Part:BBa_K2760011:Design

This construct constitutes our “Plan B” for our proposed psychobiotic Lactobachill, which consists of producing a soluble, mutated, gp80 attached to novel signal peptides for secretion to the media, upon detection of increased nitrosative stress.The production of the mutated alpha chain receptor for IL-6 (gp80) will act as an antagonist protein, it will associate with IL-6 forming the IL-6-alphaReceptorgp80 complex but it will not bind with human gp130, thus inhibiting the formation of IL-6-alphaReceptorgp80-gp130 complex which leads to IL-6 inflammation pathways, however it is not specific to trans-signaling [1].

The system is first regulated by a Pbad promoter, which allows to induce expression with L-arabinose of the nsrR repressor protein (of PyeaR promoter) and then regulate the expression of mutated gp80 with the PyeaR promoter, after it senses nitrosative stress. Reporter fluorescent proteins iLOV and M-Cherry will help us corroborate expression of both promoter systems. iLOV will be attached to the Pbad-nsrR system and M-Cherry to the PyeaR-Mutatedgp80 system. The mutated gp80 has a His-Tag (6 histidines) attached to it for purification purposes. It is also attached to a signal peptide for excretion into the media.

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 mutated sgp80 (K2760003) 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] Salvati, AL., Lahm, A., Paonessa, G., Ciliberto, G., Toniatti, C. (1995). Interleukin-6 (IL-6) Antagonism by Soluble IL-6 Receptor alpha Mutated in the Predicted gp130-binding interphase. The Journal of Biological Chemistry. Doi: 10.1074/jbc.270.20.12242

[2] Han, S., Machhi, S., Berge, M., Xi, G., Linke, T., & Schoner, R. (2017). Novel signal peptides improve the secretion of recombinant Staphylococcus aureus Alpha toxinH35L in Escherichia coli. AMB Express, 7, 93. http://doi.org/10.1186/s13568-017-0394-1