Part:BBa_K4040028

GEMS with three Alanines added Sequence and Features

Assembly Compatibility:

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  Illegal BglII site found at 2453
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  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 4
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Background of the improvement parts

This part is a mutant of GEMS designed by team SMMU in 2019. GEMS(BBa_K3032016) is a dimer of two subunits of which the scaffold is derived from EpoR (erythropoietin receptor). GEMS can be combined with a wide range of affinity domains as well as different intracellular domains for rerouting signaling to activate distinct endogenous signaling pathways [1]. In the previous paper, the authors rewired the receptors to four independent pathways (Figure. 1b), and these receptors could respond to various molecules of different molecular weights. BBa_K3032016 is a MAPK-dependent GEMS that consists of an anti-Suntag scFV and a FGFR1 intracellular domain. Therefore, this receptor is designed to specifically respond to proteins fused with SunTag and to activate MAPK pathway.

Design

In the experiment of exploring the influence of the amount of SunTag on protein activity by team SMMU in 2019, they used mCherry (BBa_J06504) as the reporter and connected it to the 8×PIR minCMVpromoter (BBa_K3132101). The cells were co-transfected with GEMS, reporter plasmid, and PIP-Elk1, and were added with the IL-Suntag proteins 24h post-transfection. Eighteen hours later, the mCherry fluorescence intensity was measured. Intriguingly, among the three ligands, only 6×SunTag showed significant activating effect (Figure. 1a). The receptor showed around 3.2-fold change at the concentration of 100ng/ml compared with negative control. However, weak fluorescence could be observed in the negative control, suggesting that this receptor possessed a low background activity (Figure. 1b). 

Figure. 1 Response of GEMS to three ligands. a, Compared with 2× and 4×SunTag proteins, GEMS only responded to the 6× ligand in a concentration-dependent way. b, mCherry expressed by cells at different 6×SunTag concentrations.***P<0.001, *P<0.05

This year we hope to change the structure of the GEMS to reduce this base activity and design experiments accordingly.

The well-studied FRB/FKBP (FKBP–rapamycin binding protein/FK506 and rapamycin binding protein) system for rapamycin-induced dimerization[2] was used for initial characterization of the GEMS system. FRB and FKBP proteins were fused to the erythropoietin receptor extracellular domain, and its transmembrane domain was C-terminally linked to the JAK/STAT signaling domain of IL-6RB. Activated JAKs phos-phorylate STAT3, which functions as transcription factor. Receptor activation can be quantified through the reporter protein SEAP (human placental secreted alkaline phosphatase) expressed from the reporter plasmid pLS13 (OStat3-PhCMVmin-SEAP-pA) containing STAT3-binding sites 5′ of a minimal promoter[3]. Up to four alanines were added between the transmembrane domain of EpoR (EpoR0-4A) and the intracellular domain of IL-6RB (IL-6RBint). Each additional alanine elongates the transmembrane helix and rotates the intracellular domain by approximately 100° relative to the extracellular domain[4,5]. This was done to identify a receptor conformation with minimal JAK activation in the off state (Fig. 2a). FRB/FKBP receptors (pTS1000–pTS1009, PhCMV-FRB/FKBP-EpoR0-4A-IL-6RBint-pA) were responsive to induction by rapamycin, and the number of alanines influenced receptor performance. Basal gene expression was highest for the variants with zero or one alanine, suggesting insufficient inhibition of preformed dimers. The highest fold change from induced to uninduced SEAP expression was observed for the variant with three alanines (Fig. 2b).

Figure. 2Receptor scaffold optimization. a, Introducing alanines C-terminally of the transmembrane domain elongates the α -helix and results in a rotation of about 100°. The ‘off’ state of the receptor can still allow a JAK interaction, depending on the number of alanines. The variant with three alanines is pictured to assume an ‘off’ conformation with low JAK interaction and an ‘on’ conformation with high JAK interaction, whereas the variant without alanines leads to a JAK interaction in the ‘off’ state. b, Rapamycin induced gene expression by different receptor scaffolds, labeled 0 to 4 to indicate the number of alanines added between the EpoR transmembrane domain and the IL-6RB intracellular domain. The variant with three alanines shows relatively low absolute values for reporter gene expression but the highest signal-to-noise ratio.

Inspired by this phenomenon, we added 0, 1, 2, 3 and 4 alanine sequences between the anti-Suntag scFV and the internal domains of FGFR1 cells respectively on the basis of GEMS (BBa_K3132016). We then verified the background expression of this receptor when the concentration of IL-6 was 0.

Process of experiment

We used mCherry (BBa_J06504) as the reporter and connected it to the 8×PIR minCMV promoter (BBa_K3132101). The cells were co-transfected with GEMS(BBa_K4040025, BBa_K4040026, BBa_K4040027, BBa_K4040028, BBa_K4040029), reporter plasmid, and PIP-Elk1 respectively. Then they were added with the IL-6*Suntag proteins at 24h post-transfection. Eighteen hours later, the mCherry fluorescence intensity was measured. The fluorescence intensity when IL-6 concentration is 100ng/ mL and 0ng/ mL is shown in Fig.3.

Figure. 3 mCherry fluorescence intensity when the IL-6 concentration is 0ng/ml and 100ng/ml respectively.***P<0.001, *P<0.05

The experimental results showed that when the concentration of IL-6 was 0, the fluorescence signal intensity gradually decreased with the increase of the inserted glutamate. But when the concentration of IL-6 was 100ng/ml, the fluorescence intensity of GEMS (BBa_K4040029) inserted with four glutamate sequences is significantly lower than that of the original GEMS and the GEMS inserted with 1-3 glutamate sequences.  For GEMS (BBa_K4040028) with an increase of 3 glutamates, the signal conduction activity can be guaranteed while the background activity is low. So we consider GEMS inserted with 3 glutamate sequences to be the best improvement part.  

Meaning of improvement

Improved GEMS (BBa_K4040028) can reduce spontaneous activation while preserving receptor activity, so this modification can improve specificity and enhance the function of GEMS receptor without reducing receptor sensitivity.  

References

[1] Scheller, L., et al., Generalized extracellular molecule sensor platform for programming cellular behavior. Nat Chem Biol, 2018. 14(7): p. 723-729.

[2]Banaszynski, L. A., Liu, C. W . & Wandless, T. J. Characterization of the FKBP .rapamycin.FRB ternary complex. J. Am. Chem. Soc. 127, 4715–4721 (2005).

[3]Schukur, L., Geering, B., Charpin-El Hamri, G. & Fussenegger, M. Implantable synthetic cytokine converter cells with AND-gate logic treat experimental psoriasis. Sci. Transl. Med. 7, 318ra201 (2015).

[4]Seubert, N. et al. Active and inactive orientations of the transmembrane and cytosolic domains of the erythropoietin receptor dimer. Mol. Cell 12, 1239–1250 (2003).

[5] Liu, W ., Kawahara, M., Ueda, H. & Nagamune, T. Construction of a fluorescein-responsive chimeric receptor with strict ligand dependency. Biotechnol. Bioeng. 101, 975–984 (2008).