Reporter Construct (Ly-Tc-) for constitutive CFP and inducible YFP
Chris Batton, Victoria Chou, Kenneth Nesmith
Reporter plasmid with Lac operator+YFP(LVA-) reporter followed by a Tet operator+CFP(LVA-) reporter
GFP - LacZ protein generator is generated with the aim of inducing a reporting reaction aftermath of the activation of EpCAM detection system. (BBa_K772100) For more information: BBa_K772006
To achieve a constitutive production, pTetR promoter is added in the upstream region of the original part.
Cleavage site, LacZ and GFP sequences are gathered from Registry.
GFP - LacZ anchor-inducer protein complex
This part contains a green fluorescent protein (GFP) as a non-reactive chemical compound (anchor) in order to neutralize the active reactive fixed to it, a reporter/inducer device or protein (enzyme etc.) and a TEV protease cleavage site between them to assemble these two proteins. BBa_772001 and BBa_K772002 are assembled to be used as the trigger of detection in the whole system which ends with the intracellular transfection (or activation) of TEV protease. After the activation, it is planned that TEV protease will cleave this anchor-inducer protein complex to release free the inducer/reporter. In conclusion, the inducer would activate a transcription pathway on the targeted inducible gene or the reporter would indicate that there is a change in the whole system.
Note that there is an RBS sequence just before the protein sequence in order to eliminate the need of addition of it.
Reporter Construct with Lac operator+YFP(LVA+) reporter followed by a Tet operator +CFP(LVA+) reporter
Reporter construct for constitutive YFP and inducible CFP
Reporter Construct with Tet operator+YFP(LVA-) reporter followed by a Lac operator +CFP(LVA-) reporter
GFP - LacZ protein complex with flag tags
This part contains a green fluorescent protein (GFP) as a non-reactive chemical compound (anchor) in order to neutralize the active reactive fixed to it, a reporter device (LacZ enzyme catalyses the reaction of the cleavage of X-Gal which results with blue color change) and a TEV protease cleavage site between them to assemble these two proteins. BBa_772001 and BBa_K772002 are assembled to be used as the trigger of detection in the whole system which ends with the intracellular transfection (or activation) of TEV protease. After the activation, it is planned that TEV protease will cleave this anchor-inducer protein complex to release free the reporter. In conclusion, in this system, the inducer would activate a transcription pathway on the targeted inducible gene or the reporter would indicate that there is a change in the whole system. This part contains flag tags extra from BBa_K772006 in the GFP region; because it is planned to be used in the Co-IP/SDS-PAGE assay to be isolated.