Part:BBa_K5317014

CcpA

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). We used this bacterial protein to evaluate the interaction with the PknB kinase.

Cloning

Theoretical Part Design

This part was codon optimised for human cell lines. This part was amplified by using the primers in table 1.

Table1: Primers used to extract the ccpA gene sequence.

Primer name	Sequence
ccpA_fw_1	tggatccccttttgtagttcctcggtattcaattctgtgag
ccpA_rv_1	TGAACCGTCAGATCCGatgacagttactatatatgatgtagcaagagaagc

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

The correct ccpA functionality was analyzed by composing a gene cassette where its placed downstream of the constitutuve CMV promoter and fused with the reporter gene mRuby2 to assess the metal-dependent localization based on the fluorescent signal. Please visit the K5317019 registry entry to view the results.

References

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