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]

Characterization

Three different experiments were conducted to characterize these biobricks:

• TEM: visualization of CsgASpyCatcherHisTag mutant biofilm
• Fluoresence Binding Test
• TEM: visualization of binding test

TEM: visualization of CsgASpyCatcherHisTag mutant biofilm

Fig. 1: Biofilms composed by CsgASpyCatcherHisTag subunits. After scrutinization, biofilm can viewed around the '''E.coli'''

Fluoresence Binding Test

In order to test the effect of binding between CsgASpyCatcherHisTag mutant and inorganic nanoparticles, we apply same amount of suspended QDs solution into M63 medium which has cultured biofilm for 72h. After 30-min incubation, we used PBS to mildly wash the well, and the result was consistent with our anticipation: On the left, CsgASpyCatcherHisTag mutant were induced and thus secreted biofilm, and firmly attached with QDS and thus show bright fluorescence. Therefore, we ensure the stable coordinate bonds between CsgASpyCatcherHisTag mutant and QDs can manage to prevent QDs from being taken away by liquid flow.

Fig. 2: Binding between CsgASpyCatcherHisTag mutant and inorganic QDs.

TEM: visualization of binding test

transmission electron microscopy(TEM) visualize the binding effect of CsgASpyCatcherHisTag mutant E.coli with AuNPs. From the picture, it shows biofilm areas are attached by AuNPs and thus confirm the viability of histag on CsgASpyCatcherHisTag mutant biofilm.

Fig. 3: Biofilms composed by CsgASpyCatcherHisTag subunits bind with AuNPs.