Team:TecMonterrey GDL/Constructs
Construct Design
Composite part Plan A:
This construct is optimized for E.coli BL21, it constitutes our “Plan A” for our psychobiotic, which consists of producing the soluble form gp130 attached to novel signal peptides, which will allow the secretion of the sgp130 to the extracellular media. In order for our construct to work the IL-6 will bind to its receptor also known as gp80, these dimer will then bind to the produced soluble sgp130 which will form a highly stable trimer that sequesters IL-6, effectively inhibiting IL-6 trans-signaling, the inflammatory signalization.
Both the IL-6 and the soluble form of the IL-6 receptor are present in the human body specifically in the gut, where our construct will work. In general, soluble receptors for several cytokines have been detected in different body fluids, these are believed to help modulate cytokine response by binding the ligand and thereby reducing its bioavailability. Since the of soluble gp130 can act as an antagonist vs the cells with membrane-bound receptor (IL-6R) [1] , the Plan A strategy was developed as a first approach towards developing our psychobiotic.
The system contains an inducible promoter that will be regulated by nitrosative stress. When this stress is present, the transcription of the soluble form of gp130 will be initiated. Additionally, a inducible pBad promoter will be activated with arabinose in order to control the production of NsrR, the protein that blocks the transcription of the Pyear promoter, these will be a form to control the amount of nitrosative stress needed for the Pyear activation. Reporter fluorescent proteins iLOV and M-Cherry will help us corroborate expression of both systems. iLOV will be attached to the Pbad-nsrR system and M-Cherry to the PyeaR-sgp130 system. The sgp30 have a His-Tag (6 histidines) attached to it for purification.