Difference between revisions of "Part:BBa K5317019"

Revision as of 14:55, 26 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). 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 (K3338001)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 cloned via HIFI assembly method.

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
INCOMPATIBLE WITH RFC[1000]
Illegal SapI.rc site found at 1669

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

@@ Line 7: / Line 7: @@
 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 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 (<span class="plainlinks">[https://parts.igem.org/Part:BBa_K3338001 K3338001]</span>)marker gene to detect expression of ccpA protein.
-=Cloning=
+===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.
+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 cloned via HIFI assembly method.
 <!-- -->
 <span class='h3bb'>Sequence and Features</span>
 <partinfo>BBa_K5317019 SequenceAndFeatures</partinfo>
 ===References===