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

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

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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

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TEAM HEIDELBERG – iGEM 2014

PCR 2.0

Heat stable proteins are valuable for…

  • Industrial processes
  • Research applications

e.g. Taq polymerase

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SLIDE 2

TEAM HEIDELBERG – iGEM 2014

PCR 2.0

PCR 2.

… maintaining DNA methylation patterns

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TEAM HEIDELBERG – iGEM 2014

PCR 2.0

DETERMINANTS OF

Two accesible ends allow many degr gree ees of

  • f freedom

Circu rcular pr proteins show remarkable resistance (Craik, Science, 2006) Existing approaches are not

  • t stand

andar ardized zed, therefore time consuming

(Vieille and Zeikus, MMBR, 2001)

PROTEINS STABILITY

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SLIDE 4

TEAM HEIDELBERG – iGEM 2014

PCR 2.0

N’

O NH

N Intein

O NH

C’

C Intein

Mechan hanism of

  • f in

intein in tra trans-sp splicing ng

Extein residues

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)

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TEAM HEIDELBERG – iGEM 2014

PCR 2.0

O NH

N’

N Intein

O NH

C’

C Intein

O N H

Mechan hanism of

  • f in

intein in tra trans-sp splicing ng

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)

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TEAM HEIDELBERG – iGEM 2014

PCR 2.0

N Intein C Intein

O N H

Protein of Interest Protein of Interest

Intein splicing can be applied for circularization.

(Iwai and Plückthun, FEBS, 1999)

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)

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TEAM HEIDELBERG – iGEM 2014

PCR 2.0

SPLIT INTEINS - A VALUABLE TOOL FOR BIOENGINEERING

N C N C

Examples of circularization: β-Lactam amase ase

(Iwai and Plückthun, FEBS, 1999)

GFP

(Iwai et al., JBC, 2001)

4.6 Å

Our target: Xylanase anase from B. subtilis

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TEAM HEIDELBERG – iGEM 2014

PCR 2.0

CIRCULAR XYLANASE FOR INDUSTRY

Introduction of terminal disulfide bonds contributed to sta tabil iliz izatio ion (Wakarchuk et al., Protein Eng. 1994) Close pro roxim imit ity of

  • f termini

allows direct circularization Biotechnological use for paper bleachin ing and biofuel produ duction

4.6 Å

Our target: Xylanase anase from B. subtilis

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SLIDE 9

TEAM HEIDELBERG – iGEM 2014

PCR 2.0

linear circular

Com

  • mplete ex

excisi sion of split inteins verifies effic icie ient splicing process

CIRCULAR XYLANASE FOR INDUSTRY

Increased ele lectro trophoretic tic mobili ility ty

  • f circular Xylanase
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SLIDE 10

TEAM HEIDELBERG – iGEM 2014

PCR 2.0

Time me [ [mi min] Fl Fluo uoresc scen ence [rfu rfu]

CIRCULAR XYLANASE FOR INDUSTRY

  • E. coli

37 37°C 63 63°C

Ce Cell lysate tes

Compa parabl ble activities at 37 37°C Circ ircula lar Xylanase remains active ve afte ter r hea heat shock hock

Linea near 3 37°C Circ ircular 37 37°C Circ ircular 63 63°C C Linea near 6 63°C

Fl Fluo uoresc scen ence [rfu rfu] Time me [ [mi min] Δ Act ctivity ty

Circ ircular 37 37°C Linea near 3 37°C Cont ntrol 37 37°C

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TEAM HEIDELBERG – iGEM 2014

PCR 2.0

THE RING OF FIRE

STANDARDIZATION OF INTEIN MECHANISM ENGINEERING HEAT-STABLE XYLANASE APPLICATION FOR METHYLATION MAINTENANCE

PCR 2.0

IN SILICO DESIGN OF CIRCULARIZATION LINKERS

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TEAM HEIDELBERG – iGEM 2014

PCR 2.0

free ends

Stability

rigid linker

Wang et. al, Nucl.

