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

Bacterial cement

Re-calcification of reefs

Metal structures formed by E. coli recombinantly expressing silicatein1.

Example of coral reef deterioration

Control expression of precipitate

Control contact of cells

Standard conditions

Minimally toxic

Less expensive

Benefits:

Variables:

www.nigels.com/cs516/

spie.org/x33929.xml?ArticleID=x33929

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, 20082

Cell Surface Display System

Automatically catalyze their own insertion and translocation across the outer membrane

2 parts: Passenger protein Carrier protein

Current applications Vaccine development Antibody production Bioremediation

www.genengnews.com

www.vaccineresistancemovement.org

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, 20111

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

StrategyMethods & 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

R. K. Iler, 1979.3 A. Rai, C. C. Perry, 2009.4

p-methylaminophenol (Metol solution)

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

ResultsWhat did we find?

Results

Standard curve generated

Linear correlation

Silicatein Assay Standard Curve

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

fusionProduction of specific shapesDirect 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

Implement a NOT-gate

www.en.wikipedia.org/wiki/Laser

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.