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

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 efficiency, cost, environmental impact � �

PROBLEM | traditional strategies 1. Incineration or burial � � 2. Reuse � 3. Steam cooking into animal feed �

SOLUTION | keratin degradation KERATINASE FEATHERS EXPRESSING BACTERIA AMINO ACIDS & SMALLER PROTEINS Highly digestible animal feed Biodegradable plastics Pharmaceuticals Detergents Fertilizers

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

BACKGROUND | keratinase Cysteine � Disulfide bond between � two cysteines �

BACKGROUND | keratinase • Serine protease � • Breaks down disulfide bonds in keratin � S ¡ S ¡ S ¡ S ¡ Keratinase � S ¡ S ¡ S ¡ S ¡ S ¡ S ¡ S ¡ S ¡ � Broken S-S bonds �

KERATINASE | sequenced bacterial keratinases Bacillus cereus � Bacillus licheniformis � Bacillus methylotrophicus � Bacillus mojavensis � Bacillus pumilus � Bacillus subtilis � Fervidobacterium pennivorans � Streptomyces fradiae � Stenotrophomonas maltophilia �

KERATINASE | why kerA? • B. subtilis PWD-1: best characterized in literature � • Can be expressed with a constitutive promoter �

B. subtilis | feather degrading system

E. coli | feather degrading system

pUB110 | original backbone

pUB110 | modified backbone

KerA BioBrick | secretion signal variant BB secretion BB kerA prefix signal suffix

KerA BioBrick | secretion signal + HIS tag variant BB secretion HIS BB kerA prefix signal tag suffix

KerA BioBrick | HIS tag variant BB secretion HIS BB kerA prefix signal tag suffix

UPSTREAM CANDIDATES | overview

UPSTREAM CANDIDATES | for B. subtilis

UPSTREAM CANDIDATES | for B. subtilis

UPSTREAM CANDIDATES | for E. coli

UPSTREAM CANDIDATES | for E. coli

ASSAY | qualitative Keratin agar plate Milk agar plate � (Wawrzkiewicz et al. 1990) �

ASSAY | quantitative 1. Lyse cell or collect surrounding media � 2. Western blot � 3. Keratin azure assay � Media Collection for KerA secreted by B. subtilis � Enzyme Activity (Abs) � Mutant kerA � WT KerA � [KeratinaseA] � E. coli cell lysis to release KerA �

KerA | optimization 50ºC � ¡ ¡ 25-37ºC �

KerA | error prone PCR PCR products � Keratin agar plates �

CONCLUSION | project summary 1) Create kerA and pUB110 BioBricks � � 2) Measure KerA activity � � � 3) Improve kerA gene through mutagenesis �

CONCLUSION | credits • 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. �

THANKS FOR YOUR ATTENTION!

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