Difference between revisions of "Part:BBa K1850005:Design"
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The SpyTag binding motif was inserted into the fusion site of ''fimH'' via site-directed mutagenesis. | The SpyTag binding motif was inserted into the fusion site of ''fimH'' via site-directed mutagenesis. | ||
| − | The | + | The stainless steel binding Metal Binding Domain (MBD) 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. | 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. | ||
Revision as of 21:44, 18 September 2015
pRha - fimH - SpyTag_225 - MBD_258
Assembly Compatibility:
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 1184
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 1184
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1184
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 1184
- 1000COMPATIBLE 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 stainless steel binding Metal Binding Domain (MBD) 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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