Part:BBa_K1850013
pAra - fim
pAra fim contains the fim operon, including the structural proteins, transporters, and transmembrane domain of Type 1 Pili under titratable control of the pBad arabinose promoter BBa_I13453. When switched into an expression backbone and induced with a correctly expressed fimH variant (see BBa_K1850000, BBa_K1850002-11), pAra fim will produce Type 1 Pili with desired fimH adhesin.
Usage and Biology
Type 1 Pili are hairlike appendages on the surface of a broad range of E. coli strains uropathogenic E. coli which mediate adhesion to eukaryotic cells. This part can be cotransformed with BBa_K1850013, which contains the fimH adhesin, to gain control over pili expression and pili-mediated binding as measured by a standard assay called an agglutination (see our [http://2015.igem.org/Team:Harvard_BioDesign/Platform wiki] for more assay info).
To express the fim operon, this part must be switched into the low copy operon expression backbone to prevent problems with gene dosing.
To characterize this part, we cotransformed BBa_K1850013 with BBa_I13453 into a strain that does not produce type 1 pili (see strain JW4275-1 from the [http://cgsc.biology.yale.edu/KeioList.php Keio Collection]) and mixed standardized volumes of induced and uninduced culture with yeast. We were able to recover pili-mediated binding activity, as indicated by the tightly-bound clump at the bottom of the tube in the induced sample compared with the uninduced and our positive and negative controls (see our [http://2015.igem.org/Team:Harvard_BioDesign/Platform wiki] for more assay info):
We also assayed for the production of the structural subunit fimA in a concentrated extract of periplasmic proteins. Induced and uninduced cultures where treated with heat to shear off pili proteins and the resulting samples were denatured and run on an SDS Page Gel. The below images show a strong band in the induced cultures at 16.5 kD., the molecular weight of the primary structural subunit protein fimA, compared to the uninduced and negative control "Delta B". "Delta E" is an overproducing strain which also shows a fim A band. The inducer of interest is arabinose, disregard the rhamnose induction as it was for the co-transformed fimH plasmid (in this case with BBa_K1850007.
This plasmid has only been used in the presence of genomic AraC, the arabinose repressor protein, as it is not plasmid-encoded.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 125
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 65
Illegal BamHI site found at 2715 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 998
Illegal AgeI site found at 1029 - 1000COMPATIBLE WITH RFC[1000]
| None |