Difference between revisions of "Part:BBa K1075025"
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pLac2-RBS34-mCherry-ssrA-TT
 
pLac2-RBS34-mCherry-ssrA-TT
  
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===Usage and Biology===
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===construction===
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The part contains the promoter plac, a ribosomal binding site, the red fluorescent protein mCherry fused to the ssrA (DAS+4) tag and a double terminator.
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 +
 
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===biology===
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plac is a IPTG inducible promoter.
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The ribosomal binding side is a sequence of mRNA where the ribosome binds to start translation.
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The ssrA tag is a short peptide sequence, which is fused to the C-terminus of proteins, which should be degraded. It relates to the protease ClpXP complex in E.coli and it also allows the adaptor proteins SspB binding and delivering substrates to the proteases in order to make the process more efficient. The mutated ssrA (DAS+4) weakens a direct binding between proteases and ssrA and increases the dependance of sspB. [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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The double terminator stops the transcription at this point.
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 +
 
 +
===application===
 +
As we want to control protein degradation by controlling the function of SspB, we tag the red fluorescent protein mCherry with ssrA (DAS+4) to measure the degradation rate.
 +
 
  
−
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>

Revision as of 17:39, 3 October 2013

pLac-RBS34-mCherry-(Ec)ssrA(DAS+4)-TT pLac2-RBS34-mCherry-ssrA-TT

construction

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


biology

plac is a IPTG inducible promoter.

The ribosomal binding side is a sequence of mRNA where the ribosome binds to start translation.

The ssrA tag is a short peptide sequence, which is fused to the C-terminus of proteins, which should be degraded. It relates to the protease ClpXP complex in E.coli and it also allows the adaptor proteins SspB binding and delivering substrates to the proteases in order to make the process more efficient. The mutated ssrA (DAS+4) weakens a direct binding between proteases and ssrA and increases the dependance of sspB. [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]

The double terminator stops the transcription at this point.


application

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


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]