Part:BBa_K3338023

IL-6 P_mut-MagA-P2A-hGLuc

Usage and Biology

IL-6 P_mut-MagA-P2A-hGLuc is an inflammatory toxin sensor comprising the IL6-Promoter downstream from the MagA-P2A-hGLuc cassette. This part combines the LPS-sensitivity of the IL6-Promoter with the simultaneous expression of the reporter genes MagA for MRI-detection and Gaussia Luciferase for bioluminescence detection in blood or urine (Goldhawk et al. 2009, Zurkiya et al. 2008, Tannous 2009). The construct displays the first step in generating a functional inflammatory toxin sensor for clinical use.

The mutation in the IL-6 promoter making it Biobrick assembly standard compatible unfortunately prevents LPS-sensitivity of the promoter. Thus, this part is not applicable as an inflammatory toxin sensor.

Cloning

Theoretical Part Design

This composite part was designed to recognize LPS in the surrounding of the cell. As a consequence, the reporter genes MagA and hGLuc should be expressed. Therefore, we used the IL-6 promoter as a signal transducer. The activation of the promoter is achieved by the cooperative binding of NF-κB and c-Jun (AP-1) (Xiao et al. 2004). AP-1- and NF-κB-translocation to the nucleus is triggered downstream of Toll like receptor (TLR) signaling cascades involving TRIF, MyD88, RIPK1 and TAK1 (Kawai and Akira 2007). The TLR family consists of more then 13 members that can detect a variety of distinct pathogen-associated molecular pattern (PAMPs) making them ideal natural inflammatory toxin sensors (Kawai and Akira 2007). In this study we used the LPS-TLR4 pathway to characterize our synthetic sensor. Since IL-6 promoter activity cannot be assessed in the clinic without suitable reporters, we added a MagA-P2A-hGLuc cassette behind the promoter. The P2A peptide represents a cleavage site between the reporters MagA and hGLuc allowing their simultaneous expression from one promoter. MagA is a transmembrane iron transporter leading to the accumulation of iron inside the cell which can be detected using MRI (Goldhawk et al. 2009, Zurkiya et al. 2008). hGLuc is a naturally secreted Luciferase. In cell culture applications it can be measured in the culture medium. In in vivo experiments it can be detected in blood or urine samples (Tannous2009).

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1347
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 220
  Illegal BamHI site found at 395
  Illegal XhoI site found at 1581
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 340
  Illegal BsaI site found at 2803
  Illegal BsaI.rc site found at 387
  Illegal BsaI.rc site found at 696
  Illegal BsaI.rc site found at 2135
  Illegal BsaI.rc site found at 2684
  Illegal SapI site found at 1825
  

Cloning

To characterize the part, we cloned it into the mammalian expression vector pEGFP-C2 (BBa_K3338020) using the NEBuilder® HiFi DNA Assembly Cloning Kit. Therefore, we linearized the vector using AseI and HindIII thus removing CMV-EGFP. The mutagenized IL-6 promoter (BBa_K3338005) with overhangs consisting of approximately 20 bp matching the ends of the linearized vector was synthesized by IDT. The plasmid was assembled following the NEBuilder® user protocol and after that sequence verified. The vector map of the construct is depicted in figure 1.

Figure 1: Vector map of the final IL-6_mut-MagA-P2A-hGLuc construct in pEGFP-C2.

References

Kawai, T., & Akira, S. (2007). Signaling to NF-kappaB by Toll-like receptors. Trends in molecular medicine, 13(11), 460–469.

Goldhawk, D. E., Lemaire, C., McCreary, C. R., McGirr, R., Dhanvantari, S., Thompson, R. T., Figueredo, R., Koropatnick, J., Foster, P., & Prato, F. S. (2009). Magnetic resonance imaging of cells overexpressing MagA, an endogenous contrast agent for live cell imaging. Molecular imaging, 8(3), 129–139.

Tannous B. A. (2009). Gaussia luciferase reporter assay for monitoring biological processes in culture and in vivo. Nature protocols, 4(4), 582–591.

Xiao, W., Hodge, D. R., Wang, L., Yang, X., Zhang, X., & Farrar, W. L. (2004). NF-kappaB activates IL-6 expression through cooperation with c-Jun and IL6-AP1 site, but is independent of its IL6-NFkappaB regulatory site in autocrine human multiple myeloma cells. Cancer biology & therapy, 3(10), 1007–1017.

Zurkiya, O., Chan, A. W., & Hu, X. (2008). MagA is sufficient for producing magnetic nanoparticles in mammalian cells, making it an MRI reporter. Magnetic resonance in medicine, 59(6), 1225–1231.