SLIDESHOW  

Charming  Carmen  

Backup  Bas1  

Tanky  Thomas  

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Building with light!

iGEM Team 2015!TU Darmstadt!

Building with light opens! up a spectrum of possibilities!

       

The Problem!

Problems in critical and poor regions:!•  Bad infrastructure!

•  Expensive materials!•  No adjustment!•  Long-term production !

“There are an estimated 11.4 million hand amputees worldwide...”!

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Our Solution: SLA Printing!

projector  mirror  

basin  stepper  motor  

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Our Solution: SLA Printing!

5  

Overview!

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Overview!

Why?!•  Stronger mechanical integrity

by SLA!•  Avoiding expensive prosthesis!

•  Enhanced biocompatibility!•  Gaining variability by

generation of a toolbox!7  

Monomer Toolbox!

Photoinduced  cross-­‐linker  (Itaconic  acid)  

Spacer  (Diol  /  Dicarbonic  acid)  

Cross-­‐linker  (Polyol)  

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Xylan Degradation!

XynA  (WLC-­‐Milwaukee,  2013)  

Aes  (ETH  Zürich,  2013)  

Ruxyn1  (HUST-­‐China,  2012)  

Side  chain  

Main  chain  Side  chain  

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In Vivo Metabolic Engineering!

Best  Composite  Part:  

CO2  

cis-­‐aconitate  decarboxylase  cadA  

Expression   Func1onality  

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In Vivo Metabolic Engineering!

Best  Composite  Part:  

GC-­‐MS  results  

Posi1ve  Control  

Sample  

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Chemistry – Polyester Synthesis!

Best  result  for  applica1on  

Itaconic  acid  

+  

PEG-­‐200   Poly(PEG-­‐itaconate)  =  Pre-­‐polymer  

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Chemistry – Printing process!

Radical  polymeriza1on  

Control  of  polymeriza1on  by  radical  quencher.  

Photoini1ator:  IRGACURE  819  Viscous   Solid  

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Chemistry - Simplification!

Posi1ve  Feedback  Loop!  

Standard  protocol  •  Exclusion  of  air  •  With  argon  

Adjusted  protocol  •  Standard  condiNons  

•  No  argon  

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Policy and Practices!

Detailed  &  inova1ve  Policy  and  Prac1ce  approach!  

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Policy and Practices!

Experts   Panel  discussion   Open  Science  Collabora1ons  (with  iGEM  Aachen  2015)  

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Biosafety!

Key   Lock  

Toxin  release  

Expression   RNA  Molecular  Dynamics  

Cell  Lysis  

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Modeling – RNA structure prediction!

In  vitro  (2D)  Ar1ficial  neural  network  (ANN)  

•  State  of  the  art  dynamic  programming  •  Database  knowledge  •  Extra:  Intermolecular  co-­‐folding  

In  silico  (1D)  

Computa1on  on  GPU  

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Modeling – Riboswitch Prediction!

Particle Swarm Algorithm

→ Trade off (Locking / Unlocking Abilities)

URL:  hfp://rsdnerf.com/   19  

Checklist!

Monomer  Toolbox   Pre-­‐polymer   Policy  and  Prac1ce  

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SLA Printing - Software!

projector  

mirror  

stepper  motor  Slicing   Controlling  

Available  open  source  on  GitHub!  

Raspberry  Pi  

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SLA Printing - Hardware!22  

Advantages!

•  Covalent binding!•  Stronger mechanical integrity!•  Seamless printing!

•  Printing progress!•  Fast, cheap & reliable!•  Costumize specific!

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Hardware!

• PrinNng  with  own  resin  • Adjustable  to  

• Purchased  resin  • Our  own  resin  

• OpNmized  for  UV-­‐light  

• Acrylic  glass  case  protects  progress  

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• Functional open source 3D printer

• Slicing and controlling software

• Photocurrable resin

• Online tool for riboswitch design

•  Wetlab 58 BioBricks

Achievements!

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Expanded Policy and Practices approach • Application scenario •  4 techno-moral scenarios • Positive feedback loops impacted project

Investigations in biodegradability • Polyester degradation • Our best basic part: Humicola insolens cutinase

Labsurfing - iGEM Networking

→  Be part of it: 4:00 pm room 201

More Achievements!

