Difference between revisions of "Part:BBa K5317014"

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__NOTOC__
 
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<partinfo>BBa_K5317014 short</partinfo>
 
<partinfo>BBa_K5317014 short</partinfo>
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===Usage and Biology===
 
===Usage and Biology===
  
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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 restricts the incorporation 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).
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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 mechanism of ccpA to evaluate the interaction with the PknB kinase when stimulated with ß-lactam antibiotics.
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=Cloning=
 
=Cloning=
  
 
===Theoretical Part Design===
 
===Theoretical Part Design===
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Placing the mRuby2 reporter gene behind ccpA allows for visualisation of expression of the protein of interest. Gene of interest was codon optimised for human cell lines.
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This part was codon-optimized and synthesized for expression in human cell lines.
  
 
===Sequence and Features===
 
===Sequence and Features===
 
<partinfo>BBa_K5317014 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5317014 SequenceAndFeatures</partinfo>
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===References===
 
  
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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
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=Characterization=
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The correct ccpA functionality was analyzed by composing a gene cassette that is placed downstream of the constitutive CMV promoter and fused with the reporter gene mRuby2 to assess by indication with ß-lactam antibiotics the ccpA localization based on the fluorescent signal. Please visit the <span class="plainlinks">[https://parts.igem.org/Part:BBa_K5317019 K5317019]</span> registry entry to view the results.
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=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
 
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

Latest revision as of 22:07, 1 October 2024

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 mechanism of ccpA to evaluate the interaction with the PknB kinase when stimulated with ß-lactam antibiotics.

Cloning

Theoretical Part Design

This part was codon-optimized and synthesized for expression in human cell lines.

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 that is placed downstream of the constitutive CMV promoter and fused with the reporter gene mRuby2 to assess by indication with ß-lactam antibiotics the ccpA 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