e.g. Taq polymerase PCR TEAM HEIDELBERG iGEM 2014 2.0 PCR 2. - PowerPoint PPT Presentation

Heat stable proteins are valuable for…  Industrial processes  Research applications e.g. Taq polymerase PCR TEAM HEIDELBERG – iGEM 2014 2.0

PCR 2. … maintaining DNA methylation patterns PCR TEAM HEIDELBERG – iGEM 2014 2.0

DETERMINANTS OF PROTEINS STABILITY Two accesible ends allow many degr gree ees of of freedom Existing approaches are not ot stand andar ardized zed, therefore time consuming (Vieille and Zeikus, MMBR, 2001 ) Circu rcular pr proteins show remarkable resistance (Craik, Science , 2006) PCR TEAM HEIDELBERG – iGEM 2014 2.0

SPLIT INTEINS - A VALUABLE TOOL FOR BIOENGINEERING Autocatalytic protei ein splic licin ing act activity ty in vitro and in vivo (Kane et al., Science ,1990) N C Intein Intein O NH NH O C’ N’ Extein residues Mechan hanism of of in intein in tra trans-sp splicing ng PCR TEAM HEIDELBERG – iGEM 2014 2.0

SPLIT INTEINS - A VALUABLE TOOL FOR BIOENGINEERING Autocatalytic protei ein splic licin ing act activity ty in vitro and in vivo (Kane et al., Science, 1990) N C Intein Intein O NH NH O O C’ N’ N H Mechan hanism of of in intein in tra trans-sp splicing ng PCR TEAM HEIDELBERG – iGEM 2014 2.0

SPLIT INTEINS - A VALUABLE TOOL FOR BIOENGINEERING Autocatalytic protei ein splic licin ing act activity ty in vitro and in vivo (Kane et al., Science , 1990) N C Intein Intein O N H Protein of Interest Protein of Interest Intein splicing can be applied for circularization. (Iwai and Plückthun, FEBS , 1999) PCR TEAM HEIDELBERG – iGEM 2014 2.0

SPLIT INTEINS - A VALUABLE TOOL FOR BIOENGINEERING Examples of circularization: Our target: Xylanase anase from B. subtilis N C β -Lactam amase ase (Iwai and Plückthun, FEBS, 1999) 4.6 Å N GFP C (Iwai et al., JBC, 2001) PCR TEAM HEIDELBERG – iGEM 2014 2.0

CIRCULAR XYLANASE FOR INDUSTRY Close pro roxim imit ity of of termini Our target: Xylanase anase allows direct circularization from B. subtilis Introduction of terminal disulfide bonds contributed to 4.6 Å sta tabil iliz izatio ion (Wakarchuk et al., Protein Eng. 1994) Biotechnological use for paper bleachin ing and biofuel produ duction PCR TEAM HEIDELBERG – iGEM 2014 2.0

CIRCULAR XYLANASE FOR INDUSTRY Increased linear circular ele lectro trophoretic tic mobili ility ty of circular Xylanase Com omplete ex excisi sion of split inteins verifies effic icie ient splicing process PCR TEAM HEIDELBERG – iGEM 2014 2.0

CIRCULAR XYLANASE FOR INDUSTRY E. coli Circ ircular 37 37°C Circ ircular 37 37°C rfu] rfu] Linea near 3 37°C Linea near 3 37°C ence [rfu ence [rfu scen scen Ce Cell lysate tes uoresc uoresc Circ ircular 63 63°C C Fluo Fluo 37°C 37 Fl Fl Δ Act ctivity ty 63°C 63 Linea Cont near 6 ntrol 37 63°C 37°C Time Time me [ me [ [mi [mi min] min] Compa parabl ble activities at 37 37°C Circ ircula lar Xylanase remains active ve afte ter r hea heat shock hock PCR TEAM HEIDELBERG – iGEM 2014 2.0

THE RING OF FIRE ENGINEERING HEAT-STABLE XYLANASE IN SILICO DESIGN OF CIRCULARIZATION LINKERS PCR APPLICATION FOR METHYLATION MAINTENANCE 2.0 STANDARDIZATION OF INTEIN MECHANISM PCR TEAM HEIDELBERG – iGEM 2014 2.0

