Composite

Part:BBa_K2760034

Designed by: iGEM18_TecMonterrey_GDL   Group: iGEM18_TecMonterrey_GDL   (2018-10-14)

Composite part Plan B (L. rhamnosus):

This is the same “Plan B” for E. coli but adapted to Lactobacillus rhamnosus GG, since it is a natural probiotic organism. Plan B consists of producing a soluble, mutated, gp80 attached to SP1.Nsp4. The production of the mutated alpha chain receptor for IL-6 (gp80) will act as an antagonist protein, due to mutations, 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 inflammation pathways [1]. The construct is codon optimized for L. rhamnosus GG, it has promoters, RBS and terminators that have been used before for L. rhamnosus.

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 mutated gp80 (mutated IL-6 receptor) human soluble protein (BBa_K2760003) 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] 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

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