  • Ac. Res, 2008

Hypothesis

flexible linker

Zhou, Acc Chem. Res., 2004

THERMOSTABILITY DEPENDS ON LINKER STRUCTURE

Restricted mobility of protein termini

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TEAM HEIDELBERG – iGEM 2014

PCR 2.0

ROD AND ANGLE LINKERS MAINTAIN PROTEIN STRUCTURE

angle rod

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TEAM HEIDELBERG – iGEM 2014

PCR 2.0 AEAAAK AEAAAK-EAAAKA AEAAAK-EAAAK-EAAAK-EAAAKA

(Arai et al., Protein Eng., 2001)

RODS ARE BUILT FROM MODULAR, STABLE α-HELICES

angle rod For every rod

  • d

a fitting helix sequence:

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SLIDE 15

TEAM HEIDELBERG – iGEM 2014

PCR 2.0

ANALYSIS OF 43,000 SUPERSECONDARY STRUCTURES REVEALS ANGLE-CREATING SEQUENCE MOTIFS

29. 29.7°, , NVL 35 35°, , LVA 36. 36.5°, , AAIAP 38. 38.7°, , KTA 60 60°, , AADGTL 74. 74.5°, , VNLTA 140 40°, , ASLPAA 160 60°, , ATGDLA

For every ang ngle a short sequence: angle rod 117 117°, , AAAHPEA

angle: sequence:

(ArchDB)

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TEAM HEIDELBERG – iGEM 2014

PCR 2.0

angle rod For every ang ngle a short sequence:

(ArchDB)

29. 29.7°, , NVL 35 35°, , LVA 36. 36.5°, , AAIAP 38. 38.7°, , KTA 60 60°, , AADGTL 74. 74.5°, , VNLTA 140 40°, , ASLPAA 160 60°, , ATGDLA 117 17°, , AAAHPEA

ANALYSIS OF 43,000 SUPERSECONDARY STRUCTURES REVEALS ANGLE-CREATING SEQUENCE MOTIFS

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SLIDE 17

TEAM HEIDELBERG – iGEM 2014

PCR 2.0

angle rod

CONFIRMATION OF LINKER MOTIF PROPERTIES

STRUCTURE SCREENING STRUCTURE MODELING

Confirmation of linker lengths

USING iGEM@HOME

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TEAM HEIDELBERG – iGEM 2014

PCR 2.0

A LINKER DESIGN SOFTWARE WORKING ON ANY PROTEIN

OF UNLINKED TERMINI CIRCULARIZATION WITH RODS AND ANGLES

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TEAM HEIDELBERG – iGEM 2014

PCR 2.0

THE CRAUT WORKFLOW

IN A NUTSHELL

  • 1. Find paths
  • 2. Translate to patterns
  • 3. Scoring
  • 4. Linker selection
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TEAM HEIDELBERG – iGEM 2014

PCR 2.0

b. Angles

  • c. Distance
  • a. Length
  • d. Active Sites

LINKERS ARE SCORED BASED ON FOUR STRUCTURAL PROPERTIES

  • 3. Scoring -
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TEAM HEIDELBERG – iGEM 2014

PCR 2.0

LINKER SCREENING

  • 1. EXPRESS WITH

LINKERS

  • 2. LYSE CELLS
  • 3. APPLY HEAT SHOCK TO

SUPERNATANT

  • 4. MEASURE SUBSTRATE

DEGRADATION

We searched for a protein that:

  • easy expression in E.coli
  • no purification needed
  • calls for a linker
  • standardized activity assay

USING LAMBDA LYSOZYME

and chose λ-Phage ge Lys ysozym zyme for the screening:

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TEAM HEIDELBERG – iGEM 2014

PCR 2.0

LINKER SCREENING USING LAMBDA LYSOZYME

  • biological and technical
  • 100 000

00

Activ tivit ity as assay ay

Time [min] Substrate [au] (Exemplary curve)

datapoints

  • 1000 substrate degradation curves
  • 9 constructs

re replicates Michaelis-Menten with product inhibition

ENYZME KINETICS MODELING

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TEAM HEIDELBERG – iGEM 2014

PCR 2.0

CRAUT - OPTIMIZED RIGID LINKER ENHANCES THERMOSTABILITY

linear lysozyme linear lysozyme linear lysozyme

ENYZME KINETICS MODELING

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SLIDE 24

TEAM HEIDELBERG – iGEM 2014

PCR 2.0

CRAUT - OPTIMIZED RIGID LINKER ENHANCES THERMOSTABILITY

linear lysozyme flexibel lysozyme linear lysozyme flexibel lysozyme linear lysozyme flexibel linker

