UNIVERSITY OF CHICAGO | iGEM 2013 PROBLEM | feather waste 2.3 billion - - PowerPoint PPT Presentation

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UNIVERSITY OF CHICAGO | iGEM 2013 PROBLEM | feather waste 2.3 billion - - PowerPoint PPT Presentation

May 18, 2023 303 likes •616 views

UNIVERSITY OF CHICAGO | iGEM 2013 PROBLEM | feather waste 2.3 billion pounds of feather waste is produced by the US commercial poultry industry annually Keratin in feather waste Difficult to degrade Limited by

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UNIVERSITY OF CHICAGO | iGEM 2013

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PROBLEM | feather waste

2.3 billion pounds of feather waste is produced by the US commercial poultry industry annually

  • Keratin in feather waste
  • Difficult to degrade
  • Limited by efficiency, cost,

environmental impact

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PROBLEM | traditional strategies

  • 1. Incineration or burial
  • 2. Reuse
  • 3. Steam cooking into animal

feed

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AMINO ACIDS & SMALLER PROTEINS Highly digestible animal feed Biodegradable plastics Pharmaceuticals Detergents Fertilizers

FEATHERS KERATINASE EXPRESSING BACTERIA

SOLUTION | keratin degradation

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PLAN | our goals

1) Create keratinase BioBrick and enzymatic expression system 2) Measure keratinase activity 3) Improve our keratinase by mutagenesis

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Disulfide bond between two cysteines Cysteine

BACKGROUND | keratinase

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  • Serine protease
  • Breaks down disulfide bonds in keratin
  • BACKGROUND | keratinase

S ¡ S ¡ S ¡ S ¡ S ¡ S ¡ S ¡ S ¡ S ¡ S ¡ S ¡ S ¡

Broken S-S bonds

Keratinase

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Bacillus cereus Bacillus licheniformis Bacillus methylotrophicus Bacillus mojavensis Bacillus pumilus Bacillus subtilis Fervidobacterium pennivorans Streptomyces fradiae Stenotrophomonas maltophilia

KERATINASE | sequenced bacterial keratinases

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  • B. subtilis PWD-1:

best characterized in literature

KERATINASE | why kerA?

  • Can be expressed

with a constitutive promoter

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  • B. subtilis | feather degrading system
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  • E. coli | feather degrading system
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pUB110 | original backbone

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pUB110 | modified backbone

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kerA secretion signal BB suffix BB prefix

KerA BioBrick | secretion signal variant

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kerA secretion signal HIS tag BB suffix BB prefix

KerA BioBrick | secretion signal + HIS tag variant

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kerA HIS tag BB suffix BB prefix

KerA BioBrick | HIS tag variant

secretion signal

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UPSTREAM CANDIDATES | overview

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UPSTREAM CANDIDATES | for B. subtilis

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UPSTREAM CANDIDATES | for B. subtilis

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UPSTREAM CANDIDATES | for E. coli

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UPSTREAM CANDIDATES | for E. coli

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ASSAY | qualitative

Milk agar plate Keratin agar plate (Wawrzkiewicz et

  • al. 1990)
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ASSAY | quantitative

  • 1. Lyse cell or collect surrounding media
  • 2. Western blot
  • 3. Keratin azure assay

[KeratinaseA] Enzyme Activity (Abs) WT KerA Mutant kerA

Media Collection for KerA secreted by B. subtilis

  • E. coli cell lysis to release KerA
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¡ ¡

50ºC 25-37ºC

KerA | optimization

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KerA | error prone PCR

PCR products Keratin agar plates

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1) Create kerA and pUB110 BioBricks

CONCLUSION | project summary

  • 2) Measure KerA activity
  • 3) Improve kerA gene through mutagenesis
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  • Thank you to all of our

graduate and faculty advisors and our sponsors:

  • Kenneth Barr
  • Jeff Bunker
  • Justin Chew
  • Karyl Kopaskie
  • Sean Crosson, Ph.D.
  • Steve Kron, Ph.D.
  • Chris Schonbaum, Ph.D.
  • James Shapiro, Ph.D.

CONCLUSION | credits

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THANKS FOR YOUR ATTENTION!

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THE END!