Difference between revisions of "Part:BBa K1850005:Design"

 
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<partinfo>BBa_K1850005 short</partinfo>
 
<partinfo>BBa_K1850005 short</partinfo>
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===Design Notes===
 
===Design Notes===
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We selected a rhamnose-inducible promoter  (<partinfo>BBa_K902065</partinfo>)  with a strong ribosome binding site (<partinfo>BBa_B0034</partinfo>), since this promoter is titratable and would allow for controlled expression of the ''fimH'' adhesin.
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We edited out an illegal PstI cut site in ''fimH'' through site-directed mutagenesis.
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The SpyTag binding motif was inserted into the fusion site of ''fimH'' via site-directed mutagenesis.
  
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The nickel binding HisTag was inserted into fusion sites of ''fimH'' via site-directed mutagenesis.
  
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We picked sites 225 and 258 since there was strong evidence in the literature that small fusions inserted at these sites such as his-tags were expressed and functional on assembled pili.
  
 
===Source===
 
===Source===

Revision as of 21:43, 18 September 2015

pRha - fimH - SpyTag_225 - MBD_258


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 1184
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1184
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1184
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1184
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

We selected a rhamnose-inducible promoter (BBa_K902065) with a strong ribosome binding site (BBa_B0034), since this promoter is titratable and would allow for controlled expression of the fimH adhesin.

We edited out an illegal PstI cut site in fimH through site-directed mutagenesis.

The SpyTag binding motif was inserted into the fusion site of fimH via site-directed mutagenesis.

The nickel binding HisTag was inserted into fusion sites of fimH via site-directed mutagenesis.

We picked sites 225 and 258 since there was strong evidence in the literature that small fusions inserted at these sites such as his-tags were expressed and functional on assembled pili.

Source

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References