Difference between revisions of "Part:BBa K1850003:Design"

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===References===
 
===References===
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Pallesen, Lars, Lars K. Poulsen, Gunna Christiansen, and Per Klemm. "Chimeric FimH Adhesin of Type 1 Fimbriae: A Bacterial Surface Display System for Heterologous Sequences." <i>Microbiology</i> 141 (1995): 2839-848. SGM Journals. <i>Society for General Microbiology</i>, 01 Nov. 1995. Web. 18 Sept. 2015.
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Bhomkar, Prasanna, Wayne Materi, Valentyna Semenchenko, and David S. Wishart. "Transcriptional Response Of <i>E. Coli</i> Upon FimH-mediated Fimbrial Adhesion." <i>Gene Regulation and Systems Biology</i> 4 (2010): 1-17. <i>NCBI</i>. Libertas Academica, 24 Mar. 2010. Web. 18 Sept. 2015.

Revision as of 21:38, 18 September 2015


pRha - fimH - SpyTag_225 - HisTag_258


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 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

The fimH gene was amplified from the E. coli K-12 genome.

References

Pallesen, Lars, Lars K. Poulsen, Gunna Christiansen, and Per Klemm. "Chimeric FimH Adhesin of Type 1 Fimbriae: A Bacterial Surface Display System for Heterologous Sequences." Microbiology 141 (1995): 2839-848. SGM Journals. Society for General Microbiology, 01 Nov. 1995. Web. 18 Sept. 2015.

Bhomkar, Prasanna, Wayne Materi, Valentyna Semenchenko, and David S. Wishart. "Transcriptional Response Of E. Coli Upon FimH-mediated Fimbrial Adhesion." Gene Regulation and Systems Biology 4 (2010): 1-17. NCBI. Libertas Academica, 24 Mar. 2010. Web. 18 Sept. 2015.