ENYZME KINETICS MODELING

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SLIDE 25

TEAM HEIDELBERG – iGEM 2014

PCR 2.0

CRAUT - OPTIMIZED RIGID LINKER ENHANCES THERMOSTABILITY

linear lysozyme linear lysozyme flexibel linker linear lysozyme linear lysozyme linear lysozyme linear lysozyme linear lysozyme flexibel lysozyme rigid linker (low score) linear lysozyme flexibel lysozyme rigid linker (low score) linear lysozyme flexibel linker rigid linker (low score)

ENYZME KINETICS MODELING

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SLIDE 26

TEAM HEIDELBERG – iGEM 2014

PCR 2.0

CRAUT - OPTIMIZED RIGID LINKER ENHANCES THERMOSTABILITY

linear lysozyme linear lysozyme flexibel linker linear lysozyme flexibel lysozyme rigid linker (low score) linear lysozyme flexibel linker rigid linker (low score) linear lysozyme flexibel lysozyme rigid linker (high score) rigid linker (low score) linear lysozyme flexibel lysozyme rigid linker (low score) linear lysozyme flexibel linker rigid linker (high score) rigid linker (low score)

Δ

ENYZME KINETICS MODELING

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TEAM HEIDELBERG – iGEM 2014

PCR 2.0

OUR IN SILICO LINKER DESIGN SOFTWARE WAS BASED

in p publico co

STRUCTURE SCREENING LYSOZYME ASSAY STRUCTURE MODELING

in n silic lico in vi vitro tro

ON A NOVEL MODEL AND VALIDATED IN VITRO

ENYZME KINETICS MODELING

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SLIDE 28

TEAM HEIDELBERG – iGEM 2014

PCR 2.0

OUR IN SILICO LINKER DESIGN SOFTWARE WAS BASED ON A NOVEL MODEL AND VALIDATED IN VITRO

in p publico co

LYSOZYME ASSAY STRUCTURE MODELING

in n silic lico in vi vitro tro

STRUCTURE SCREENING ENYZME KINETICS MODELING

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TEAM HEIDELBERG – iGEM 2014

PCR 2.0 Supports Java and Py nd Pyth thon Open source software  Available to all iGEM teams!

1294

Active Computers $ 28,190 Cluster/month

BOINC NC project Feedback angle structure

iGEM@HOME FOR DISTRIBUTED COMPUTING

www. w.ige gemathom

  • me.or
  • rg
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TEAM HEIDELBERG – iGEM 2014

PCR 2.0

User Activity

GLOBAL HUMAN PRACTICE APPROACH iGEM@HOME FOR A

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TEAM HEIDELBERG – iGEM 2014

PCR 2.0

Evaluation

(6) Did you know iGEM before participating in iGEM@home?

No No [63. 63.4% 4%] N.A.[7.3%] Yes, I knew [29.3%]

GLOBAL HUMAN PRACTICE APPROACH iGEM@HOME FOR A 400

active users

40,000

slides shown

iGEM@home

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TEAM HEIDELBERG – iGEM 2014

PCR 2.0

Discussion evening: Synthetic Biology, Ethics and Religion Laboratory course for High school students

