Part:BBa_K4803108

CD4signal-FKBP-CD28TMD-TEVp

Introduction

This biobrick is MESA Part created by combining CD4 signal (BBa_K4803005), FKBP(BBa_K4803004), CD28 transmembrane domain (BBa_K4803006), and TEV protease (BBa_K2969000).

Usage and Biology

This Part is based on the mechanism of MESA, a synthetic receptor. FKBP and FRB dimerize in the presence of rapamycin. When ectodomain rapamycin is detected by this Part and BBa_K4803109, the two Parts dimerize. Upon dimerization, TEV Protease of this Part is activated, which cleaves the intracellular TEV Protease cleavage site of BBa_K4803109 and releases the lower tTA.

Figure 1: Overview of MESA system

Characterization

Wet Design

In UTokyo2023 project, we tested 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 2: Completed plasmid of YT82

Figure 3: Completed plasmid of iU-73

Figure 4: 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 this part, the MESA Target Chain (BBa_K4803109) and a plasmid encoding coordinated expression of the green fluorescent protein under the TRE3G promoter along with tTA (BBa_K4803100).

Three days after transfection, rapamycin was added at concentrations of 100 nM, 10 nM, 1 nM, 0.1 nM, and 0 nM.

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

Figure 5: 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 6: Mean fluorescence intensity for each concentration of rapamycin at 24 h after the addition of rapamycin. Error bar indicates standard error.

Figure 7: Histogram of fluorescence intensity for each concentration of rapamycin at 24 h 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 8: 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.

Modeling

Model validation and development by wet measurement.

The results from the Wet Lab showed that MESA leak is about 50% even in the absence of ligand.

Figure 8: Equations of the developed model

Figure 9: Relationship between ligand concentration and fluorescence intensity (Theoretical curve (blue), experimental value (red))

Reference

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

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

Rivera, V. M., Wang, X., Wardwell, S., Courage, N. L., Volchuk, A., Keenan, T., ... & Clackson, T. (2022). 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

Voigt, C. A. and Fernandez-Rodriguez, J. , Post-translational control of genetic circuits using Potyvirus proteases Jesus, 2016, 44(13): 6493-6502

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 475
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 925
  Illegal SapI.rc site found at 1273