Construct Design ![decoration1](https://static.igem.org/mediawiki/2018/3/3a/T--TecMonterrey_GDL--decoration1.png)
Composite part Plan A:
![](https://static.igem.org/mediawiki/2018/b/bf/T--TecMonterrey_GDL--ConstructEColi.png)
![](https://static.igem.org/mediawiki/2018/9/91/T--TecMonterrey_GDL--ConstructIL-6.png)
![](https://static.igem.org/mediawiki/2018/f/f1/T--TecMonterrey_GDL--Construct3.png)
![](https://static.igem.org/mediawiki/2018/e/ea/T--TecMonterrey_GDL--Construct4.png)
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 (E. coli BL21):
![](https://static.igem.org/mediawiki/2018/9/91/T--TecMonterrey_GDL--ConstructIL-6.png)
![](https://static.igem.org/mediawiki/2018/b/bf/T--TecMonterrey_GDL--ConstructEColi.png)
![](https://static.igem.org/mediawiki/2018/f/f1/T--TecMonterrey_GDL--Construct3.png)
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.
![](https://static.igem.org/mediawiki/2018/4/4e/T--TecMonterrey_GDL--Construct23.png)
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.
![](https://static.igem.org/mediawiki/2018/c/c3/T--TecMonterrey_GDL--Constructpt2.png)
![](https://static.igem.org/mediawiki/2018/6/6c/T--TecMonterrey_GDL--ConstructFinal.png)