Part:BBa_K3338000

Iron transporter MagA from Magnetospirillum magneticum

Usage and Biology

MagA is a transmembrane iron transporter originating from the aquatic magnetotactic bacterium Magnetospirillum magnetotacticum. In the original host the protein is involved in the formation of intracellular magnetic particles of magnetite (Fe₃O₄) (Nakamura et al. 1995, Uebe et al. 2012). These particles are the core components of magnetosomes in aquatic magnetotactic bacteria allowing them to passively align and swim along the earth's magnetic field lines (Lower and Bazylinski 2013). When expressed in procaryotic or eukaryotic cells it leads to the accumulation of intracellular iron (Goldhawk et al. 2009, Zurkiya et al. 2008). In the last years its capability as a reporter gene in mammalian cells using the non-invasive imaging technique magnetic resonance imaging (MRI) was recognized (Goldhawk et al. 2009, Zurkiya et al. 2008). It was shown that cells expressing MagA that are transplanted in animals can be detected using MRI without being incubated with iron supplement prior to transplantation (Zurkiya et al. 2008, Rohani 2014). However, not all cells types are suitable for MagA expression because it sometimes induces strong cytotoxic effects (Pereira 2016). In the HeLa cell system, we used in our study, MagA was properly expressed.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1182
  Illegal BsaI.rc site found at 514
  Illegal BsaI.rc site found at 1063
  Illegal SapI site found at 204
  

Characterization

In order to test whether MagA is expressed in HeLa cells without inducing cytotoxic effects and to ensure that it is properly localized to the cell membrane it was expressed as N-terminal EGFP-fusion under the control of the constitutive CMV-promoter. Therefore, MagA was PCR-amplified from MS-1 magA (Addgene plasmid #21751, Bertani et al. 2001) using the specific primers MagA_fw and Mag_A_rv (see table 1).

Table1: Primers used to design the fragments.

Primer name	Sequence
MagA_fw	CGAGCTGTACAAGTCCGGCCGGACTCAGATCTCGAGCTCAatggacctgcatcatcccg
MagA_rv	TGGATCCCGGGCCCGCGGTACCGTCGACTGCAGAATTCGAtcagattccagtgccaggtccggcattg

The primers were designed with approximately 20 bp 5’ overhangs overlapping with the ends of the HindIII-linearized pEGFP-C2 vector (BBa_K3338020). An agarose gel of the PCR product is shown in figure 1A. The plasmid backbone and the PCR product were assembled using the NEBuilder® HiFi DNA Assembly Cloning Kit. The sequence was verified by DNA-sequencing. The vector map is displayed in figure 1B.

Figure 1: Construction of a vector for EGFP-MagA expression in Hela cells under control of the CMV-enhancer/promoter. A, 1 % agarose gel of the PCR product of MagA. DNA was stained with Midori Green fluorescent dye. The plasmid MS-1 magA from Addgene (plasmid #21751, Bertani et al. 2001) was used as template. The primers are listed in table 1.B, Vector map of the final expression plasmid containing the composite part BBa_K3338012 based on the pEGFP-C2 vector for characterization of MagA.

The vector was transfected into HeLa cells using the lipofection technique with ViaFect transfection reagent or the electroporation technique. The constitutive expression of the EGFP-MagA fusion protein was analyzed by fluorescent microscopy 24 hours after transfection. EGFP-MagA expression was indicated by a fluorescent signal that was not localized in the area of the nucleus or the cytoplasm, but rather in membrane regions (see figure 2).

Figure 2: 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 expected for MagA the results indicate membrane localization of the fusion protein. Furthermore, it was clearly shown that MagA expression of HeLa cells does not cause cell death making it suitable as a reporter in a HeLa-cell system.

Conclusions

Here we describe the new part MagA as a reporter gene for use in mammalian cells. We showed that it is properly expressed in HeLa cells without inducing cytotoxic effects even when expressed under control of the strong constitutive CMV-promoter. As expected for the transmembrane iron transporter, it is properly localized to the cell membrane indicating correct protein folding. The iron transport activity of MagA when expressed in eukaryotic cells was experimentally proven in many studies (Pereira et al. 2016). However, the accumulation of iron inside transfected HeLa cells in our system still has to be experimentally confirmed although it is quite likely. Due to the corona restrictions there was no possibility and time to perform MRI measurements.

References

Bertani, L. E., Weko, J., Phillips, K. V., Gray, R. F., & Kirschvink, J. L. (2001). Physical and genetic characterization of the genome of Magnetospirillum magnetotacticum, strain MS-1. Gene, 264(2), 257–263.

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.

Lower, B. H., & Bazylinski, D. A. (2013). The bacterial magnetosome: a unique prokaryotic organelle. Journal of molecular microbiology and biotechnology, 23(1-2), 63–80.

Nakamura, C., Burgess, J. G., Sode, K., & Matsunaga, T. (1995). An iron-regulated gene, magA, encoding an iron transport protein of Magnetospirillum sp. strain AMB-1. The Journal of biological chemistry, 270(47), 28392–28396.

Pereira, S. M., Williams, S. R., Murray, P., & Taylor, A. (2016). MS-1 magA: Revisiting Its Efficacy as a Reporter Gene for MRI. Molecular imaging, 15, 1536012116641533.

Rohani, R., Figueredo, R., Bureau, Y., Koropatnick, J., Foster, P., Thompson, R. T., Prato, F. S., & Goldhawk, D. E. (2014). Imaging tumor growth non-invasively using expression of MagA or modified ferritin subunits to augment intracellular contrast for repetitive MRI. Molecular imaging and biology, 16(1), 63–73.

Uebe, R., Henn, V., & Schüler, D. (2012). The MagA protein of Magnetospirilla is not involved in bacterial magnetite biomineralization. Journal of bacteriology, 194(5), 1018–1023.

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.