Composite part Plan B (E. coli BL21):

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

Composite part Plan A:

For L. rhamnosus

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 the soluble form gp130 attached to novel signal peptides for secretion to the media, this proteins will form a dimer with gp80 found in the gut, which will then attach to IL-6 and form a stable trimer capable of blocking interleukin inflammatory signalization. The construct is codon optimized for L. rhamnosus GG, it has promoters, RBS and terminators that have been used before for L. rhamnosus. It also contains two restriction sites, between the iGEM prefix and suffix, to ligate the Lactobacillus ORI site.

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 gp80 and 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-gp80-gp130 system. Both glycoproteins (gp) have a His-Tag (6 histidines) attached to themselves for purification.

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 gp80 and 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-gp80-gp130 system. Both glycoproteins (gp) have a His-Tag (6 histidines) attached to themselves for purification.

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

Composite part Plan B:

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 also contains two restriction sites, between the iGEM prefix and suffix, to ligate the Lactobacillus ORI site.

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 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.

Construct for SP1-Nsp4 characterization in E. coli.

This construct is meant to be used in characterizations of the signal peptide SP1-Nsp4, it should be excreted into the media by the Sec-dependent (Sec) secretion pathway, the general secretion E. coli route. When this signal peptides are linked to the N-terminus of recombinant proteins, they are critical for translocation and secretion of proteins [1].

This plasmid expression vector has a T7 promoter (BBa_I712074) which will be induced by IPTG, it contains a specific strong RBS for E. coli, the signal peptide SP1-Nsp4, iLOV (BBa_K660004) as the fluorescent with a His-Tag (6 histidines) attached to it; finally an E. coli specific terminator (BBa_I712074).

Construct for SP2-Nsp2 characterization in E. coli.

This construct is meant to be used in characterizations of the signal peptide SP2-Nsp2, obtained from the same study as the SP1-Nsp4 signal peptide, this causes the attached protein to be excreted into the media by the Sec-dependent (Sec) secretion pathway, the general secretion E. coli route. When this signal peptides are linked to the N-terminus of recombinant proteins, they are critical for translocation and secretion of proteins [1].

This plasmid expression vector has a T7 promoter (BBa_I712074) which will be induced by IPTG, it contains a specific strong RBS for E. coli, the signal peptide SP2-Nsp2, iLOV (BBa_K660004) as the fluorescent with a His-Tag (6 histidines) attached to it; finally an E. coli specific terminator (BBa_I712074).

References: [1] 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

SP (signal peptide) negative control characterization construct (E.coli)

This construct is meant to be used as a negative control in characterizations of the signal peptides SP1-Nsp4 and SP2-Nsp2, it lacks signal peptides but is composed from the same elements as the previous constructs.

This plasmid expression vector has a T7 promoter (BBa_I712074) which will be induced by IPTG, it contains a specific strong RBS for E. coli, iLOV (BBa_K660004) as the fluorescent reporter with a His-Tag (6 histidines) attached to it; finally an E. coli specific terminator (BBa_I712074).

SP (signal peptide) positive control characterization construct (E. coli)

This construct is meant to be used as a positive control in characterizations of the signal peptides SP1-Nsp4 and SP2-Nsp2, it contains the DsbAss signal peptide but the other elements remain the same from the previous constructs.

This plasmid expression vector has a T7 promoter (BBa_I712074) which will be induced by IPTG, it contains a specific strong RBS for E. coli, DsbAss signal peptide, iLOV (BBa_K660004) as the fluorescent reporter with a His-Tag (6 histidines) attached to it; finally an E. coli specific terminator (BBa_I712074).

For L. rhamnosus

These series of constructs were codon optimized for L. rhamnosus as a way to test whether this signal peptides for secretion that work on E. coli, via the Sec secretion pathway and SRP pathway. They are expression plasmids that contain L. rhamnosus spaCBA promoter, RBS and loop terminator, they also contain HindIII restriction site between the iGEM prefix and SacI restriction to join with the Lactobacillus ORI, and fluorescent reporter M-cherry.