapping

Homologs

Source organism Target organism

Putative Interactions Known Interactions

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Enabling Hydrocarbon Conversion in Aqueous Environments

Conversion Regulation Survival Solubility

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Solubility

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!"#!$

AlnA AlnA

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Solubility

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!"#!$

AlnA AlnA

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Solubility

18 0.05 0.10 0.15 0.20

Absorbance (493nm)

E.coli WT AlnA strain

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Solubility

18 0.05 0.10 0.15 0.20

Absorbance (493nm)

E.coli WT AlnA strain

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Enabling Hydrocarbon Conversion in Aqueous Environments

Conversion Regulation Survival Solubility

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Scientific Achievements

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Added 16 BioBricks to the Toolkit Developed methods for characterization Proof of principle for all 4 sub-parts

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Future prospects

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!"#$ !"#$%&&' !"#

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Oil contaminated water Clean water

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Human Practice

Education Perception Awareness

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Awareness Perception Collaboration

Interviews Wiki Media

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Education

Game SynBio Workshops BioTech Room

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Synthetic Biology Workshops

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Awareness Perception Collaboration

Interviews Wiki Media

Education

Game SynBio Workshops BioTech Room

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26 Saturday, November 6, 2010

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Awareness Perception Collaboration

Interviews Wiki Media

Education

Game SynBio Workshops BioTech Room

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Dutch National News

Media Attention

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Awareness Perception Collaboration

Interviews Wiki Media

Education

Game SynBio Workshops BioTech Room

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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.

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And our Sponsors:

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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 Bioengineering91(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

  • leovorans 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)

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References (2)

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  • S. Tanaka,K. Ikeda, H. Miyasaka, Enhanced Tolerance Against Salt-Stress and Freezing-Stress of Escherichia coli Cells Expressing Algal bbc1 Gene.!Current

Microbiology, 42:173-177 (2001)

  • Y. Suda,T. Yoshikawa,Y. Okuda,M. Tsunemoto,S. Tanaka,K. Ikeda,H. Miyasaka,M. Watanabe,K. Sasaki,K. Harada,T. Bamba,K.

Hirata, Isolation and characterization of a novel antistress gene from!Chlamydomonas sp. W80.!Journal of Bioscience and Bioengineering, 107(4) 352-354 (2009)

  • Y. Hase, S. Yokoyama, A. Muto, et al.!, Removal of a ribosome small subunit-dependent GTPase confers salt resistance on Escherichia coli cells.!RNA Society,

15:1766-1774 (2009) Mihaela Marilena Lăzăroaie,Investigation of saturated and aromatichydrocarbon resistance mechanismsin!Pseudomonas aeruginosa!IBBCent. Eur. J. Biol.!4(4) 469-481 (2009)

  • M. Okochi, K. Kanie, M. Kurimoto ,M. Yohda & Hiroyuki Honda!Overexpression of prefoldin from the hyperthermophilic archaeumPyrococcus

horikoshii!OT3 endowed!Escherichia coli!with organic solvent tolerance!Appl Microbiol Biotechnol!79:443-449 (2008) Walzer, G., Rosenberg, E. and Ron, E.Z.!The Acinetobacter outer membrane protein A (OmpA) is a secreted emulsifier.!Environmental Microbiology.!8:1026-1032.(2006) Toren, A., Segal, G., Ron, E.Z. and Rosenberg, E.!Structure--function studies of the recombinant protein bioemulsifier AlnA.Environmental Microbiology.!4:257-261.(2002) Navon-Venezia, S., et al.!Alasan, a new bioemulsifier from Acinetobacter radioresistens.!Applied and Environmental Microbiology.!61:3240-3244.(1995) Toren, A., Orr, E., Paitan, Y., Ron, E.Z. and Rosenberg, E.!The active component of the bioemulsifier alasan from Acinetobacter radioresistens KA53 is an OmpA-like protein.!The Journal of Bacteriology.!184:165-170.(2002) Toren, A., Navon-Venezia, S., Ron, E.Z. and Rosenberg, E.!Emulsifying activities of purified Alasan proteins from Acinetobacter radioresistens KA53.!Applied and Environmental Microbiology.!67:1102-1106. Ron, E.Z. and Rosenberg, E.!(2002) Biosurfactants and oil bioremediation.!Current Opinion in Biotechnology.!13:249-252.(2001) Suresh Kumar, A., Mody, K. and Jha, B.!Evaluation of biosurfactant/bioemulsifier production by a marine bacterium.!Bulletin of Environmental Contamination and Toxicology.!79:617-621.(2007)

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