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Measuring multiple metabolite concentration in one sample is
difficultTraditional methods of analysing chemical compounds are
expensive and labour intensive
Example: HPLC (High Performance Liquid Chromatography)
- Separates chemical and biological compounds that are non-volatile
- Commonly used analytical method in metabolic engineering.
Background Objective Wetlab Drylab Future Application Collaboration Human Practices Conclusion
$5k - $17k
$35.00 - $90.00 per hour
HPLC Limitations: (i) Price
Background Objective Wetlab Drylab Future Application Collaboration Human Practices Conclusion
• Not able to continuously analyse samples
• Must be constantly monitored
HPLC Limitations: (ii) Tedious
• Multiple runs must be performed Cost increases
Frequent iGem use
Background Objective Wetlab Drylab Future Application Collaboration Human Practices Conclusion
Principles Combined to Reach Solution
Proof of Concept: Violacein Pathway
+Synthetic Biology =
Autonomous control system
Electrical Engineering
Background Objective Wetlab Drylab Future Application Collaboration Human Practices Conclusion
We’ve created an automated system made of :-
(i) Culture Management System
(ii) Image Processing and Response System
Alternative to HPLC
<$150
Background Objective Wetlab Drylab Future Application Collaboration Human Practices Conclusion
Function of Each Automated System
Culture Management SystemControls the growth rate of the yeast culture
-Determines metabolite level with images
Image Processing and Response System
Sends feedback signals to Culture Management System
Background Objective Wetlab Drylab Future Application Collaboration Human Practices Conclusion
Benefits of Our Automated System
Small sample required
Can be carried out continuously
Inexpensive!
Background Objective Wetlab Drylab Future Application Collaboration Human Practices Conclusion
Project Objectives
Modification of Violacein
Pathway
Decomposition into RGB
(RedGreenBlue) values
Raspberry Pi computes
difference in values
Release of inducible
chemicals for correction
and optimisation
Background Objective Wetlab Drylab Future Application Collaboration Human Practices Conclusion
Wet Lab: Choosing the Violacein Pathway
5-Enzyme Violacein Pathway
COLORED METABOLITES ACT AS
COLOR INDICATORS
4 Color Indicators
PURPLE
PINK
TEAL
GREEN
Wetlab: Violacein Pathway Plasmid Design Plasmid Results Future Directions BioBricks
Wet Lab: Original Violacein Pathway
VIO A VIO B VIO EVIO A VIO B VIO E VIO C VIO D PURPLEVIO C VIO D
Violacein
In nature, all 5 enzymes are expressed
We decided to modify the violacein pathway in order create more
color indicators
Wetlab: Violacein Pathway Plasmid Design Plasmid Results Future Directions BioBricks
Wet Lab: Modified Violacein Pathway
VIO A VIO B VIO E VIO C VIO D TEAL
Proviolacein
VIO A VIO B VIO E VIO C VIO D PINK
Deoxyviolacein
Cup1
Cup1
Gal1
Gal1
VIO A VIO B VIO EVIO A VIO B VIO E VIO C GREENVIO D
ProdeoxyviolaceinCup1 Gal1
VIO A VIO B VIO E
VIO A VIO B VIO E
Wetlab: Violacein Pathway Plasmid Design Plasmid Results Future Directions BioBricks
2 kb Gal1+VioD
2 kb Cup1+VioC6kb Vector Backbone
6kb VioA+B+E Total Plasmid Size: 16kb
Wet Lab: Plasmid Design
4 part, 16kb Gibson Assembly
Fragments Expected Plasmid
Wetlab: Violacein Pathway Plasmid Design Plasmid Results Future Directions BioBricks
Wet Lab: Plasmid Results
6kb VioA+B+E 6kb Vector Component
2 kb Cup1+VioC 2 kb Gal1+VioD
Results
Improvement Points
Successfully built all of the fragments needed for Gibson Assembly
Actual Gibson Assembly of plasmid proved unsuccessful possibly due to size of plasmid
Increase concentration of fragments for Gibson Assembly
Reduce number of fragments in Gibson Assembly
Conduct additional Overlap Extension PCRs to determine annealing temperature for fragments
Wetlab: Violacein Pathway Plasmid Design Plasmid Results Future Directions BioBricks
VioABE
Gal1+VioDCup1+VioC
Wet Lab: Future Directions
Modification of the Violacein pathway, through two different methods:
• Completing the singular plasmid with our entire Violacein gene cluster via Gibson Assembly
• Making a three plasmid system with VioABE transformed into the chassis strain followed by a double transformation
Wetlab: Violacein Pathway Plasmid Design Plasmid Results Future Directions BioBricks
Wet Lab: BioBricks Submitted
VioC VioD
(VioC and VioD Codon Optimized for
S. cerevisiae)
pCup1
BBa_K2165004BBa_K2165000
Gal1+VioD (Codon
optimized) + ADH1
BBa_K2165001 BBa_K2165002 BBa_K2165003
pCup1+VioC (Codon
optimized) + ADH1
Wetlab: Violacein Pathway Plasmid Design Plasmid Results Future Directions BioBricks
Controlling Our Metabolite Products
Using Control Theory
Drylab: Automation Processing Chemostat Raspberry Pi Data
Theory: Relating Inducer Concentration with Product
ConcentrationMachine Learning
Galpromoter
Repressor⇒Galactose
VioD Expression
Product
Drylab: Automation Processing Chemostat Raspberry Pi Data
Colorimetric analysis
Theory: Relating Product Concentrationwith RGB Values
Drylab: Automation Processing Chemostat Raspberry Pi Data
Our Drylab Hardware Setup
Culture Management
Image Processing + Response
Drylab: Automation Processing Chemostat Raspberry Pi Data
Culture Management using an automatically diluted culture growth system
Chemostat: Maintains yeast growth in constant log phase by controlling dilution rate and thus the growth rate.
