Part:BBa_K5317020

CMV-GraR-mRuby2

Usage and Biology

Contained in the CMV-GraR-mRuby2 casette are two key genes next to the constitutively active CMV promoter: mRuby2, a red fluorescent protein for live-cell imaging, and GraR, a regulator that might bind to the PknB-kinase upon detecting beta-lactams and gets activated by phosphorylation. 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).

Our aim was its utilization as a signal transmitter in our cell-based antibiotic sensor. When phosphorylated by the detection unit PknB (K5317013), GraR translocates into the nucleus and binds to specific DNA sequences in our engineered 3xCre3xAP1-miniCMV promoter ((K5317017)), leading to the expression of the downstream located miRFP670 (K5317002).

Cloning

Theoretical Part Design

The GraR sequence (K5317015) was codon-optimized for mammalian expression systems and synthesized before fusing the mRuby2 fluorescent marker (K5317001) C-terminally to visualize localisation of GraR when stimulated with ß-lactam antibiotics. To ensure a constitutive expression of the transcription factor, the CMV of the pEGFP-C2 backbone was utilized.

Sequence and features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 889
  
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 889
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 889
  
• 1000
  INCOMPATIBLE 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, creating matching approx. 20 bp-long overhangs between both inserts and the backbone ensuring the correct order of CMV-GraR-mRuby2. In mammalian systems, we used this plasmid to study the potential interactions between GraR and PknB. The inserts were amplified by using the primers in table 1.

Table1: Primers used to extract the GraR gene sequence.

Primer name	Sequence
GraR_fw	TGAACCGTCAGATCCGatgcaaatactactagtagaagatgacaatactttgt
GraR_rev	tggatccccttcatgagccatatatccttttcctacttttgt
mRuby2_fw	tggatccccttcatgagccatatatccttttcctacttttgt
mRuby2_rev	TCAGTTATCTAGATCCGGTGttacttgtacagctcgtccatcccacc

Figure 1: Assembled vector map with GraR-mRuby2 integrated into the pEGFP-C2 backbone.

Characterisation

Transfection experiments were conducted of CMV-GraR-mRuby2 in mammalian HEK293T cells to show successful expression and localization of GraR under unstimulated conditions.

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.

The representative images of figure 2 depict HEK293T cells after transfection with CMV-GraR-mRuby2. Even under unstimulated conditions, the HEK293T cells emitt low mRuby2 fluorescent signals, indicating a successful codon optimization and transfection. No further experiments were performed using this transcription factor, since the intracellular assembly of all sensor parts was performed using the transcription factor ATF2 (K5317016) as the signal transmitter.

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