Part:BBa_K5317015
GraR
Usage and Biology
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-transporters (El-Halfawy et al., 2020; Yang et al., 2012; Meehl et al., 2007). The GraSR system is a two-component regulatory system that controls the expression of many genes involved in stress response, cell wall metabolism and virulence pathways in Staphylococcus aureus (Falord et al., 2011).
Accordingly, GraR functions as a transcription factor and our cell-based antiobiotics sensor utilises it as such by aiming for its PknB-dependent phyosphorylation (K5317013).
Cloning
Theoretical Part Design
This part was codon optimised for human cell lines and was synthesised by Eurofins.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 235
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 235
- 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 235
- 1000COMPATIBLE WITH RFC[1000]
Characterization
The GraR functionality was analyzed by composing a gene cassette fused with the reporter gene mRuby2 K5317001 to assess GraR cell localization based on the fluorescent signal with unstimulated ampicillin conditions. Please visit the K5317020 registry entry to view the results.
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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