Split Luciferase Complementation Assay

Advantages of luciferase for our project :

• Fast and reversible signal (quasi-immediate)
• Light excitation not needed (minimal autofluorescence) => simplification of the signal detection and processing mechanisms
• Signal highly correlated with concentration of substrate => very modulable
• Small concentrations of substrate are needed

Our constructs :
N terminal part of Luciferase fused to CheZ and C terminal part of Luciferase fused to CheY.

In absence of chemoattractant, CheZ and CheY are interacting and the two parts of the split reassemble to oxidize luciferin and thus luminescence is triggered. After addition of a chemoattractant (e.g. Glucose), CheZ and CheY don't interact anymore and luminescence is shut down.

We created two different constructs, one with split Firefly luciferase and one with split Renilla luciferase, to test different possibilities with different substrates (Firefly luciferase oxidizes d-luciferin and Renilla luciferase oxidizes coelenterazine)

Data For Our Favorite New Parts

Characterization of New Parts Submitted to the Registry

Documented & submitted:

• BBa_K1486001 (Ara CpxR http://parts.igem.org/Part:BBa_K1486001 )
• BBa_K1486002 (Ara GFPCpxR http://parts.igem.org/Part:BBa_K1486002 )
• BBa_K1486005 (Ara CpxrGFP http://parts.igem.org/Part:BBa_K1486005 )
• BBa_K1486043 (LeuZ + rLuc http://parts.igem.org/Part:BBa_K1486043 )
• BBa_K1486049 (CpxR reporter promoter FW http://parts.igem.org/Part:BBa_K1486049 )
• BBa_K1486050 (CpxR reporter promoter RV http://parts.igem.org/Part:BBa_K1486050 )
• BBa_K1486056 (Ga1Mut http:/parts.igem.org/Part:BBa_K1486056 )

Works as expected:

• BBa_K1486002 (Ara GFPCpxR http://parts.igem.org/Part:BBa_K1486002 )
• BBa_K1486005 (Ara CpxRGFP http://parts.igem.org/Part:BBa_K1486005 )
• BBa_K1486008 (GA1 TedCécile http://parts.igem.org/Part:BBa_K1486008 )
• BBa_K1486012 (CpxR IFP1 http://parts.igem.org/Part:BBa_K1486012 )
• BBa_K1486013 (cpxR IFP2 http://parts.igem.org/Part:BBa_K1486013 )
• BBa_K1486014 (IFP1 CpxR rLuc http://parts.igem.org/Part:BBa_K1486014 )
• BBa_K1486015 (IFP2 CpxR rLuc http://parts.igem.org/Part:BBa_K1486015 )
• BBa_K1486018 (Ara split fLuc http://parts.igem.org/Part:BBa_K1486018 )
• BBa_K1486021 (Ara split rLuc http://parts.igem.org/Part:BBa_K1486021 )

Works as expected & submitted:

• BBa_K1486002 (Ara GFPCpxR http://parts.igem.org/Part:BBa_K1486002 )
• BBa_K1486005 (Ara CpxrGFP http://parts.igem.org/Part:BBa_K1486005 )
• BBa_K1486018 (Ara split fLuc http://parts.igem.org/Part:BBa_K1486018 )
• BBa_K1486021 (Ara split rLuc http://parts.igem.org/Part:BBa_K1486021 )
• BBa_K1486049 or BBaK1486050 (CpxR reporter promoter FW or RV http://parts.igem.org/Part:BBa_K1486050 )

Further Characterization and Improvement of Parts Already in the Registry

• Documented & submitted: BBa_K1486056 (CpxR reporter (Calgary) http://parts.igem.org/Part:BBa_K1486056 ) We repaired it
• Submitted the two parts of the split of EPIC Firefly luciferase (N-terminal part (BBa_K1486016) and C-terminal part (BBa_K1486017)) from Cambridge 2010. The plasmid (BBa_K1486018) containing the two parts of the split separated by a spacer can be very useful as a negative control or to establish a background noise for a complementation assay experiment.
• Compared the EPIC Firefly luciferase from Cambridge 2010 team to the renilla luciferase (BBa_K1486022) in the same conditions, to determine which one is best suited for a complementation assay experiment. The full and split luciferases has been compared. Renilla luciferase (full and splits(BBa_K1486021)) have been submitted.

Microfluidics

• Design of SmashColi - a testing chip to analyse the effects of different mechanical stresses on cells
• Design of FilterColi - a testing chip to analyse the effects of different osmotic stresses on cells
• Design of the BioPad - a large-scaled chip implemented to be the touch-senstive interface of our final trackpad
• Design of CleanColi - blabla

	MITOMI	MITOMI modified	SmashColi	BioPad	FilterColi	CleanColi
Full chip
Unit Cell
Designed
Mold fabrication
Fabrication of the chip
Application
Reference	MITOMI paper

Modelling

Human Practices

• Met with a journalist from the biggest newspaper of our region (Le Temps) and got an article about our project.
• Our work was commented by Bent Stumpe, inventor of the touchscreen, as well as Rolf Heuer, the current director of the CERN, in Geneva
• Organized an outreach event with 80 highschool students at EPFL, teaching them about synthetic biology as well as laboratory techniques and made them participate in a game called « mini iGEM ».
• Presented our work at the Hackuarium in Renens