Part:BBa_K4803106

CD4signal-IFNGRex-CD28TMD-TEV CS-tTA

Introduction

This biobrick is Part of MESA (Modular Extracellular Sensor Architecture) created by combining CD4 signal (BBa_K4803005), IFNGR1 extracellular domain (BBa_K4803002), 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. When IFN-γ is detected by this Part and another receptor part of the MESA system (BBa_K4803105), the two Parts dimerize. Upon dimerization, the TEV protease of BBa_K4803105 is activated, which cleaves the intracellular TEV protease cleavage site of this Part and releases the lower tTA.

Figure 1: Overview of MESA system

Characterization

We checked the responsiveness of the MESA system by varying the concentration of IFN-γ. HEK293A was co-transfected with this part, the MESA Protease Chain (BBa_K4803105) and a plasmid encoding coordinated expression of the green fluorescent protein under the TRE3G promoter along with tTA (BBa_K4803100). Three days after transfection, DW only, IFN-γ 1 pM, 1 nM, 10 nM and 100 nM were added and incubated at 37 °C for 24 h. Fluorescence intensity was measured using BD FACS Melody. Results are as follows.

Analysis by flow cytometry (BD FACS Melody) was performed 24 h after IFN-γ addition.

Figure 2: Percentage of cells with fluorescence stronger than the maximum fluorescence intensity of non-transfected cell lines (293a) for each IFN-γ concentration at 24 h after IFN-γ addition.

Figure 3: Mean fluorescence intensity for each IFN-γ concentration at 24 h after IFN-γ addition. The error bars indicate standard error.

Figure 4: Histogram of fluorescence intensity by IFN-γ concentration at 24 h after IFN-γ addition.

Orange→NC, Blue→0 nM, Red→10 nM

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 IFN-γ.

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

Schroder, K., Hertzog, P. J., Ravasi, T., & Hume, D. A. (2004). Interferon-γ: an overview of signals, mechanisms and functions. Journal of Leucocyte Biology, 75(2), 163-189. https://doi.org/10.1189/jlb.0603252

https://www.ncbi.nlm.nih.gov/nuccore/NM_000416.3?report=genbank

Sequence and Features

Assembly Compatibility:

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

  Illegal BamHI site found at 1516
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]