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

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

May 10, 2023 186 likes •450 views

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)

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SLIDE 1
  • E. coli Based

Biotemplating

Team Minnesota

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SLIDE 2

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)

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SLIDE 3

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

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SLIDE 4
  • 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

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SLIDE 5

Background Information

System Components

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SLIDE 6

Biotemplating System Overview

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SLIDE 7

Biotemplating System Overview

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SLIDE 8

Biotemplating System Overview

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SLIDE 9

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

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SLIDE 10

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

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SLIDE 11

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

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SLIDE 12

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

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SLIDE 13

Strategy

Methods & Process

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SLIDE 14

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
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SLIDE 15

Silicatein Functional Assay

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

p-methylaminophenol (Metol solution)

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SLIDE 16

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

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SLIDE 17

Biotemplating System Overview

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SLIDE 18

Results

What did we find?

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SLIDE 19

Results

  • Standard curve

generated

  • Linear correlation

Silicatein Assay Standard Curve

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SLIDE 20

Results

  • Quantify total cell

associated silicatein

  • Cells with INP-

silicatein

  • Cells with only

INP

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SLIDE 21

Conclusions & Future Directions

More Possibilities

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SLIDE 22

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

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SLIDE 23

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

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SLIDE 24

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!

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SLIDE 25

Thank You!

The End

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SLIDE 26

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.