Difference between revisions of "Part:BBa K1850004:Design"

Latest revision as of 21:41, 18 September 2015

pRha - fimH - SpyTag_N

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.

We picked the N-terminus as a new insertion site to try since prior work did not show if peptide fusions could be expressed and properly assembled at this site.

Source

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

References

Zakeri, Bijan, Jacob O. Fierer, Emrah Celik, Emily C. Chittock, Ulrich Schwarz-Linek, Vincent T. Moy, and Mark Howarth. "Peptide Tag Forming a Rapid Covalent Bond to a Protein, through Engineering a Bacterial Adhesin." Proceedings of the National Academy of the United States of America 109.12 (2012): E690-697. PNAS. National Academy of the Sciences. Web. 18 Sept. 2015.

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.

@@ Line 12: / Line 12: @@
 The SpyTag binding motif was inserted into the fusion site of ''fimH'' via site-directed mutagenesis.
+We picked the N-terminus as a new insertion site to try since prior work did not show if peptide fusions could be expressed and properly assembled at this site.
 ===Source===