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Thank you!!Prof. Dr. Katja Schmitz!Prof. Dr. Kay Hamacher!Prof. Dr. Wolf-Dieter Fessner!Prof. Dr. Gerhard Thiel!Our experts:!•  Prof. Dr. Stefan Jockenhövel!•  Prof. Dr. Chichkov!•  Dr. Philipp Urban!•  Dr. Harald König!•  Reinhard Heil!•  Christoph Schneider!AG Warzecha!Dr. Melanie Kern!Barbara Wolf!Anne Einhäupl!Karl Schuller!Prof. Dr. Alfred Nordmann!Ana Maria Delgado Aleman!Charlotte Kaspar!

Poster  145  

Acknowledgements!

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projector  mirror  

basin  stepper  motor  

SLA Printing Process!

Radical  polymeriza1on  

Viscous  

Solid  

CO  2  

cis-­‐aconitate  decarboxylase  (BBa_K1602003)  

-­‐  cadA  gene  encoded  by  Aspergillus  terreus  

-­‐  Molecular  weight:  52,8  kDa  

-­‐  pH-­‐OpNmum:  6,2  

-­‐  Temperature  OpNmum:  37  °C  

cis-­‐aconitate  decarboxylase  cadA  

Itaconic Acid Production!

Itaconic Acid Production!

Posi1ve  control  

Biological  sample  

GC-­‐MS  

cis-aconitate decarboxylase (cadA)!

CO        +    H    O                                            H    CO                                          2  

cis-­‐aconitate  decarboxylase  

cis-­‐aconitate   itaconic  acid  

2   2              3  

-­‐  pH  indicator:  bromothymol  blue  

-­‐  AbsorpNon  maximum:  620  nm  

aldose  reductase  GRE3  

aldose  reductase  (BBa_K1602004)  

-­‐  GRE3  gene  encoded  by  Saccharomyces  cerevisiae  

-­‐  Molecular  weight:  37,1  kDa  

-­‐  NADPH  dependent    

-­‐  Temperature  OpNmum:  28  °C  

NADPH   NADP  +  

Xylitol Production!

aldose  reductase  GRE3  

aldose  reductase  (BBa_K1602004)  

-­‐  GRE3  gene  encoded  by  Saccharomyces  cerevisiae  

-­‐  Molecular  weight:  37,1  kDa  

-­‐  NADPH  dependent    

-­‐  Temperature  OpNmum:  28  °C  

NADPH   NADP  +  

Xylitol Production!

NADPH  assay,  340  nm  

Xylan Degradation!

XynA  (WLC-­‐Milwaukee,  2013)  

Aes  (ETH  Zürich,  2013)  

Ruxyn1  (HUST-­‐China,  2012)  

Side  chain  

Main  chain  Side  chain  

9  

Immobilized  protein  scaffold  on  a  silica  surface  

In Vitro Metabolic Channeling!

Silica  

In Vitro Bioreactor!

Surface  enlargement   In  vitro  degrada1on  

Posttranslational Regulated Killswitch!

Key  &  lock  principle  

Key   Lock  

Toxin  release  

Cell  Lysis  

Posttranslational Regulated Killswitch!

Promoter    valida1on   Key  &  lock  interac1on  

Posttranslational Regulated Killswitch!

Conclusion:  2  possible  scenarios  

• Leaky  expression  of  hokD  leads  to  cell  death  

• No  interacNon  between  taRNA  and  crRNA  

Stability  simula1on  at  600K:  

all-­‐atom-­‐RMSD   all-­‐atom-­‐RMSF  

3D-­‐Structure  predicNon  

RNA Molecular Dynamics!

RNA Structure Prediction!Primary  structure   Secondary  structures  

folds  to  

Riboswitch Prediction!

Particle Swarm Algorithm

→ Trade off (Locking / Unlocking Abilities)

• the  acachment  plate  is  made  out  of  brushed  aluminum  • the  resin  basin  is  coated  with  a  polypropylene  foil  (or  fluorinated  ethylene  propylene)  

Hardware!

Hardware!• Basic  idea  originates  from  instrucables.com  

• Adapted  to  our  own  needs  from  the  original  instrucNon  by  TristramBudel  

THANK  YOU!