OUTREACH ACTIVITIES ON A LOCAL LEVEL

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TEAM HEIDELBERG – iGEM 2014

PCR 2.0

THE RING OF FIRE

STANDARDIZATION OF INTEIN MECHANISM ENGINEERING HEAT-STABLE XYLANASE APPLICATION FOR METHYLATION MAINTENANCE

PCR 2.0

IN SILICO DESIGN OF CIRCULARIZATION LINKERS PCR 2.0

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TEAM HEIDELBERG – iGEM 2014

PCR 2.0

C

CH3

DNA methylation

Epigenetic regulation in development and disease

Jaenisch et al., Nat. Genet., 2003

REVOLUZIONIZE PCR

Circular

DNA methyl- transferase

Maintaining methylation patterns in

PCR 2.0 EMPOWER EPIGENETICS

PCR 2.0

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TEAM HEIDELBERG – iGEM 2014

PCR 2.0

DNMT1 MAINTAINS METHYLATION

C G G C

CH3 CH3

AFTER REPLICATION

C G G C

CH3 CH3

Fully methylated DNA

C G G C

CH3 CH3

Hemi-methylated DNA

C G G C

CH3

Parental strand Newly synthetized strand

C G G C

CH3

Replication Methylation by DNMT1 PCR 2.0

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TEAM HEIDELBERG – iGEM 2014

PCR 2.0

TRUNCATED DNMT1 NOW

Trun uncat ated m mDNMT1 T1 (731 31-160 602) Circular D DNMT NMT1

Linker

provided by P. Bashtrykov

CIRCULARIZED

48Å

PCR 2.0

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TEAM HEIDELBERG – iGEM 2014

PCR 2.0

CIRCULAR DNMT1 WORKS

Methylated undigested Unmethylated

37°C

Activity of DNMT1 assayed after heat shock

using methylation sensitive restriction enzymes: Sau3AI, HpaII

(Bashtrykov et al., Chem. Biol., 2012)

AT HIGH TEMPERATURES

72°C 65°C

PCR 2.0

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TEAM HEIDELBERG – iGEM 2014

PCR 2.0

CIRCULAR DNMT1 IS MORE HEAT-STABLE THAN LINEAR DNTM1

∆

Linear DNMT1 Circular DNMT1

PCR 2.0

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SLIDE 39

TEAM HEIDELBERG – iGEM 2014

PCR 2.0

MOVING THE FIRST STEP TOWARDS

PCR2.0 with 6 cycles as simple proof of concept

each cycle Methylated Unmethylated

THE PCR 2.0

  • nce

DNMT1 added amplified

Unmet. Fully met Specificity control

PCR PCR 2.0

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TEAM HEIDELBERG – iGEM 2014

PCR 2.0

THE RING OF FIRE

STANDARDIZATION OF INTEIN MECHANISM ENGINEERING HEAT-STABLE XYLANASE APPLICATION FOR METHYLATION MAINTENANCE

PCR 2.0

IN SILICO DESIGN OF CIRCULARIZATION LINKERS

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TEAM HEIDELBERG – iGEM 2014

PCR 2.0

THE INTEIN TOOLBOX PROTEIN SPLICING FOR EVERYONE

BBa_K1362000 BBa_K1362001 BBa_K1362002 BBa_K1362003 BBa_K1362054 BBa_K1362055 BBa_K1362056 BBa_K1362057 BBa_K1362058 BBa_K1362059 BBa_K1362060 BBa_K1362000 BBa_K1362001 BBa_K1362141 BBa_K1362142 BBa_K1362061 BBa_K1362110 BBa_K1362050 BBa_K1362051 BBa_K1362170 BBa_K1362171 BBa_K1362172 BBa_K1362173 BBa_K1362174

Circularization Purification Oligomerisation Fusion&Tagging On / Off favouri rite te pa part

BBa_K1362000

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TEAM HEIDELBERG – iGEM 2014

PCR 2.0

GUIDES AND STANDARDS OUR TOOLBOX COMES TO YOU CLONING STANDARD

DNA overhang C I S G ... C L S G ... C L T G ... C F S G ... C L T Y ... I V H N V V H N L V H N F V H N I A S N N-intein Ssp DnaB Ter ThyX Ssp DnaX Ssp GyrB Ter DnaE3

TGCT CAAC

TOOLBOX GUIDE

C-intein

Find the right parts!

