The Team Northern Alberta Tar Sands * Images courtesy of Syncrude - PowerPoint PPT Presentation

The Team

Northern Alberta Tar Sands * Images courtesy of Syncrude Inc. and Shell Scotford

Northern Alberta Tailings Ponds *Image courtesy of Syncrude Inc .

Analogy

Motility

The “ Bacuum ” Cleaner • Locomotion • Regulation • Degradation • Identification

Overview • Locomotion • Regulation • Degradation • Identification

Chemotaxis Amino Acids Carbohydrates Running

Chemotaxis Amino Acids Carbohydrates Running

Chemotaxis Amino Acids Carbohydrates P Tumbling

Chemotaxis Amino Acids Carbohydrates P Tumbling

Chemotaxis Amino Acids Carbohydrates P Tumbling

Overriding chemotaxis Amino Acids Carbohydrates P

Controlling CheZ expression 12 Protein (  M) 8 CheY 4 CheY •P 0 0 2000 4000 6000 8000 CheZ molecules per cell Bray, D., Levin, M. D., and Lipkow, K. 2007. Curr. Biol. 17, 12-19 .

Controlling CheZ expression 12 1.0 Motility Protein (  M) Motility bias 0.8 8 CheY 0.6 0.4 4 CheY •P 0.2 0 0.0 0 2000 4000 6000 8000 CheZ molecules per cell Bray, D., Levin, M. D., and Lipkow, K. 2007. Curr. Biol. 17, 12-19.

Motility assay Non-motile Motile E. coli D c heZ E. coli WT

Biobrick parts • cheZ o BBa_K147000 Promoter + control + cheZ + T 1 + T 2

Overview • Locomotion • Regulation • Degradation • Identification

Riboswitch • UTR of mRNA o Short (can be <100 nt) • Binds target molecule , conformational change • Regulates transcription termination, translation initiation or alternative splicing • Fast response time

Types of riboswitches • Widely diverse in nature • Vary in ligands bound, methods of regulation • Bacteria, archaea, eukarya • Still a growing field of research Winkler and Breaker. 2003. Chem. Biochem. 4, 1024-1032.

Riboswitch • Theophylline aptamer - + CheY-P CheZ CheY Topp, S. and Gallivan, J. 2007. J. Am. Chem. Soc. 129, 6807-6811.

Biobrick parts Low [ligand] High [ligand] Promoter + RS + gene + T 1 + T 2

Overview • Locomotion • Regulation • Degradation • Identification

2-chlorobenzoate • Elimination of chloro substituents o Potential step in PCB degradation ohb operon xylE P. aeruginosa P. putida catechol 2-chlorobenzoate 2-hydroxymuconic ( ortho -chlorobenzoate) semialdehyde (coloured!) 1 Ingram et al. 1989 . J. Bacteriol. 171, 6617-6624. 2 Tsoi et al. 1999. Appl. Environ. Microbiol. 65, 2151-2161.

Biobrick parts • ohb operon ( ohbA, ohbB, ohbC, ohbR ) o BBa_K147002 (ohbA) • xylE o BBa_K147003 Promoter + RS + ohb + T 1 + T 2 + RBS + xylE + T 1 + T 2

Overview • Locomotion • Regulation • Degradation • Identification

Identification • Continually monitor modified bacterium • Problem with reporter genes? o Expression limited under stressed conditions • Solution? o Translation of essential genes o Initiated by unique sequence

rpsA TIR • Initiates translation of S1 ribosomal protein • Lacks Shine-Dalgarno and requirement of S1 • Unique watermark Hellen, C. U. T. 2007. Structure. 15, 4-6.

Biobrick parts • rspA TIR Promoter + rspA TIR + GFP + T 1 + T 2

Conclusions Locomotion Degradation Identification Regulation

Progress • Modeling of CheZ function! • Motility assay of WT vs. D cheZ (RP1616) • Submitted Biobricks cheZ , xylE , ohbA o Characterized cheZ part • Isolated rpsA TIR • Constructed theophylline riboswitch in Biobrick format (Nov. 6!)

Future work • Continue with construct formation • Modeling of reprogrammed bacuum motility • Generate novel aptamer using SELEX

SELEX Systematic Evolution of Ligands through Exponential Enrichment Random nucleic acid synthesis Counter- selection Tetracycline with Ni 2+ / selection Optimization Nucleic acid with selected binding

Future work (cont’d) • Characterize novel riboswitch function • Assemble “ Bacuum ” Cleaner • Test the system!

Acknowledgements • Dr. Wieden, Dr. Selinger • Gallivan Lab, Emory University • Tiedje Lab, University of Michigan