Difference between revisions of "Part:BBa K5317002"

 
 
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<partinfo>BBa_K5317002 short</partinfo>
 
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PknB is a eukaryote-like serine/threonine kinase in Staphylococcus aureus that consists of an N-terminal cytosolic kinase domain, a central transmembrane domain and three C-terminal extracellular PASTA domains.2 When this extracellular domain recognises beta-lactam compounds, it transmits the signal by autophosphorylating the N-terminal kinase domain, thereby activating downstream signalling cascades.3 In S. aureus, this mechanism normally enables early recognition and adaptation to antibiotic stress.4 In our project, we aim to introduce PknB into HEK cells, together with three proteins that can be phosphorylated by PknB. This targeted phosphorylation increases DNA binding activity 1 and is expected to induce the expression of a specific gene, which generates a fluorescent signal.
 
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===Usage and Biology===
 
===Usage and Biology===
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The monomeric near-infrared (NIR) fluorescent protein miRFP670 was developed by Shcherbakova and colleagues in 2016.
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The excitation maxima of miRFP670 is at 642 nm and the emission maxima at 670 nm. miRFP670 was genetically engineered from bacterial phytochrome photoreceptors (BphP) to generate reporters using the optical window with low autofluorescence, decreased light scattering, and minimal absorption of biological substances such as hemoglobin, water, and melanin. Of the BphP-based fluorophores, miRFP is particularly suitable for investigations in mammalian cell lines due to a fluorescence quantum yield of 14 % and maintaining brightness in the physiological pH range from 5 to 8 (Shcherbakova ''et al.'', 2016).
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miRFP670 was employed in our mammalian cell-based antibiotic detection sensor downstream of the ATF2-3xCre3xAP1 promoter (<span class="plainlinks">[https://parts.igem.org/Part:BBa_K5317017 K5317017]</span>) visualizing the antibiotic presence-dependent phosphorylation of ATF2 (<span class="plainlinks">[https://parts.igem.org/Part:BBa_K5317016 K5317016]</span>).
  
 
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<partinfo>BBa_K5317002 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5317002 SequenceAndFeatures</partinfo>
  
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=Reference=
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Shcherbakova, D. M., Baloban, M., Emelyanov, A. V., Brenowitz, M., Guo, P., & Verkhusha, V. V. (2016). Bright monomeric near-infrared fluorescent proteins as tags and biosensors for multiscale imaging. Nature communications, 7, 12405. https://doi.org/10.1038/ncomms12405
  
 
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Latest revision as of 21:22, 1 October 2024


miRFP670

Usage and Biology

The monomeric near-infrared (NIR) fluorescent protein miRFP670 was developed by Shcherbakova and colleagues in 2016. The excitation maxima of miRFP670 is at 642 nm and the emission maxima at 670 nm. miRFP670 was genetically engineered from bacterial phytochrome photoreceptors (BphP) to generate reporters using the optical window with low autofluorescence, decreased light scattering, and minimal absorption of biological substances such as hemoglobin, water, and melanin. Of the BphP-based fluorophores, miRFP is particularly suitable for investigations in mammalian cell lines due to a fluorescence quantum yield of 14 % and maintaining brightness in the physiological pH range from 5 to 8 (Shcherbakova et al., 2016).

miRFP670 was employed in our mammalian cell-based antibiotic detection sensor downstream of the ATF2-3xCre3xAP1 promoter (K5317017) visualizing the antibiotic presence-dependent phosphorylation of ATF2 (K5317016).

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 763
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 763
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 419
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 763
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 763
    Illegal NgoMIV site found at 82
    Illegal NgoMIV site found at 370
    Illegal NgoMIV site found at 405
  • 1000
    COMPATIBLE WITH RFC[1000]

Reference

Shcherbakova, D. M., Baloban, M., Emelyanov, A. V., Brenowitz, M., Guo, P., & Verkhusha, V. V. (2016). Bright monomeric near-infrared fluorescent proteins as tags and biosensors for multiscale imaging. Nature communications, 7, 12405. https://doi.org/10.1038/ncomms12405