EXPERIMENTS

Parts

Part Type Description Length (bp)

BBa_I3401 		Reporter GFP reporter for RHS of library test constructs 857

BBa_K1159307 		Terminator 35S Terminator of Cauliflower Mosaic Virus (CaMV) 217

BBa_K1825004 		Regulatory CaMV 35s promoter, small constitutive promoter 390

pSB1C3 		Plasmid Backbone High copy number BioBrick assembly plasmid with chloramphenicol resistance. 2070

BBa_K1465228 		Translational Unit Sedoheptulose 1,7-bisphosphatase (SBPase) from Bacillus methanolicus. A Calvin Cycle enzyme which converts Sedoheptulose-1,7-bisphosphate to Sedoheptulose-7-phosphate. 975

BBa_K2649014 		Device iRFP 951

BBa_K4296000 		Regulatory TaHsp70d promoter 1251

BBa_K4296001 		Other P2A Linker 57

BBa_K592100 		Coding Codes for the bright blue fluorescent protein mTagBFP which emits light at a maximum at 456 nm. 705

BBa_K2384005 		DNA Codes for ACC deaminase, an enzyme that catalyzes the breakdown of ethylene precursor, ACC. 885


Parts Added to the iGEM Registry

To the iGEM registry we added part BBa_K4296000, which is the TaHsp70d promoter from T. aestivum (wheat). TaHsp70d is a heat shock protein and its promoter is heat-inducible. In our project, we used BBa_K4296000 to create a genetic circuit that was heat-inducible. Our enzymes of interest would only be expressed at a higher-than-normal cell temperature, which is 37°C for this promoter.

We showed that our enzymes would only be expressed under heat stress conditions by creating fusion proteins with each of our enzymes of interest (ACCD, SBPase, CMO) with a different fluorescent protein (RFP, BFP, YFP). Measuring the fluorescence of our transfected protoplasts showed that only after being treated with the temperature and time conditions required for our promoter, which in this case is 37°C for 2 hours, is there a detectable amount of fluorescence, indicating that only after being heat shocked are our proteins of interest expressed. More information and our data can be found on our Experimental Results page.

The BBa_K4296000 part can be used by other teams to create a heat-inducible system in wheat, since TaHsp70 is only active in wheat. The part is also compatible with golden gate assembly.