Composite

Part:BBa_K4803109

Designed by: Kotaro MURAI   Group: iGEM23_UTokyo   (2023-10-10)

CD4signal-FRB-CD28TMD-TEV CS-tTA

Introduction

This biobrick is MESA (Modular Extracellular Sensor Architecture) Part created by combining CD4 signal (BBa_K4803005), FRB(BBa_K4803003), CD28 transmembrane domain (BBa_K4803006), TEV cleavage site (BBa_K1362453), and tTA (BBa_K2696013).

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 extracellular rapamycin is detected by this Part and BBa_K4803108, the two Parts dimerize. Upon dimerization, TEV protease of BBa_K4803108 is activated, which cleaves the intracellular TEV protease cleavage site of this part and releases the lower tTA.

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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: Protease Chain (PC) part and Target Chain (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 TRE-ZsGreen.

Plasmids are shown below.

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Figure 2: Completed plasmids of YT82
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Figure 3: Completed plasmids of iU-73
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Figure 4: Completed plasmids 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 Protease Chain (BBa_K4803108) and the 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).

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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.
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Figure 6: Mode fluorescence intensity for each concentration of rapamycin at 24 h after the addition of rapamycin. error bar indicates standard error.
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Figure 7: Histogram of fluorescence intensity for each concentration of rapamycin at 24 h after the addition of rapamycin; Normalize 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.
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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.

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Figure 8. Equations of the developed model.
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Figure 9. Relationship between ligand concentration and fluorescence intensity (Theoretical curve (blue), experimental value (red))

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


Reference

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

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

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

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