E. coli Based Biotemplating Team Minnesota Biotemplating The - PowerPoint PPT Presentation

E. coli Based Biotemplating Team Minnesota

Biotemplating  The production of complex, 3-D shapes using bacteria www.material.kemi.uu.se  Biotemplating in nature: – Bone (hydroxyapatite) – Coccolith (calcite) – Coral (calcite) – Spicules (silica)

Applications  Creation of biomimetic structures  Polymerization of metals on the surface of cells Example of coral reef deterioration  Bacterial cement  Re-calcification of reefs Metal structures formed by E. coli recombinantly expressing silicatein 1 .

Variables:  Control expression of precipitate  Control contact of cells spie.org/x33929.xml?ArticleID=x33929 Benefits:  Standard conditions  Minimally toxic  Less expensive www.nigels.com/cs516/

Background Information System Components

Biotemplating System Overview

Biotemplating System Overview

Biotemplating System Overview

Silicatein  Isolated from marine sponge Suberites domuncula  Protein responsible for spicule formation in sponges  Nucleates silica polymerization and metal crystallization  Can cause the formation of metal sheets when expressed in E. coli  Obtained Silicatein gene from Korshev lab, Johannes Gutenberg- University, Mainz, Germany. Native silicatein filaments,Brutchey and Morse, 2008 2

Cell Surface Display System  Automatically catalyze their own insertion and translocation across the outer membrane  2 parts:  Passenger protein  Carrier protein www.vaccineresistancemovement.org  Current applications  Vaccine development  Antibody production  Bioremediation www. genengnews.com

Ice Nucleation Protein  Outer membrane protein from Pseudomonas syringae  Catalyze formation of ice crystals  3 domains:  N-terminal  C-terminal  Central domain  Very stable  Able to carry high molecular weight proteins Bloois, E. et al, 2011 1

Regulatory system  Coliroid Light Induction System designed by University of Texas at Austin and UCSF iGEM team in 2004 www.partsregistry.org/Coliroid  Control the expression of a target protein into specific 2D structure www.partsregistry.org/Coliroid

Strategy Methods & Process

INP-Silicatein Fusion  Used Silicatein gene sent by Korshev lab  Obtained a truncated version of INP (BBa_K265009 )  Fusion of INP and Silicatein  Cloned into BioBrick vector pMCS5BB under lacP promoter

Silicatein Functional Assay p-methylaminophenol (Metol solution) R. K. Iler, 1979. 3 A. Rai, C. C. Perry, 2009. 4

Light Regulatory System  Cloned into BioBrick vector pucBB-pBAD and pucBB-pTET  Obtained PcyA gene from Synechocystis PCC6803 genomic DNA,  Obtained ho1 from plasmid library  Obtained chimera protein Cph8 from Voigt lab, UCSF

Biotemplating System Overview

Results What did we find?

Results  Standard curve Silicatein Assay Standard Curve generated  Linear correlation

Results  Quantify total cell associated silicatein  Cells with INP- silicatein  Cells with only INP

Conclusions & Future Directions More Possibilities

Conclusions  Fully functional surface silicatein expressed  The coliroid light-sensitive system assembled  In progress:  Integrating regulatory system with INP-SIL fusion  Production of specific shapes  Direct evidence for surface display of INP- silicatein

Future Directions  Use of IR/Heat-Shock induction system  Use of urease or other nucleation proteins  Formulation of media composition www.en.wikipedia.org/wiki/Laser  Implement a NOT-gate

Acknowledgements  Advisors: David Babson, Sarah Bloch, Tanhia Gonzales, Maureen Quin, Poonam Srivastava, Ian Windsor  Instructors: Jeff Gralnick, Claudia Schmidt- Dannert Thank You To Our Sponsors!

Thank You! The End

References 1.) van Bloois E, Winter RT, Kolmar H, Fraaije MW. (2011) Decorating microbes: surface display of proteins on Escherichia coli. Trends in Biotechnology 29:79-86. 2.) Brutchey RL and Morse DE. (2008) Silicatein and the translation of its molecular mechanism of biosilicification into low temperature nanomaterial synthesis. American Chemical Scoiety . 108 (11): 4915-4934. 3.) Iler, RK. The Chemistry of Silica: Solubility, Polymerization, Colloid and Surface Properties, and Biochemistry. New York: Wiley, 1979. 4.) Rai A, Perry CC. (2009) Facile fabrication of uniform silica films with tunable physical properties using silicatein protein from sponges. Languir 26(6): 4152-4159.