Our BioBricks

New Parts

• Submitted a BioBrick consisting of CpxR under Arabinose Promoter (BBa_K1486001). Reporter sfGFP was fused to CpxR's N terminus (BBa_K1486002) and C terminus (BBa_K1486005), to evaluate the expression of our construct and characterize the Arabinose promoter in E.Coli.


• Submitted four BioBricks coding for CpxR fused to one of the two fragments of the split reporter Infrared Fluorescent Protein (IFP). Fragments IFP1 and IFP2 (corresponding to the N and C terminal of the IFP respectively) were fused at the N or C terminal of the CpxR, leading two four different constructs: CpxR-IFP1 (BBa_K1486012), CpxR-IFP2 (BBa_K1486013), IFP1-CpxR (BBa_K1486014) and IFP2-CpxR (BBa_K1486015). Sequences are under arabinose promoter. These Biobricks are construction intermediates to build our four final constructs below.


• Submitted four BioBricks, which are the final constructs of the IFP subproject. Each BioBrick codes for two proteins: CpxR fused to IFP1, and CpxR fused to IFP2. The different combinations with the split at the C ot N terminal of CpxR lead to four constructs: CpxR-IFP1 & CpxR-IFP2 (BBa_K1486008), IFP1-CpxR & IFP2-CpxR (BBa_K1486009), IFP1-CpxR & Cpxr-IFP2 (BBa_K14860010), CpxR-IFP1 & IFP2-CpxR (BBa_K14860011). Sequences are under arabinose promoter. These BioBricks aimed at first to study the orientation of CpxR's homodimerization by observing the IFP signal measured from the four strains under stress. We discovered that only the orientations with both parts of the split are at the C terminal of the CpxR lead to an IFP signal when cells are stressed. Since all of these Biobricks are incompatible with RFC[10], we removed illegal restriction sites from BBa_K1486008 and created BBa_K1486056 (version without PstI illegal restriction enzyme sites). The experiment mentioned above was repeated with BBa_K1486056 and identical were reproduced. All further experiments were done with BBa_K1486056.


• Submitted a BioBrick (BBa_K1486056) identical to the BioBrick BBa_K1486008 coding for two proteins CpxR-IFP1 & CpxR-IFP2, which is the orientations of the split leading to en IFP signal when cells are under stress. The difference is that this BioBrick contains two silent points mutation removing illegal restriction sites (PstI).


• CpxR reporters were made with the promoter in forward and reverse direction, respectively BBa_K1486049 and BBa_K1486050.


• Submitted the two parts of the split yeast optimized superfolder GFP (N-terminal part (BBa_K1486029) and C-terminal part (BBa_K1486035)) created from plasmids pFA6a-link-yoSuperfolderGFP-Kan (44901) and pFA6a-link-yoSuperfolderGFP-Ura (44873) we ordered to Addgene, we . We attached them to the ADH1 terminator, regulating the transcripion of our fusion proteins and to the selection markers Kan and Ura3. We stressed our cells under various conditions to trigger the HOG pathway and were able to show that interaction of Hog1 and Pbs2 in response to osmotic stress allowed the re-assembly of the full GFP protein.


• Submitted a BioBrick (BBa_K1486043) containing two leucine zipper sequences, each fused with one moiety of renilla Luciferase, to test the efficiency of the split renilla luciferase in order to use it for a complementation assay.

Further Characterization and Improvement of Parts Already in the Registry

• We realized that Calgary's CpxR reporter BioBrick was missing the regulatory part of the sequence, so we repaired it and sent it as our BBa_K1486048. The BioBrick was further developed by testing the native CpxR target sequence that is found in front of CpxA in the E.coli genome (as Calgary's part did not include the whole sequence). These are BioBricks BBa_K1486049 and BBa_K1486050, with the promoter in forward and reverse direction respectively.


• 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 have 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 - an "on-chip waste treatment" unit that can be integrated at the end of any chip to decontaminate GMOs or pathogens



To find out more about what we did for each chip, click here

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 iGEM and our work at the Hackuarium, the new BioHackerspace in Lausanne.