Composite

Part:BBa_K5317019

Designed by: Vanessa Bruhn   Group: iGEM24_Hannover   (2024-09-22)

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. 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 our specifically engineered promoter 3xCre3xAP1-miniCMV (K5317017). We, therefore, fused a mRuby2 marker gene (K5317001) to detect localization of the CcpA protein in HEK292T cells.

Cloning

Theoretical Part Design

CcpA was codon-optimized for the expression in mammalian systems and synthesized with the correct approx. 20 bp overhangs for introduction into a backbone plasmid. Placing the CcpA (K3338014) upstream of the reporter gene mRuby2 (K5317001) allows for visualization of expression and localization of CcpA.

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

Cloning

We linearized the mammalian expression vector pEGFP-C2 with NheI and BamHI, providing a CMV promoter, and inserted the genes CcpA (K33380014) and mRuby2 (K3338001) downstream, generation a fusion protein. The correct order in the plasmid of CcpA and mRuby2 was guided by approx. 20 bp long overhangs at each 5' and 3' and of the amplicon, following the NEBBuilder® HIFI user protocol. This composite part was cloned by using the primers in table 1.

HTML Table Caption Table1: Primers used to extract and clone the ccpA gene sequence.

Primer name Sequence
CcpA_fw TGAACCGTCAGATCCGatgacagttactatatatgatgtagcaagagaagc
CcpA_rev tggatccccttttgtagttcctcggtattcaattctgtgag
mRuby_fw actacaaaaggggatccaccggtcg
mRuby2_rev TCAGTTATCTAGATCCGGTGttacttgtacagctcgtccatcccacc

Figure 1: Vector map of the assembled CMV-CcpA-mRuby cassette in the C2 backbone plasmid.

Characterisation

We conducted transfection experiments of CMV-CcpA-mRuby2 in mammalian HEK293T cells to show the localization of CcpA under unstimulated conditions.

Single-transfection experiments

Figure 2: Depicted HEK293T cells transfected with CMV-CcpA-mRuby2 containing plasmid. Scale bar = 10 µm.

The CMV-CcpA-mRuby2-transfected HEK293T cells in the representative images in figure 2 depicted no fluorescent signal and therefore no further experiments were conducted with this transcription factor.

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

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