Part:BBa_K2760010

This construct constitutes our “Plan A” for our psychobiotic proposal Lactobachill, which consists of producing the soluble form of gp130 attached to novel signal peptide 4 (SP1-NSP4), (which will allow the secretion of the sgp130 to the extracellular media), upon sensing an increase in nitrosative stress. In order for our construct to work the IL-6 will bind to its receptor also known as sgp80, in the extracellular media, this dimer will then bind to the produced and secreted sgp130 which will form a highly stable trimer, whose objective is to selectively inhibit IL-6 trans-signaling.

Both the IL-6 and the soluble form of the IL-6 receptor (gp80) are present in the human body specifically in the gut, where our Lactobachill should, in theory, act. 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 gp130 before they bind with soluble gp80-IL-6 dimer [1] , it is meant to target trans-signaling.

The system contains a strong inducible promoter that will be regulated by arabinose in order to produce nsrR (repressor protein) in order to control the PyeaR promoter, so the system can respond to nitrosative stress. When this stress is present, the transcription of the soluble form of gp130 will be initiated. Reporter fluorescent proteins iLOV and M-Cherry will help us corroborate expression of both systems respectively. 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.

It contains the Pbad strong promoter (BBa_K206000), an E. coli strong ribosome binding site (registered before as Part:BBa_J34801), then the sequence for protein nsrR (BBa_K2760023) which is the PyeaR promoter repressor, two stop codons and then another ribosome binding site (BBa_J34801). After this ribosome binding site, follows a codon optimized for L. rhamnosus and E.coli iLOV sequence (BBa_K2760024), two stop codons and a terminator (BBa_B0015). A PyeaR promoter follows (BBa_K216005), the same ribosome binding site BBa_J34801, an attached signal peptide for excretion into the media (BBa_K2760000) in the gp130 (receptor IL-6 complex) human soluble protein (BBa_K2760002) and a His-Tag (BBa_K2760012), then two stop codons, the RBS (BBa_J34801) and finally the M-cherry (BBa_K2760029), stop codon, and a double terminator (BBa_B0015).

References:

[1] Müller-Newen, G., Küster, A., Hemmann, U., Keul,R., Horsten, U., Martens, A., Graeve, L., Wijdenes, J., Heinrich, P. (1998) Soluble IL-6 Receptor Potentiates the Antagonistic Activity of Soluble gp130 on IL-6 Responses. The Journal American of Association Immunology. ISSN: 1550-6606.

[2] Han,S., Machhi, S., Berge,H., Xi, G., Linke, T., and Schoner, R. (2017). Novel signal peptides improve the secretion of recombinant Staphylococcus aureus Alpha toxinH35L in Escherichia coli. AMB Express. Doi: https://doi.org/10.1186/s13568-017-0394-1