Team:TecMonterrey GDL/Composite Part
Constructs
For E. coli BL21
This construct was optimized for E.coli BL21. Strategy A consists of producing the soluble form of the transducer gp130 (sgp130) attached to novel signal peptides, which will allow the secretion of sgp130 to the extracellular media. In order for our strategy to work, Interleukin-6 (IL6) needs to bind to its soluble receptor (sgp80) forming a dimeric complex capable of binding to the secreted sgp130; this is going to result in a highly stable trimer. The trimer will kidnap IL-6, and thus, transignaling pathway will be effectively inhibited.
Composite part Plan A : BBa_K2760010
Both, the IL-6 and the soluble form of the IL-6 receptor, are presented in the human body, specifically, in the gut, where our construct will work. 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 soluble gp130 can act as an antagonist against 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 contains an inducible promoter regulated by nitrosative stress. When nitrosative stress is presented, the transcription of the soluble form of gp130 will be initiated. Additionally, a inducible pBAD promoter will be activated with arabinose in order to control the production of NsrR, the protein that blocks the transcription of the PyeaR promoter, these will be a form to control the amount of nitrosative stress needed for the PyeaR activation. Reporter fluorescent proteins iLOV and mCherry will help us corroborate expression of both systems. iLOV will be attached to the pBAD-NsrR system and mCherry to the PyeaR-sgp130 system. The sgp30 have a His-Tag (6 histidines) attached to it for purification.
Plan B
The construct was optimized for E. coli BL21. Strategy B consists of producing a soluble, mutated alpha chain receptor gp80 attached to novel signal peptides for secretion to the media. The mutated receptor will act as an antagonist protein. It will associate with IL-6 forming the IL6-alpha receptor gp80 complex but it will not bind with human gp130, thus, blocking the inflammation pathway by inhibiting the formation of IL-6-alpha receptor gp80-gp130 complex [3].
Composite part Plan B : BBa_K2760011
The system is first regulated by the pBAD promoter, which allows to induce expression with L-arabinose of the NsrR repressor protein (of PyeaR promoter) and regulate then, the expression of mutated gp80 with the PyeaR promoter, after it senses nitrosative stress. Reporter fluorescent proteins iLOV and mCherry will help us corroborate expression of both promoter systems. iLOV will be attached to the pBAD-NsrR system and mCherry to the PyeaR-mutated gp80 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. This peptide should be excreted into the media by the Sec-dependent (Sec) secretion pathway, the general secretion E. coli route. When these signal peptides are linked to the N-terminus of recombinant proteins, they are critical for translocation and secretion of proteins [1].
Construct for SP1-NSP4 : BBa_K2760019
This plasmid expression vector has a T7 promoter (BBa_I712074),induced by IPTG, and contains a T7 medium strength RBS for E. coli, the signal peptide SP1-NSP4, iLOV (BBa_K2760024) as the fluorescent marker with a His-Tag (6 histidines) attached to it; and a double terminator (BBa_B0015).
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 [2].
Construct for SP2-NSP2 : BBa_K2760021
This plasmid expression vector has a T7 promoter (BBa_I712074) ,induced by IPTG, and contains a a T7 medium strength RBS for E. coli, the signal peptide SP2-NSP2, iLOV (BBa_K2760024) as the fluorescent marker with a His-Tag (6 histidines) attached to it; finally a double terminator (BBa_B0015).
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 of signal peptides but it is composed from the same elements as the previous constructs.
SP negative control : BBa_K2760020
This plasmid expression vector has a T7 promoter (BBa_I712074) induced by IPTG, it contains a a T7 medium strength RBS for E. coli, iLOV (BBa_K2760024) as the fluorescent reporter with a His-Tag (6 histidines) attached to it; finally a double terminator (BBa_B0015).
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; the other elements remain the same from the previous constructs.
This plasmid expression vector has a T7 promoter (BBa_I712074) induced by IPTG, a T7 medium strength RBS for E. coli, DsbAss signal peptide, iLOV (BBa_K2760024) as the fluorescent reporter with a His-Tag (6 histidines) attached to it; finally a double loop terminator (BBa_B0015).
Future experiments
For L. rhamnosus
Since our desired host is L. rhamnosus, we have designed some parts to assemble future constructs for this specific host. This is the same “Plan A” for E. coli but adapted to Lactobacillus rhamnosus GG, since it is a natural probiotic organism. Plan A consists of producing the soluble form gp130 attached to novel signal peptides for secretion to the media. This proteins will form a dimer with sgp80 found in the extracellular media, 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 sgp80 can act as an antagonist versus 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 mutated-sgp80 and sgp130 with the PyeaR promoter, after it senses nitrosative stress. Reporter fluorescent proteins iLOV and mCherry will help corroborate expression of both systems. iLOV will be attached to the Pbad-nsrR system and mCherry to the PyeaR-gp80-gp130 system. Both glycoproteins (gp) have a His-Tag (6 histidines) attached to themselves for purification.
Plan A : BBa_K2760033
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 soluble IL-6-alpha receptor gp80 complex but it will not bind with human gp130, thus inhibiting the formation of IL-6-alphaReceptorgp80-gp130 complex which leads to inflammation pathways [4]. 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.
Plan B : BBa_K2760034
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 mCherry will help us corroborate expression of both promoter systems. iLOV will be attached to the pBAD-NsrR system and mCherry 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.
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 mCherry.
Construct for SP1-Nsp4 characterization L. rhamnosus
This construct is meant to be used in characterizations of the signal peptide SP1-NSP4 in L. rhamnosus, which would 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 in E. coli [2].
This plasmid expression vector has a spaCBA promoter, it contains a specific RBS for L. rhamnosus GG, the signal peptide SP1-NSP4, mCherry (BBa_K2760024) as the fluorescent reporter with a His-Tag (6 histidines) attached to it; and the terminator 667.
Construct for SP2-Nsp2 characterization in L. rhamnosus
This construct is meant to be used in characterizations of the signal peptide SP2-NSP2 in E. coli, which would 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 in E. coli [2].
SP2-NSP2 : BBa_K2760031
This plasmid expression vector has a spaCBA promoter, it contains a specific RBS for L. rhamnosus GG, the signal peptide SP2-NSP2, mCherry (BBa_K2760029) as the fluorescent reporter with a His-Tag (6 histidines) attached to it; and the terminator 667.
SP (signal peptide) negative control characterization construct in L. rhamnosus
This construct is meant to be used in characterizations of the signal peptide SP2-NSP2 in E. coli, which would 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 in E. coli [2].
SP negative control : BBa_K2760032
This plasmid expression vector has a spaCBA promoter, it contains a specific RBS for L. rhamnosus GG, the signal peptide SP2-NSP2, mCherry (BBa_K2760029) as the fluorescent reporter with a His-Tag (6 histidines) attached to it; and the terminator 667.
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
[3] 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
[4] 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