Purpose: Keep image quality good and consistent by maintaining nearly identical cultureSimulated image of our Chemostat
Chemostat Vial Media
Bottle
Effluent Bottle
Drylab: Automation Processing Chemostat Raspberry Pi Data
Chemostat vial
Image Processing + Response using a
compact computer and camera
Raspberry Pi: Compact and portable $35 computerCamera: Simple + Immediate use HD camera
Purpose: Analyze image, determine concentration of metabolites, and automate changes
Inducer Syringe
Raspberry Pi
Raspberry Pi Computer Raspberry Pi Camera Motorized inducer syringe
Drylab: Automation Processing Chemostat Raspberry Pi Data
Preliminary Data on our Setup
Drylab: Automation Processing Chemostat Raspberry Pi Data
Analysing a colourless solution
A
B
D
E
C Cup1
Gal
Cup1
Gal
None
L-Trp
Teal
Cup1
Gal
Cup1
Gal
None
Magenta
Green
Violet
Violacein Pathway Metabolic Pathway A
Background Objective Wetlab Drylab Future Application Collaboration Human Practices Conclusion
Future Applications
Background Objective Wetlab Drylab Future Application Collaboration Human Practices Conclusion
Wetlab: Determine rate of expression of VioC and VioD with its respective promoters
Drylab: Translate image analysis system into mobile app with cell phone cameraOptimise platform to use all types of mediaLearn the relationship between inducer input + protein expression of specific
modelFull control over metabolite production entirely through automated systemTrack real-time process and concentration of metabolite through color-
independent methodsUtilize colour signals for error analysis and industrial applications
CollaborationFRET Detection System- Helped troubleshoot their DIY system by advising them on Arduino programming
Background Objective Wetlab Drylab Future Application Collaboration Human Practices Conclusion
Istanbul Technical University
Xiamen University-Participated in XMU-China’s information exchange platform, the iGem Newsletter for 2016.
Human Practices
Comic Series
Outreach Events
Background Objective Wetlab Drylab Future Application Collaboration Human Practices Conclusion
Outreach Events
Shoreline Stem Festival
Engineering Discovery
Days
Bennet Elementary School
Background Objective Wetlab Drylab Future Application Collaboration Human Practices Conclusion
Attributions: Principal Investigator Support
Dr.Liangcai Gu
Dr. Alex Merz
Dr. Eric Klavins Dr. Maitreya DunhamFor advise with and
providing equipmentfor chemostats
For providing emergency cells,
reagents, and enzymes
For assisting with official iGEM
business, fundraising, and administration
Background Objective Wetlab Drylab Future Application Collaboration Human Practices Conclusion
For support, advice, and guidance with troubleshooting on a weekly basis
Advisors
Tom DuanJustin JenkinsChris Choe
Rashmi RavichandranKevin Li
Background Objective Wetlab Drylab Future Application Collaboration Human Practices Conclusion
Wet Lab Members
• Cameron Roots• Anita Elnathan• Natalie
Johnston• Texia Loh• Anne Bania
• Brandy Tang• Sun Jung Park• Vivian Tang• George Sun• Anastasia
Nicolov
Dry Lab Members
• George Sun• Yoshitaka
Goto• Yang Ping
Kuo• Austin Hsu*• Aman Arya*• Ameya
Phansalkar*
*Special thanksBackground Objective Wetlab Drylab Future Application Collaboration Human Practices Conclusion
Sponsors
Background Objective Wetlab Drylab Future Application Collaboration Human Practices Conclusion
THANK YOU!