Post on 22-Jul-2020
transcript
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!
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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
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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!