Difference between revisions of "Part:BBa K1075025"
 
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<partinfo>BBa_K1075025 short</partinfo>
 
<partinfo>BBa_K1075025 short</partinfo>
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pLac2-RBS34-mCherry-ssrA-TT
 
  
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===Usage and Biology===
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The part contains the pLac promoter, a ribosomal binding site, the red fluorescent protein mCherry fused to the E. coli(ec)ssrA (DAS+4) tag and a double terminator.
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===biology===
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The (ec)ssrA tag is a short peptide sequence that can be fused to the C-terminus of proteins which should be degraded. The adaptor protein SspB binds to it and delivers the substrates to the protease ClpXP complex in E. coli. The mutation (DAS+4) in (ec)ssrA prevents the degradation of the ssrA-tagged protein without the presence of sspB and therefore makes the system more specific and controllable. [http://www.ncbi.nlm.nih.gov/pubmed/16762842]
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mCherry is a red fluorescent protein with the excitation maximum at 587 nm and the Emission maximum  at 610 nm. [http://www.ncbi.nlm.nih.gov/pubmed/15558047]
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===application===
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As we want to control protein degradation by controlling the function of ecSspB, we tagged the red fluorescent protein mCherry with ecssrA(DAS+4) to measure the degradation rate.
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The part was designed for proof of principle. Application as a bacterial fotographic film might be possible as well.
  
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The ssrA tag is a sequence, which allows proteolytic enzymes to degrade them. It relates to the protease ClpXP  complex in E.coli and it also allows adaptor proteins such as sspB binding and delivering substrates to the proteases in order to make the process more efficient.
 
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The mutation ssrA (DAS+4) prevents a direct binding to the protease and ensures the dependance of sspB.
 
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The tagged protein in this case is mCherry, a red fluorescing protein. Degradation can be measured by the decrease of light intensity.
 
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Prefixed is RBS, the binding site for a ribosome and the pLac promoter. It also includes a double terminator 'TT', which interrupts the translation.
 
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Latest revision as of 01:25, 5 October 2013

pLac-RBS34-mCherry-(Ec)ssrA(DAS+4)-TT

The part contains the pLac promoter, a ribosomal binding site, the red fluorescent protein mCherry fused to the E. coli(ec)ssrA (DAS+4) tag and a double terminator.

biology

The (ec)ssrA tag is a short peptide sequence that can be fused to the C-terminus of proteins which should be degraded. The adaptor protein SspB binds to it and delivers the substrates to the protease ClpXP complex in E. coli. The mutation (DAS+4) in (ec)ssrA prevents the degradation of the ssrA-tagged protein without the presence of sspB and therefore makes the system more specific and controllable. [http://www.ncbi.nlm.nih.gov/pubmed/16762842]

mCherry is a red fluorescent protein with the excitation maximum at 587 nm and the Emission maximum at 610 nm. [http://www.ncbi.nlm.nih.gov/pubmed/15558047]

application

As we want to control protein degradation by controlling the function of ecSspB, we tagged the red fluorescent protein mCherry with ecssrA(DAS+4) to measure the degradation rate.

The part was designed for proof of principle. Application as a bacterial fotographic film might be possible as well.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1717
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]