University of Toronto & University of Waterloo Cells-See-Us Thermometer

Project Overview Introduction: Limitations to using other thermometersLimitations to using other thermometers –size 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 micrometers –temperature of an organism of any size Cells-See-Us Thermometer: a bio-synthetic device that detects changes in temperature and are reflected by colour.a bio-synthetic device that detects changes in temperature and are reflected by colour. red when hot, orange when warm and green when coldred when hot, orange when warm and green when cold

Dimers break LacI monomers do not bind onto repressing sites – Rfp and CI are formed. CI inhibits Gfp HotCold LacI binds to Rfp repressing site (R0010) and CI repressing site (R0011). No Rfp or CI is made LacI forms dimer Gfp is formed Project Model

Construction 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 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 testing

Testing Phase Entire construct! Intermediary constructs used for testing

Test Results Module 1:

Test Results Module 1:

Test Results Module 1:

Future Work Complete tests for Modules 2 and 3Complete tests for Modules 2 and 3 Additional Tests: Surface response of reporter genes to varying amounts of inducersSurface response of reporter genes to varying amounts of inducers Absorbance of Arabinose, and Absorbance/Emission Spectra of reporter genesAbsorbance of Arabinose, and Absorbance/Emission Spectra of reporter genes

Conclusions - Further tests need to be performed to ensure proper functionality of the Cells-See-Us Thermometer Lessons learned: -Xba1 restriction enzyme performs slower than EcoRI, SpeI, PstI enzymes Limitations to the device: -Threshold values of repressors limit temperature range -Experiments involving radiation Improvements to the device: -Functionality for larger temperature ranges

Acknowledgements Active Members: - Natalie Yeung (Toronto) - Farshid Mirrahimi (Toronto) - Elliott Sales de Andrade (Toronto) - Jessica Yang (Toronto) - Jovan Lukovich (Toronto) - Konstantin Savitsky (Toronto) - HoKwon Kim (Toronto) - George Ye (Toronto) - Irena Mahdavi (Toronto) - Michael Leung (Toronto) - Ting Zhu (Toronto) - Ram Puri (Toronto) - Melinda Yeung (Toronto) iGEM Trained: - Niladri Chattopadhyay (Toronto) - Mohammed Hasan (Toronto) - Hari Vijayendran (Toronto) - Saba Mortazavi (Toronto) - Sara M Khojasteh (Toronto) - Vahe Akbarian (Toronto) - Dave Puri (Toronto) - Arash Mirrahimi (Toronto) - Linda Chen (Waterloo) - Christina Lucey (Waterloo) - Chris Herriot (Waterloo) - Rohan Gidvani (Waterloo) - Jeffrey Wong (Waterloo) Faculty Advisors: - Prof. Stephen Davies (Toronto) - Prof. Brian Ingalls (Waterloo) - Seema Nagaraj (Toronto) - Lance DaSilva (Waterloo) - Patrick Tsui (Waterloo) Team Members: Thank you iGEM organizers and MIT for hosting! Sponsors: University of Waterloo Mathematics Endowment Fund university of toronto engineeringsociety Construction & Testing Team Leads: - Charles Yoon (Toronto) - Andy Hung (Toronto) Finance Directors: - Andy Hung (Toronto) - Wing Yee Cheung (Toronto) - Kathy Lam (Waterloo) Communication Leads: - Charles Yoon (Toronto) - Farshid Mirrahimi (Toronto) - Nancy Xu (Waterloo) Design Committee Leads: - Herak Sjkder (Toronto) - Sam Leung (Waterloo) Waterloo Engineering Endowment Fund Special thanks to: - University of Toronto Davies’ Lab for their generous donations of lab space, equipment, and supplies