THERMOSTABILITY DEPENDS ON LINKER STRUCTURE Hypothesis Stability rigid linker Wang et. al, Nucl. Ac. Res, 2008 flexible linker Zhou, Acc Chem. Res ., 2004 free ends Restricted mobility of protein termini PCR TEAM HEIDELBERG – iGEM 2014 2.0

ROD AND ANGLE LINKERS MAINTAIN PROTEIN STRUCTURE angle rod PCR TEAM HEIDELBERG – iGEM 2014 2.0

RODS ARE BUILT FROM MODULAR, STABLE α -HELICES For every rod od angle a fitting helix sequence: AEAAAK rod AEAAAK-EAAAKA AEAAAK-EAAAK-EAAAK-EAAAKA (Arai et al., Protein Eng ., 2001) PCR TEAM HEIDELBERG – iGEM 2014 2.0

ANALYSIS OF 43,000 SUPERSECONDARY STRUCTURES REVEALS ANGLE-CREATING SEQUENCE MOTIFS For every ang ngle angle a short sequence: (ArchDB) 29. 29.7°, , NVL 35 35°, , LVA 36.5°, 36. , AAIAP 38. 38.7°, , KTA rod 60 60°, , AADGTL 74. 74.5°, , VNLTA angle: sequence: 117 117°, , AAAHPEA 140 40°, , ASLPAA 160 60°, , ATGDLA PCR TEAM HEIDELBERG – iGEM 2014 2.0

ANALYSIS OF 43,000 SUPERSECONDARY STRUCTURES REVEALS ANGLE-CREATING SEQUENCE MOTIFS For every ang ngle angle a short sequence: (ArchDB) 29. 29.7°, , NVL 35 35°, , LVA 36. 36.5°, , AAIAP 38. 38.7°, , KTA rod 60 60°, , AADGTL 74. 74.5°, , VNLTA 117 17°, , AAAHPEA 140 40°, , ASLPAA 160 60°, , ATGDLA PCR TEAM HEIDELBERG – iGEM 2014 2.0

CONFIRMATION OF LINKER MOTIF PROPERTIES USING iGEM@HOME angle Confirmation of linker lengths rod STRUCTURE MODELING STRUCTURE SCREENING PCR TEAM HEIDELBERG – iGEM 2014 2.0

A LINKER DESIGN SOFTWARE WORKING ON ANY PROTEIN CIRCULARIZATION WITH RODS AND ANGLES OF UNLINKED TERMINI PCR TEAM HEIDELBERG – iGEM 2014 2.0

THE CRAUT WORKFLOW IN A NUTSHELL 2. Translate to patterns 1. Find paths 3. Scoring - 4. Linker selection PCR TEAM HEIDELBERG – iGEM 2014 2.0

LINKERS ARE SCORED BASED ON FOUR STRUCTURAL PROPERTIES d. Active Sites c. Distance b. Angles 3. Scoring - a. Length PCR TEAM HEIDELBERG – iGEM 2014 2.0

LINKER SCREENING USING LAMBDA LYSOZYME We searched for a protein that: • calls for a linker • easy expression in E.coli • no purification needed and chose λ -Phage ge • standardized activity assay Lys ysozym zyme for the screening: 1. EXPRESS WITH 3. APPLY HEAT SHOCK TO 4. MEASURE SUBSTRATE LINKERS 2. LYSE CELLS SUPERNATANT DEGRADATION PCR TEAM HEIDELBERG – iGEM 2014 2.0

LINKER SCREENING USING LAMBDA LYSOZYME Activ tivit ity as assay ay datapoints 100 000 00 • (Exemplary curve) 1000 substrate degradation curves • 9 constructs • Substrate [au] re replicates biological and technical • Michaelis-Menten ENYZME KINETICS MODELING Time [min] with product inhibition PCR TEAM HEIDELBERG – iGEM 2014 2.0

CRAUT - OPTIMIZED RIGID LINKER ENHANCES THERMOSTABILITY linear lysozyme linear lysozyme linear lysozyme ENYZME KINETICS MODELING PCR TEAM HEIDELBERG – iGEM 2014 2.0

