Jamboree Presentation

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

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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

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Outreach Events

Shoreline Stem Festival

Engineering Discovery

Days

Bennet Elementary School

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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!