Revision as of 00:28, 27 October 2020

CMV-EGFP-MagA

The described part was used to express a EGFP-MagA fusion protein under control of the constitutively active CMV-promoter.

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.

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 1. The plasmid backbone and the PCR product were assembled using the NEBuilder® HiFi DNA Assembly Cloning Kit. The sequence was verified by DNA-sequencing.

Figure 1: Construction of a vector for EGFP-MagA expression in Hela cells under control of the CMV-enhancer/promoter. A, PCR product of MagA. 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.

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.