Step 1: Find 3D structure Step 2: Generate linker sequence Step 3: Use BBa_K1362000

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TEAM HEIDELBERG – iGEM 2014

PCR 2.0

THE INTEIN TOOLBOX PROTEIN SPLICING FOR EVERYONE

On / Off Circularization Purification Oligomerisation Fusion&Tagging favouri rite te pa part

BBa_K1362000 BBa_K1362001 BBa_K1362002 BBa_K1362003 BBa_K1362054 BBa_K1362055 BBa_K1362056 BBa_K1362057 BBa_K1362058 BBa_K1362059 BBa_K1362060 BBa_K1362000 BBa_K1362001 BBa_K1362141 BBa_K1362142 BBa_K1362061 BBa_K1362110 BBa_K1362050 BBa_K1362051 BBa_K1362170 BBa_K1362171 BBa_K1362172 BBa_K1362173 BBa_K1362174 BBa_K1362000

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TEAM HEIDELBERG – iGEM 2014

PCR 2.0

PROTEIN ACTIVATION VIA INTEINS RECONSTITUTING SPLIT INDC

splicing inteins non-splicing control

+

Readout via blue pigment Split indigoidine synthetase (130kD kDa)

IndCN IndCC On / Off

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SLIDE 45

TEAM HEIDELBERG – iGEM 2014

PCR 2.0

THE RING OF FIRE

STANDARDIZATION OF INTEIN MECHANISM ENGINEERING HEAT-STABLE XYLANASE APPLICATION FOR METHYLATION MAINTENANCE

PCR 2.0

IN SILICO DESIGN OF CIRCULARIZATION LINKERS

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TEAM HEIDELBERG – iGEM 2014

PCR 2.0

ACHIEVEMENTS

BRO ROUGH UGHT C T CIRCU CULAR ARIZATI ZATION FROM NATURE TO iGEM DEVEL ELOPED PED I IN SILICO CO D DESIGN GN C CON ONCE CEPT FOR PROTEIN LINKERS AP APPLI LIED CIRCULAR DNMT1 FOR METH THYL YLATI TION M N MAINTAI TAINING NG PCR STANDAR NDARDI DIZE ZED THE USE OF INTEINS

67 parts

LYS LYSOZYME DNMT MT1 XYLAN ANAS ASE

PCR 2.0

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TEAM HEIDELBERG – iGEM 2014

PCR 2.0

Acknowledgements

External Cooperations

  • Prof. Dr. Henning Mootz
  • Dr. Pavel Bashtrykov
  • Prof. Albert Jeltsch
  • Prof. Dinshaw Patel
  • Prof. Jikui Song
  • Prof. Dr. Rebecca Wade

Xiaofeng Yu

  • Dr. Ben Webb
  • Prof. Dr. Matthias Mayer
  • Dr. Andrea Gumiero

Johanna Klughammer DKFZ Life-Science Lab

Bioquant/DKFZ

  • Prof. Dr. Robert Russell
  • Dr. Oliver Wichmann

Mathias Utz Ute Koch

  • Prof. Dr. Frank Lyko
  • Dr. Dieter Weichenhan
  • Dr. Karsten Rippe

Stephanie Trauth

  • Dr. Martina Schnölzer
  • Dr. Uwe Warnken

Dominik Niopek Tim Heinemann

Students

Konrad Herbst Florian Schmidt Ilja Kats Fanny Georgi Nikos Ignatiadis Stefan Holderbach Stefan Huber Lisa Theobald Leon Binder Michael Blessenohl

iGEM Teams

Team Tuebingen Team Aachen Team Freiburg Team Marburg

All iGEM@home Users!!!

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TEAM HEIDELBERG – iGEM 2014

PCR 2.0

ACKNOWLEDGMENTS WE THANK YOU ALL

PCR 2.0

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TEAM HEIDELBERG – iGEM 2014

PCR 2.0

ACKNOWLEDGMENTS WE THANK YOU ALL

PCR 2.0

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TEAM HEIDELBERG – iGEM 2014

PCR 2.0

PROTEIN ACTIVATION VIA INTEINS RECONSTITUTION OF SPLIT GFP

Splic licin ing int ntei eins

5 µm 5 µm

fluorescence (497 - 522 nm)

No Non-splic licin ing co contr trol

fluorescence (497 - 522 nm) events events

On / Off

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TEAM HEIDELBERG – iGEM 2014

PCR 2.0

CIRCULAR DNMT1 IS MORE HEAT-STABLE

THAN LINEAR DNTM1 ∆

Linear DNMT1 Circular DNMT1