Part:BBa_K3338023
IL-6 Pmut-MagA-P2A-hGLuc
Usage and Biology
IL-6 Pmut-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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1347
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 220
Illegal BamHI site found at 395
Illegal XhoI site found at 1581 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE 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-6mut-MagA-P2A-hGLuc construct in pEGFP-C2.
Characterization
To characterize the final part all individual components (basic parts) were examined. First, we could show that the IL-6 promoter exhibits significant LPS-sensitivity (see figure 2) and a relatively low basal activity as compared with the wildtype CMV and our two synthetic CMV promoters (see figure 3). Only the mutated IL-6 promoter exhibits a lower basal activity but unfortunately no LPS-sensitivity.
Figure 2: Relative activity of IL6 promoter in HeLa cells 24 hours (A) and 48 hours (B) after treatment with different concentrations of LPS for 3 hours, normalized to untreated control (0 µg/mL LPS). Data shown represents mean ± SEM of n=4 biological replicates. Statistical analysis was performed by unpaired t-test in comparison to untreated control, significance level: 10 %, significance is indicated by asterisk.
Figure 3: Relative basal activity of all tested promoters in HeLa cells 48 hours (A) and 72 hours (B) after transfection. Data was normalized to CMV promoter which served as reference. Data shown represents mean ± SEM of n=4 biological replicates.
All together the IL-6 promoter is well-suited for our sensor. Secondly, we were able to prove that the reporter MagA can be expressed in HeLa cells without inducing cytotoxic effects and is furthermore localized to the cell membrane indicating proper folding of the protein (see figure 4). Iron accumulation following MagA expression wasn’t shown explicitly in our system but was previously shown for mammalian cells (Pereira et al. 2016).
Figure 4: Representative microscopy image of eGFP-MagA expressing HeLa cells. Both fluorescence (left) and brightfield channel (middle) as well as a merge (right) are shown. Scale bar: 10 µm.
As hGLuc was used to determine the IL-6 promoter activity (see figure 2) we also showed its applicability as a reporter. We also assessed that the use of the P2A peptide enables the simultaneous and strong expression of two proteins from one promoter with comparable expression levels making it ideal for our purpose (see figure 5).
In contrast IRES leads to an uneven expression of both proteins which would not be beneficial for our inflammatory toxin sensor. Due to the corona-restrictions and thus the limited lab time we only cloned the final part into pEGFP-C2 but had no time to transfect it into HeLa cells and to characterize it as a whole.
Summary
Here we describe our newly developed inflammatory toxin sensor. It allows the sensing of LPS in the surrounding of the transfected cells. The function of every single subpart was experimentally proven and the final part was assembled in the pEGFP-C2 vector.
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.
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