Part:BBa_K2677000

RFP-FimH

FimH is an adhesion protein that lies at the end of type 1 pili structures. These are hairlike appendages found on certain bacteria that assist in epithelial adhesion. This protein allows the bacterial to bind on several membrane structures in the body and lends ability to withstand higher shear forces present in the gut. This composite part is an extension of Harvard BioDesign 2015, BBa_K1850011. Our addition is a red fluorescent marker (RFP) from BBa_ BBa_J04450. We implemented this detection device in order to capture bacterial binding with a fluorescent microscope. Our main goal was to create an engineered probiotic that could bind to cancer cell surfaces. We thus chose Harvard's part because it contained the RPMrel colon cancer specific binding peptide. This composite part is characterized to be functional by the assay below.

Fig. A shows Caco-2 cells alone, and Fig. B and Fig. C show Caco-2 cells infected with engineered Nissle 1917. The 137 isoform (Fig. B) failed to result in binding to Caco-2 cells. Meanwhile the 49 isoform (Fig. C)successfully bound to Caco-2 cells in suspension, concluding that the expression of RFP and fimH 49 did not interfere with each other, and robustly allowed for the visualization of engineered Nissle 1917 using our method. As an extra control, figure D shows wild type Nissle 1917 and Caco-2 cells. Binding was not seen in this group either.

The order of assembly was confirmed through sequencing, however quality of the results was very poor (likely due to primer misbinding). We deduced fimH added first because fractions of its sequence were present in the first 1000 bases of the sequence but and RFP was not.

References:

1. Sauer MM, Jakob RP, Eras J, Baday S, Eriş D, Navarra G, Bernèche S, Ernst B, Maier T, Glockshuber R. “Catch-bond mechanism of the bacterial adhesin FimH” Nat Commun. 2016 Mar

2. Kelly KA and Jones DA. Isolation of a colon tumor specific binding peptide using phage display selection. Neoplasia. 2003 Sep-Oct;5(5):437-44.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 781
  Illegal AgeI site found at 893
  
• 1000
  COMPATIBLE WITH RFC[1000]