CRAUT - OPTIMIZED RIGID LINKER ENHANCES THERMOSTABILITY linear lysozyme linear lysozyme linear lysozyme flexibel lysozyme flexibel lysozyme flexibel linker ENYZME KINETICS MODELING PCR TEAM HEIDELBERG – iGEM 2014 2.0

CRAUT - OPTIMIZED RIGID LINKER ENHANCES THERMOSTABILITY linear lysozyme linear lysozyme linear lysozyme flexibel lysozyme flexibel lysozyme flexibel linker rigid linker (low score) rigid linker (low score) linear lysozyme linear lysozyme linear lysozyme linear lysozyme linear lysozyme linear lysozyme rigid linker (low score) flexibel linker ENYZME KINETICS MODELING PCR TEAM HEIDELBERG – iGEM 2014 2.0

CRAUT - OPTIMIZED RIGID LINKER ENHANCES THERMOSTABILITY linear lysozyme linear lysozyme linear lysozyme flexibel lysozyme flexibel lysozyme flexibel linker rigid linker (low score) rigid linker (low score) linear lysozyme linear lysozyme linear lysozyme linear lysozyme rigid linker (low score) rigid linker (high score) flexibel lysozyme flexibel linker flexibel linker rigid linker (high score) rigid linker (low score) rigid linker (low score) Δ ENYZME KINETICS MODELING PCR TEAM HEIDELBERG – iGEM 2014 2.0

OUR IN SILICO LINKER DESIGN SOFTWARE WAS BASED ON A NOVEL MODEL AND VALIDATED IN VITRO in vi vitro tro in n silic lico in p publico co LYSOZYME ASSAY ENYZME KINETICS STRUCTURE MODELING MODELING STRUCTURE SCREENING PCR TEAM HEIDELBERG – iGEM 2014 2.0

OUR IN SILICO LINKER DESIGN SOFTWARE WAS BASED ON A NOVEL MODEL AND VALIDATED IN VITRO in vi vitro tro in n silic lico in p publico co LYSOZYME ASSAY ENYZME KINETICS STRUCTURE MODELING MODELING STRUCTURE SCREENING PCR TEAM HEIDELBERG – iGEM 2014 2.0

iGEM@HOME FOR DISTRIBUTED COMPUTING Feedback angle structure BOINC NC project Supports Java and Py nd Pyth thon www. w.ige gemathom ome.or org Open source software  Available to all iGEM teams! Active Computers $ 28,190 1294 Cluster/month PCR TEAM HEIDELBERG – iGEM 2014 2.0

iGEM@HOME FOR A GLOBAL HUMAN PRACTICE APPROACH User Activity PCR TEAM HEIDELBERG – iGEM 2014 2.0

iGEM@HOME FOR A GLOBAL HUMAN PRACTICE APPROACH Evaluation (6) Did you know iGEM before participating in iGEM@home iGEM@home? No No [63. 63.4% 4%] N.A.[7.3%] Yes, I knew [29.3%] 40,000 400 slides shown active users PCR TEAM HEIDELBERG – iGEM 2014 2.0

OUTREACH ACTIVITIES ON A LOCAL LEVEL Discussion evening: Laboratory course for Synthetic Biology, Ethics High school students and Religion PCR TEAM HEIDELBERG – iGEM 2014 2.0

THE RING OF FIRE ENGINEERING HEAT-STABLE XYLANASE IN SILICO DESIGN OF CIRCULARIZATION LINKERS PCR APPLICATION FOR METHYLATION MAINTENANCE 2.0 STANDARDIZATION OF INTEIN MECHANISM PCR PCR TEAM HEIDELBERG – iGEM 2014 2.0 2.0

REVOLUZIONIZE PCR EMPOWER EPIGENETICS CH 3 Circular DNA C methyl- Maintaining methylation DNA transferase patterns in methylation PCR 2.0 Epigenetic regulation in development and disease Jaenisch et al., Nat. Genet., 2003 PCR PCR TEAM HEIDELBERG – iGEM 2014 2.0 2.0