Part:BBa_K2132001

CsgASpyCatcherHisTag

This is the subunit of the biofilm of E. Coli Curli system linked to SpyCatcher and HisTag. The two added parts can be used for various functions with SpyTag and other His tag-binding materials like quantum dots.

Sequence and Features

Assembly Compatibility:

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

  Illegal XhoI site found at 841
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

1. Congo Red：successful secretion and expression

After CR dye, the figure indicates that the His-CsgA-SpyCatcher-Histag mutant induced by 0.25 μg ml-1 of aTc successfully secreted a thin-layer biofilm on the plate which are stained to brown-red color by CR, compared to the negative control with no inducer. (Because the ratio between Congo Red dye and Brilliant Blue dye is not in the best state which leads to the unapparent phenomenon through the lens, the brown red biofilm is easy to be identified visually.) This assay also proved that the new and challenging construction of appending a large protein onto CsgA subunits will work accurately and effectively.

2. Quantum dots fluorescence test: successful binding test of Histag with nanomaterials Then comes to the next part: we want to check if SpyCatcher protein will be too large to cause steric hindrance effect on Histag peptide. The best approach to verify is the fluorescence assay of binding with nanomaterials.

After applying the same steps as introduced above, the bottom of left well show a large area of bright fluorescence, manifesting His-CsgA-SpyCatcher-Histag mutant secreted biofilms under the control of inducer and Histag on it is not blocked, what is more, it is firmly attached with QDs. From this assay, we assure that the SpyCatcher will not impose negative effect on the binding between inorganic material and biofilm. The picture was snapped by ChemiDoc MP, BioRad.

3. TEM: visualization of binding test