Part:BBa_K5317020
CMV-GraR-mRuby2
Usage and Biology
Contained within are two key genes: mRuby2, a red fluorescent protein for live-cell imaging, and graR, a regulator that might bind to PknB upon phosphorylating GraR. . GraR is known for its role in β-lactam resistance by upregulating cell wall biosynthesis genes, altering cell wall composition, and increasing expression of ABC-transporter (El-Halfawy et al., 2020),(Yang et al., 2012),(Meehl et al., 2007). The GraSR system was found to control genes involved in stress response, cell wall metabolism and virulence pathways, in addition to playing an important role in CAMP resistance (Falord et al., 2011). When activated by pknB, GraR binds to specific DNA sequences to regulate gene expression, in our case it presumably binds to a specific engineered promotor.
Cloning
Theoretical Part Design
We placed the mRuby2 fluorescent marker (K5317001) downstream behind graR to visualize localisation of graR when stimulated with ß-lactam antibiotics . This gene was codon optimised for human cell lines.
Sequence and features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 889
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 889
- 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 889
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 1342
Cloning
This part was engineered with NEBBuilder® HIFI assembly method. First the backbone was linearized with NheI and BamHI and matching ends of gene and backbone ensured seamless cloning of GraR. In mammalian systems, this part is useful for studying the potential interactions between GraR and PknB. This part was amplified by using the primers in table 1.
| Primer name | Sequence |
|---|---|
| graR_fw_1 | TGAACCGTCAGATCCGatgcaaatactactagtagaagatgacaatactttgt |
| graR_rv_2 | tggatccccttcatgagccatatatccttttcctacttttgt |
| graR_fw_3 | tggatccccttcatgagccatatatccttttcctacttttgt |
| graR_rv_4 | TCAGTTATCTAGATCCGGTGttacttgtacagctcgtccatcccacc |
Figure 1: Assembled vector map with graR-mRuby2 integrated into the pEGFP-C2 backbone.
Characterisation
Transfection experiments of GraR in mammalian HEK cells to show localisation and activation of graR in unstimulated ampicillin conditions. This is was one of three possible transcription factors we analysed within this project.
Single-transfection experiments
Figure 2: Single-transfected HEK293T cells with the CMV-graR-mRuby2 plasmid depicted low mRuby2-signal under unstimulated conditions. Scale bar = 20 µm.
Depicted HEK cells show transfected CMV-GraR-mRuby2. Low mRuby2 fluorescent signals are recognisable. However, did not lead to further experiments.
References
El-Halfawy, O. M., Czarny, T. L., Flannagan, R. S., Day, J., Bozelli, J. C., Kuiack, R. C., Salim, A., Eckert, P., Epand, R. M., McGavin, M. J., Organ, M. G., Heinrichs, D. E., & Brown, E. D. (2020). Discovery of an antivirulence compound that reverses β-lactam resistance in MRSA. Nature Chemical Biology, 16(2), 143–149. https://doi.org/10.1038/s41589-019-0401-8
Falord, M., Mäder, U., Hiron, A., Débarbouillé, M., & Msadek, T. (2011). Investigation of the Staphylococcus aureus GraSR Regulon Reveals Novel Links to Virulence, Stress Response and Cell Wall Signal Transduction Pathways. PLoS ONE, 6(7), e21323. https://doi.org/10.1371/journal.pone.0021323
Meehl, M., Herbert, S., Götz, F., & Cheung, A. (2007). Interaction of the GraRS Two-Component System with the VraFG ABC Transporter To Support Vancomycin-Intermediate Resistance in Staphylococcus aureus. Antimicrobial Agents and Chemotherapy, 51(8), 2679–2689. https://doi.org/10.1128/AAC.00209-07
Yang, S.-J., Bayer, A. S., Mishra, N. N., Meehl, M., Ledala, N., Yeaman, M. R., Xiong, Y. Q., & Cheung, A. L. (2012). The Staphylococcus aureus Two-Component Regulatory System, GraRS, Senses and Confers Resistance to Selected Cationic Antimicrobial Peptides. Infection and Immunity, 80(1), 74–81. https://doi.org/10.1128/IAI.05669-11
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