University of TorontoUniversity of Toronto &&

University of WaterlooUniversity of Waterloo

Cells-See-Us Cells-See-Us ThermometerThermometer

Project OverviewProject Overview

Introduction:Introduction:• Limitations to using other thermometersLimitations to using other thermometers

– size of the models may be too large for certain conditionssize of the models may be too large for certain conditions

• Applications of a bio-synthetic thermometerApplications of a bio-synthetic thermometer– map temperature gradients with spatial resolution down to map temperature gradients with spatial resolution down to

micrometersmicrometers– temperature of an organism of any sizetemperature of an organism of any size

Cells-See-Us Thermometer:Cells-See-Us Thermometer:• a bio-synthetic device that detects changes in temperature a bio-synthetic device that detects changes in temperature

and are reflected by colour. and are reflected by colour. • redred when when hothot, , orangeorange when when warmwarm and and greengreen when when coldcold

Dimers breakDimers breakLacI monomers do not bind onto LacI monomers do not bind onto repressing sites – Rfp and CI are repressing sites – Rfp and CI are formed.formed.

CI inhibits GfpCI inhibits Gfp

HotHot

ColdCold

LacI binds to Rfp repressing site (R0010) LacI binds to Rfp repressing site (R0010) and CI repressing site (R0011). No Rfp or and CI repressing site (R0011). No Rfp or CI is madeCI is made

LacI forms dimerLacI forms dimerGfp is formedGfp is formed

Project ModelProject Model

Construction PhaseConstruction Phase

• Constructed many new partsConstructed many new parts

• cI repressor replaced with tetracycline repressor (tetR)cI repressor replaced with tetracycline repressor (tetR)– less leaky and has an external control pointless leaky and has an external control point

• We have 3 control points: I0500 (by Arabinose), LacI (by IPTG), tetR (by aTc) We have 3 control points: I0500 (by Arabinose), LacI (by IPTG), tetR (by aTc) – Allows for more complex biasing and modular testingAllows for more complex biasing and modular testing

Testing PhaseTesting Phase

Entire construct!Entire construct!

Intermediary constructs used for testingIntermediary constructs used for testing

Test ResultsTest Results

Module 1:Module 1:

Test ResultsTest ResultsModule 1:Module 1:

Test ResultsTest ResultsModule 1:Module 1:

Future WorkFuture Work

• Complete tests for Modules 2 and 3Complete tests for Modules 2 and 3

Additional Tests:Additional Tests:• Surface response of reporter genes to varying Surface response of reporter genes to varying

amounts of inducersamounts of inducers

• Absorbance of Arabinose, and Absorbance of Arabinose, and Absorbance/Emission Spectra of reporter genes Absorbance/Emission Spectra of reporter genes

ConclusionsConclusions- Further tests need to be performed to ensure proper - Further tests need to be performed to ensure proper

functionality of the Cells-See-Us Thermometerfunctionality of the Cells-See-Us Thermometer

Lessons learned:Lessons learned:- Xba1 restriction enzyme performs slower than EcoRI, Xba1 restriction enzyme performs slower than EcoRI,

SpeI, PstI enzymesSpeI, PstI enzymes

Limitations to the device:Limitations to the device:- Threshold values of repressors limit temperature rangeThreshold values of repressors limit temperature range- Experiments involving radiationExperiments involving radiation

Improvements to the device:Improvements to the device:- Functionality for larger temperature rangesFunctionality for larger temperature ranges

AcknowledgementsAcknowledgements

Active Members:Active Members:- Natalie Yeung (Toronto)- Natalie Yeung (Toronto)- Farshid Mirrahimi (Toronto)- Farshid Mirrahimi (Toronto)- Elliott Sales de Andrade (Toronto)- Elliott Sales de Andrade (Toronto)- Jessica Yang (Toronto)- Jessica Yang (Toronto)- Jovan Lukovich (Toronto)- Jovan Lukovich (Toronto)- Konstantin Savitsky (Toronto)- Konstantin Savitsky (Toronto)- HoKwon Kim (Toronto)- HoKwon Kim (Toronto)- George Ye (Toronto) - George Ye (Toronto) - Irena Mahdavi (Toronto)- Irena Mahdavi (Toronto)- Michael Leung (Toronto)- Michael Leung (Toronto)- Ting Zhu (Toronto)- Ting Zhu (Toronto)- Ram Puri (Toronto)- Ram Puri (Toronto)- Melinda Yeung (Toronto)- Melinda Yeung (Toronto)

iGEM Trained:iGEM Trained:- Niladri Chattopadhyay (Toronto)- Niladri Chattopadhyay (Toronto)- Mohammed Hasan (Toronto)Mohammed Hasan (Toronto)- Hari Vijayendran (Toronto)- Hari Vijayendran (Toronto)- Saba Mortazavi (Toronto)- Saba Mortazavi (Toronto)- Sara M Khojasteh (Toronto)- Sara M Khojasteh (Toronto)- Vahe Akbarian (Toronto)- Vahe Akbarian (Toronto)- Dave Puri (Toronto)- Dave Puri (Toronto)- Arash Mirrahimi (Toronto)- Arash Mirrahimi (Toronto)- Linda Chen (Waterloo)Linda Chen (Waterloo)- Christina Lucey (Waterloo)Christina Lucey (Waterloo)- Chris Herriot (Waterloo)- Chris Herriot (Waterloo)- Rohan Gidvani (Waterloo)- Rohan Gidvani (Waterloo)- Jeffrey Wong (Waterloo)- Jeffrey Wong (Waterloo)

Faculty Advisors:Faculty Advisors:- Prof. Stephen Davies (Toronto)- Prof. Stephen Davies (Toronto)- Prof. Brian Ingalls (Waterloo)- Prof. Brian Ingalls (Waterloo)- Seema Nagaraj (Toronto)- Seema Nagaraj (Toronto)- Lance DaSilva (Waterloo)- Lance DaSilva (Waterloo)- Patrick Tsui (Waterloo)- Patrick Tsui (Waterloo)

Team Members:Team Members:

Thank you iGEM organizers Thank you iGEM organizers and MIT for hosting!and MIT for hosting!

Sponsors:Sponsors:

University of WaterlooUniversity of WaterlooMathematics Endowment FundMathematics Endowment Fund

university of toronto

engineeringsociety

Construction & Testing Team Leads:Construction & Testing Team Leads:- Charles Yoon (Toronto)- Charles Yoon (Toronto)- Andy Hung (Toronto)- Andy Hung (Toronto)

Finance Directors: Finance Directors: - Andy Hung (Toronto)- Andy Hung (Toronto)- - Wing Yee Cheung (Toronto)Wing Yee Cheung (Toronto)- Kathy Lam (Waterloo)- Kathy Lam (Waterloo)

Communication Leads: Communication Leads: - Charles Yoon (Toronto)- Charles Yoon (Toronto)- Farshid Mirrahimi (Toronto) - Farshid Mirrahimi (Toronto)

- Nancy Xu (Waterloo)- Nancy Xu (Waterloo)

Design Committee Leads:Design Committee Leads:- Herak Sjkder (Toronto)- Herak Sjkder (Toronto)- Sam Leung (Waterloo)- Sam Leung (Waterloo)

Waterloo Engineering Waterloo Engineering Endowment FundEndowment Fund

Special thanks to:Special thanks to:

- University of Toronto Davies’ Lab for their generous - University of Toronto Davies’ Lab for their generous donations of lab space, equipment, and suppliesdonations of lab space, equipment, and supplies