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

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, millions of people are infected with deseases related to protease activity • cyclic peptides • stable • unique potential • use as therapeutic agent 3

Microviridin Family • cyclization over side chain residues – no cysteins or any backbone structure involved • cyclic peptide with two ester and one amide bond 4

The Project Microcystis aeruginosa mdn cluster Heterologous Expression in E. coli Library Generation Selection System Optimized Protease Inhibitor

Modularization of the Cluster mdnA mdnB mdnC mdnD mdnE mdnA mdnB mdnC mdnD mdnE BBa_K627001 BBa_K627002 BBa_K627003 BBa_K627004 BBa_K627005 ligase acetyltransferase ABC transporter ligase • 6.5 kbp cluster with 5 important elements: • mdnA pro-peptide sequence • 4 different enzymes for processing of the pro peptide 6

Characterization mdnA mdnB mdnC mdnD mdnE 700000 YGGTFKYPSDWEDY 600000 1734.12 Da 500000 mAU at 210 nm 400000 300000 200000 +22 Da +38 Da 100000 -766 Da -368 Da 0 900 1200 1500 1800 2100 2400 3 8 13 18 m/z t in min HPLC Mass Spectrometry  successfull detection and characterization 7

Design of mdnA Libraries 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 Y G G F Y V A A A V aa diversity F D D … F W 10 diversity 5 0  Total Diversity: 810 possible mutants  • Library successfully generated 8

The Progress Microcystis aeruginosa  mdn cluster  Library Generation Selection System Optimized Protease Inhibitor

Phage Display coating binding washing elution infect E.coli 10

Preparing the Phage microviridin gene III mdnA-myc-tag-geneIII mdnA-myc-geneIII mdnB mdnC mdnD mdnE M13 Phage myc-tag 11

Establishing Phage Display Generated BioBricks mdnA-myc-gene III myc-gene III BBa_K627006 BBa_K627007 1,2 • myc tag detected 1 extinction at 492 nm  anti-geneVIII Ab-HRP 0,8 0,6 0,4 9E10 (anti-myc) Ab 0,2 0 control mdnA ELISA 12

Test Panning 7 mdnA-Phage in % 6 5 4 3 2 1 0 pre-panning post panning Phage Display – Panning on Trypsin  • microviridin on phage 13

Panning with Different Proteases 120 clone number after panning 100 80 60 40 20 0  • microvirdin binds papain 14

The Progress Microcystis aeruginosa  mdn cluster  Library Generation  Selection System Optimized Protease Inhibitor

In Vivo Selection • Basic Idea • β – lactamase is only active in periplasm • signal sequence of TorA serves as a export signal ssTorA ampicillin 16

Results 140% R 120% P 100% Surviving CFU 80% BioBr 60% 40% 20% 0 0%  • functional export of β -lactamase into the periplasm worked  cells were able to survive up to 400 µg/ml ampicillin 17

In Vivo Selection proteolytic cleavage • cells will die protease ampicillin 18

Protease Detector at Work ? Generated BioBricks TorA-14 3C cleavage- β -lactamase TorA-TEV cleavage- β -lactamase BBa_K627012 BBa_K627013 AraC_14 3C TEV 14 3C AraC_TEV BBa_K627011 BBa_K627017 BBa_K627008 BBa_K627016 120 100 Surviving colonies (%) 80 60 • 40 Kill switch worked  20 0 0 0 0 0 0 0 0 0 25 50 75 100 150 200 300 400 600 800 Ampicillin concentration in µg/ml TorA-TEV cleavage- β -lactamase TorA-TEV cleavage- β -lactamase AraC_TEV 19

In Vivo Selection INHIBITION TorA ampicillin 20

Modeling of in vivo Selection can be modeled… 21

Fitting the Model to Data  Model fitted to data 22

The Progress Microcystis aeruginosa  mdn cluster  Library Generation   Selection System Optimized Protease Inhibitor

Let‘s test the Libary - Results + mdnA libary Cells survive up to 400 µg/ml ampcillin  libary works 24

The Progress Microcystis aeruginosa  mdn cluster  Library Generation   Selection System  Optimized Protease Inhibitor

Human Practice and Xtras 26

Parliament Survey Synthetic Biology: Medicine and Healthcare POTENTIAL RISK Asked: >600 Answers: 10  Invitation to German Parliament high medium low abstention high medium 27

Visit the German Consulate Boston, 4th November 2011 All German finalist iGEM-teams at the German Consulate in Boston (Bielefeld, Munich and Potsdam ) 28

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 29

Achievments and Future Plans … and another big achievment: The BioLog App 30

A(pp)tention Please … 31

Thank you for your attention! Dank: Instruktoren und Sponsore Refrences 32

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