Boston Presentation
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Transcript of Boston Presentation
![Page 1: Boston Presentation](https://reader036.fdocuments.us/reader036/viewer/2022062406/55cae255bb61ebfa5d8b45fa/html5/thumbnails/1.jpg)
2014 Michigan Synthetic Biology TeamUniversity of Michigan
Ann Arbor, MI
ScFV Antibody Secretion in E. coli
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Eukaryote Prokaryote
Purifying Mammalian Proteins in E. coli is Problematic
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Prokaryotic Periplasm mimics the Reducing Environment of ER
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E. coli OsmY Secretes into the Periplasm
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Utilization of OsmY as a Secretion-Based Protein Purification Method
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Human Practice:Project Implications
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Antibodies and the Economy
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Meeting with Covance to Determine Market Viability for Antibody Detection
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• Fluorescence Microscopy of Secreted mCherry
• Western Blot Analysis of Secreted mCherry
• Western Blot Analysis of Secreted ScFV Antibody
Results
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Expression Vector Creates Functioning Polypeptide
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Expression Vector is Capable of Secreting Protein Fusion
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Functional Antibodies can be Secreted from the Construct
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Future Directions• Optimize overexpression conditions for construct
• Perform ELISA with antibody-rich supernatant
• Compare OsmY construct with industry standards
• Identify effects of cleaving OsmY from ScFV on binding efficiency
• Quantify construct secretion rates, providing models with data (ELISA)
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Modeling
[C]=k-1k1[P]+Sk1(1-e-k1t) [P]=1k-1+k2(k1[C]+k-2[M]) [M]=1k-2+D[M](k2[P]) [P]=[C]k1(k-2+D[M])k-1(k-2+D[M])+k2D[M] [P]=S(1-e-k1t)(k-2+D[M]k2D[M]) [M]=SD[M](1-e-k1t)
[C]=S(k-1(k-2+D[M])+k2D[M]k1k2D[M])(1-e-k1t) [C]ss=S+k-1[P]ssk1 [P]ss=k-2[M]ss+k1[C]ssk-1+k2 [M]ss=k2[P]ssk-2+D[M] [P]ss=S(k-2+D[M])k2D[M], k1(k-1+k2)(k-2+D[M])0[M]ss=SD[M] [C]ss=S(k2D[M]+k-1(k-2+D[M])k1k2D[M])
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Modeling:Secretion of OsmY-Fusion
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Solving the System
Assumptions:• Volume of Cytoplasm, Periplasm, and Media are constant
• Synthesis rate is independent of time
• Transfer reactions are first order
• Periplasm and Cytosol quickly reach a steady state
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Secreted Protein Concentration:Predicting the Steady State
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Next Step: Test and Develop
Perform ELISA,Get Data
Compare Model to Data
Adjust ModelPredict new outcomes
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We Can Check One Assumption Now:
[P] and [C] quickly reach a steady state
Mol L
Time
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Future Directions• Quantify periplasmic and media
concentrations of OsmY-Fusion via ELISA• Utilize ELISA data to adjust parameters,
improving robustness of models
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Monetization: The Real Driving Force
• OsmY can be used to secrete diverse proteins
• We can predict how such proteins will secrete
• OsmY can be used in a bioreactor
• Useful tool for industries
STEP 1: MAKE PROTEIN
SECRETION SYSTEM
Step 2:Predict rate of Synthesis and
Secretion
STEP 3: PROFIT
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Carnegie Mellon Regional Meet-Up
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Collaborations
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Outreach
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OutreachOutreach
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Acknowledgements
University of Michigan• Marcus Ammerlaan, Ph.D, Advisor• Anuj Kumar, Ph.D, Advisor• Kaitlin Flynn, Advisor• Victor DiRita, Ph.D• James Bardwell, Ph.D• Ursula Jacob, Ph.D• Ming Liu, Ph.D
TU-Braunschweig• Michael Hust, Ph.D
Covance• Christine Gwinn, Microbiology Supervisor
Thank You, Sponsors!
Acknowledgements