Part:BBa_K2760033

Composite part Plan A (L. rhamnosus GG):

This is the same “Plan A” for E. coli but adapted to Lactobacillus rhamnosus GG, since it is a natural probiotic organism. Plan A which consists of producing soluble gp130 attached to novel signal peptides for secretion to the media, this protein will form a dimer with soluble gp80, present in the extracellular media, gp80-gp130 which will then attach to IL-6 and form a stable trimer capable of blocking interleukin inflammatory signaling. The construct is codon optimized for L. rhamnosus GG, it has promoters, RBS and terminators that have been used before for L. rhamnosus.

Soluble forms of both receptor IL-6 subunits are present in human blood, 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 combination of soluble gp130 and gp80 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 is first regulated by a spaCBA promoter, which allows the expression of the nsrR repressor protein (of PyeaR promoter) and then the cassette regulates the expression of gp130 with the PyeaR promoter, after it senses nitrosative stress. Reporter fluorescent proteins iLOV and M-Cherry will help corroborate expression of both systems. iLOV will be attached to the Pbad-nsrR system and M-Cherry to the PyeaR-gp130 system. The glycoprotein (gp) has a His-Tag (6 histidines) attached to it for purification purposes.

It contains the the spaCBA promoter (BBa_K2760005), a L. rhamnosus GG ribosome binding site (BBa_K2760006), then the codon optimized sequence for protein nsrR (BBa_K2760023) which is the PyeaR promoter repressor, two stop codons and then another ribosome binding site (the same as earlier). After this ribosome binding site, follows a modified and optimized iLOV sequence (BBa_K2760024 ), two stop codons and a loop terminator (Terminator 667). A PyeaR promoter follows (BBa_K216005), the same ribosome binding site, the signal peptide 1, SP1-NSP4 (BBa_K2760000), afterwards, the gp130 (receptor IL-6 complex) human soluble protein (BBa_K2760002) and a His-Tag (BBa_K2760012), then two stop codons, the RBS and finally an optimized M-cherry (BBa_K2760029), stop codon, loop terminator 908 (BBa_K2760008).

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