Modification, Selection and Production of Cyclic Peptides iGEM 2011 - - PowerPoint PPT Presentation

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Modification, Selection and Production of Cyclic Peptides iGEM 2011 - - PowerPoint PPT Presentation

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Modification, Selection and Production of Cyclic Peptides iGEM 2011 World Championship Jamboree at MIT in Boston Sunday, 6th November 2011 Outline Introduction Laboratory Outreach & Xtras 2 Potential of Cyclic Peptides every year,

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iGEM 2011 World Championship Jamboree at MIT in Boston Sunday, 6th November 2011

Modification, Selection and Production of Cyclic Peptides

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Outline

Laboratory Introduction Outreach & Xtras

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Potential of Cyclic Peptides

  • every year, millions of people are infected with

deseases related to protease activity

  • cyclic peptides
  • stable
  • unique potential
  • use as therapeutic agent
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Microviridin Family

  • cyclization over side chain residues

– no cysteins or any backbone structure involved

  • cyclic peptide with two ester and one amide

bond

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The Project

Microcystis aeruginosa

mdn cluster

Library Generation Selection System Optimized Protease Inhibitor Heterologous Expression in E. coli

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Modularization of the Cluster

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  • 6.5 kbp cluster with 5 important elements:
  • mdnA pro-peptide sequence
  • 4 different enzymes for processing of the

pro peptide

mdnC mdnB mdnA mdnD BBa_K627005 mdnE BBa_K627004 mdnD BBa_K627002 mdnB BBa_K627001 mdnA BBa_K627003 mdnC mdnE

ligase ligase acetyltransferase ABC transporter

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Characterization

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100000 200000 300000 400000 500000 600000 700000 3 8 13 18 mAU at 210 nm t in min 1800 m/z 2100 1500 1200 900 2400 1734.12 Da +22 Da +38 Da

  • 368 Da
  • 766 Da

Mass Spectrometry HPLC

mdnC mdnB mdnA mdnD mdnE

YGGTFKYPSDWEDY

successfull detection and characterization

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Design of mdnA Libraries

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5 10 diversity

  • Library successfully generated

wt aa Y G G T F K Y P S D W E D Y modified tht gvt gvt ACC nkk AAA TAC CCT TCT GAC TGG GAA GAT tht aa diversity Y G G F Y V A A A V F D D … F W

  • Total Diversity: 810 possible mutants
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The Progress

Microcystis aeruginosa

mdn cluster

Library Generation Selection System Optimized Protease Inhibitor

 

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coating

Phage Display

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binding washing elution infect E.coli

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Preparing the Phage

mdnA-myc-tag-geneIII M13 Phage

gene III microviridin myc-tag

mdnC mdnB mdnA-myc-geneIII mdnD mdnE

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Establishing Phage Display

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BBa_K627006 mdnA-myc-gene III BBa_K627007 myc-gene III

Generated BioBricks ELISA

  • myc tag detected

0,2 0,4 0,6 0,8 1 1,2

control mdnA extinction at 492 nm

9E10 (anti-myc) Ab anti-geneVIII Ab-HRP

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Test Panning

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Phage Display – Panning on Trypsin

1 2 3 4 5 6 7

pre-panning post panning mdnA-Phage in %

  • microviridin on phage
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Panning with Different Proteases

20 40 60 80 100 120

clone number after panning

  • microvirdin binds papain
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The Progress

Microcystis aeruginosa

mdn cluster

Library Generation Selection System Optimized Protease Inhibitor

 

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In Vivo Selection

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ssTorA

  • Basic Idea
  • β–lactamase is only active in

periplasm

  • signal sequence of TorA serves

as a export signal

ampicillin

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Results

R P BioBr

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  • functional export of β-lactamase into the periplasm worked

 cells were able to survive up to 400 µg/ml ampicillin

0% 20% 40% 60% 80% 100% 120% 140% Surviving CFU

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In Vivo Selection

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proteolytic cleavage

  • cells will die

ampicillin protease

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BBa_K627017 14 3C

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Protease Detector at Work ?

Generated BioBricks

BBa_K627012 TorA-14 3C cleavage-β-lactamase BBa_K627011 AraC_14 3C BBa_K627013 TorA-TEV cleavage-β-lactamase BBa_K627008 AraC_TEV

  • Kill switch worked

TorA-TEV cleavage-β-lactamase AraC_TEV TorA-TEV cleavage-β-lactamase

20 40 60 80 100 120 25 50 75 100 150 200 300 400 600 800 Surviving colonies (%) Ampicillin concentration in µg/ml

BBa_K627016 TEV

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In Vivo Selection

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TorA

ampicillin INHIBITION

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Modeling of in vivo Selection

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can be modeled…

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Fitting the Model to Data

Model fitted to data

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 

The Progress

Microcystis aeruginosa

mdn cluster

Library Generation Selection System Optimized Protease Inhibitor

 

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Let‘s test the Libary - Results

Cells survive up to 400 µg/ml ampcillin libary works mdnA libary

+

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 

The Progress

Microcystis aeruginosa

mdn cluster

Library Generation Selection System Optimized Protease Inhibitor

  

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Human Practice and Xtras

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Parliament Survey

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high medium low abstention

high medium

Synthetic Biology: Medicine and Healthcare

POTENTIAL RISK

Asked: >600 Answers: 10  Invitation to German Parliament

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Visit the German Consulate

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Boston, 4th November 2011 All German finalist iGEM-teams at the German Consulate in Boston (Bielefeld, Munich and Potsdam)

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Achievments and Future Plans

  • achievments:

– 13 BioBricks

  • 5 for mdn modularization
  • 2 for phage display
  • 6 for in vivo selection

– proof of principle for all systems – talk with German parliamentarian

  • future plans:

– using the libary against different proteases – inhibition studies for new Microviridins

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Achievments and Future Plans

… and another big achievment: The BioLog App

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A(pp)tention Please…

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Thank you for your attention!

Dank: Instruktoren und Sponsore Refrences

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iGEM 2011 World Championship Jamboree at MIT in Boston Sunday, 6th November 2011

Questions?