Part:BBa_K4803003

FRB

Usage and Biology

FRB (FKBP-rapamycin binding domain of FKBP12-rapamycin associated protein) is a 100-amino acid domain of the mammalian target of rapamycin (mTOR). FRB is known to form a heterodimer with FK506 binding protein(FKBP) in the presence of rapamycin [1]. In UTokyo2023 project, it was used as the receptor domain for MESA(Modular Extracellular Sensor Architecture) system [2]. The sequence was obtained by codon optimization (IDT codon optimization tool) for FRB described in [3].

Figure 1: Overview of MESA system

Characterization

Figure 2: Stereoscopically predicted FRB in AlphaFold2

Wet Design

Testing the MESA system to cause signaling by rapamycin.

Three plasmids were transfected into HEK293A: PC part and TC part of MESA, and YT82, which shows green fluorescence in response to the tTA signal.

PC part uses iU-73 plasmid, which encodes CD4signal-FKRB-CD28TMD-TEVprotease.

TC part uses iU-74 plasmid, which encodes CD4signal-FRB-CD28TMD-TEVcs-tTA.

YT82 encodes TRE3G-ZsGreen.

Plasmids are shown below.

Figure 3: Completed plasmid of YT82

Figure 4: Completed plasmid of iU-73

Figure 5: Completed plasmid of iU-74

Wet Result

We checked the responsiveness of the MESA system by varying the concentration of rapamycin. HEK293A was co-transfected with the MESA Target Chain(BBa_K4803109), the MESA Protease Chain (BBa_K4803108) and the plasmid encoding coordinated expression of the green fluorescent protein under the TRE3G promoter along with tTA (BBa_K4803100).

Rapamycin was added at concentrations of 100 nM, 10 nM, 1 nM, 0.1 nM, and 0 nM.

Fluorescence data after 24h were analyzed using flow cytometry(BD FACS Melody).

Figure 6: Percentage of cells with fluorescence stronger than the maximum fluorescence intensity of non-transfected cell lines (293A) for each concentration of rapamycin at 24 h after the addition of rapamycin.

Figure 7: Mean fluorescence intensity for each concentration of rapamycin at 24h after the addition of rapamycin. The error bars indicate standard error.

Figure 8: Histogram of fluorescence intensity for each concentration of rapamycin at 24h after the addition of rapamycin; Normalized to mode represents relative value with mode as 100. Orange →NC, Blue →rapamycin 0 nM, Red → rapamycin 10 nM.

In actual applications using SWIFT or the MESA system, cell lines that show the desired results are sorted and the clones are used. Therefore, to ensure that only cell lines expressing ZsGreen are selected, cell lines showing fluorescence greater than the maximum fluorescence intensity of 293A were extracted and the average fluorescence intensity was shown on the graph.

Figure 9: Mean of fluorescence intensity after gating

Although tTA leaks to some extent when MESA parts are added, the amount of tTA that migrates into the nucleus increases with the addition of rapamycin.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 219
  Illegal XhoI site found at 4
  Illegal XhoI site found at 85
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

References

[1] Banaszynski, L. A., Liu, C. W., & Wandless, T. J. (2005). Characterization of the FKBP・Rapamycin・FRB ternary complex. Journal of the American Chemical Society, 127(13), 4715-4721. https://doi.org/10.1021/ja043277y

[2] Daringer, N. M., Dudek, R. M., Schwarz, K. A., & Leonard, J. N. (2014). Modular extracellular sensor architecture for engineering mammalian cell-based devices. ACS synthetic biology, 3(12), 892-902. https://doi.org/10.1021/sb400128g

[3] Rivera, V. M., Wang, X., Wardwell, S., Courage, N. L., Volchuk, A., Keenan, T., ... & Clackson, T. (2000). Regulation of protein secretion through controlled aggregation in the endoplasmic reticulum. Science, 287(5454), 826-830. https://doi.org/10.1038/s41467-022-28971-9