Part:BBa_K3831033:Design
Construct D for testing the function of modified Pgrac promoter for expression of cI repressor
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 2699
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 2898
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 228
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
This composite part consists of two expression units. One expression unit contains the IPTG-inducible Phyperspank (BBa_K143015) which controls the production of the cI repressor (BBa_K3831024) and the reporter protein m-Scarlet-I (BBa_K3831021). The other expression unit contains the newly designed Pgrac OcI (BBa_K3831025) which controls the expression of the reporter protein sfGFP (BBa_K3831029). This system as a whole should work like a switch. Without the presence of IPTG, B. subtilis cells should produce GFP. When induced, the cells should start producing the cI repressor and m-Scarlet-I. m-Scarlet-I fluorescence would serve to confirm the expression of the two proteins. The cI repressor should bind itself to its operator located in Pgrac OcI and stop the expression of GFP. The fall of GFP fluorescence would thus serve as a reporter of the cI mediated repression. To ensure the reversibility of the switch as well as a quick reaction of the system, all proteins contain a degradation tag (BBa_K3831019). The whole composite part is flanked by two terminators (BBa_B0010 and BBa_K780000) to prevent reciprocal influence on the transcription of both the composite part and the plasmid backbone (and later the B. subtilis chromosome). This composite part is designed in a way which allows its further modification in order to create the composite part Z.
Source
Original sequence