Revision as of 17:01, 23 September 2024

CMV-CcpA-mRuby2

Usage and Biology

The regulatory functions of CcpA are modulated by phosphorylation by serine/threonine kinases, which can affect its DNA-binding activity and thus its ability to regulate target genes. This phosphorylation-dependent mechanism enables S. aureus to adapt to different environmental conditions, thereby increasing its survivability and virulence (Liao et al., 2022). The ccpA inactivation impairs biofilm formation and restrictstheincorporation of extracellular DNA (eDNA) into the biofilm matrix, while codY inactivation leads to a structured but robust biofilm bound to eDNA and intercellular adhesin polysaccharide (PIA) in S. aureus.(Bulock et al., 2022) We aim to use this mechanism to detect ß-lactams, which can bind to pknB, potentially leading to phosphorylation of ccpA, which could then bind to a specifically engineered promoter. We therefore used an mRuby2 marker gene to detect expression of ccpA protein.

Cloning

We linearized the mammalian expression vector pEGFP-C2 with NheI and BamHI and inserted the both genes ccpA and mRuby, which were fused bevorhand together with matching overhangs. The ccpA gene was acquired from S. aureus. Following the NEBBuilder® user protocol this vector was synthesized with HIFI assembly method.

References

Bulock, L. L., Ahn, J., Shinde, D., Pandey, S., Sarmiento, C., Thomas, V. C., Guda, C., Bayles, K. W., & Sadykov, M. R. (2022). Interplay of CodY and CcpA in Regulating Central Metabolism and Biofilm Formation in Staphylococcus aureus. Journal of Bacteriology, 204(7), e00617-21. https://doi.org/10.1128/jb.00617-21

Liao, X., Li, H., Guo, Y., Yang, F., Chen, Y., He, X., Li, H., Xia, W., Mao, Z.-W., & Sun, H. (2022). Regulation of DNA-binding activity of the Staphylococcus aureus catabolite control protein A by copper (II)-mediated oxidation. Journal of Biological Chemistry, 298(3), 101587. https://doi.org/10.1016/j.jbc.2022.101587