Part:BBa_K1583113

pAra + fusion protein BFP_Spycatcher_His

This device expresses the fusion protein BFP_SpyCatcher_His under arabinose control.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 131
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 71
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

This device expresses the fusion protein BFP_SpyCatcher_His under arabinose control.

It was created as a fusion protein from the following biobricks:

• Blue Fluorescent Protein BBa_K592100
  
• SpyCatcher BBa_K1583113
  
• His tag BBa_K1583059
  

The SpyTag SpyCatcher couple reacts under physiological conditions spontaneously to form a covalent isopeptide bond between both proteins linking them irreversibly together.

CsgA is a protein monomer which can aggregate to form amyloid nanowires in natural biofilms of E.coli. This protein is transported as an unfolded protein out of the cell. Outside the cell CsgA proteins self-assemble into nanowires after nucleation on the membrane protein CsgB. CsgC prevents CsgA proteins from self-assembling inside the cell and the transport is ensured by the proteins CsgEFG.

In our project we created Biobricks where we added specific tags to the CsgA protein in order to produce a functionalised amyloid nanowire. These modifications aim at increasing adhesive properties towards a certain surface or material. Examples are CsgA_His or the device CsgA_Hydroxyapatite affinity with hydroxyapatite being the main component of teeth.

With this device, we were planning to prove the possibility of modifying our amyloid nanowires with a functionality such as a fusion protein.

Unfortunately, due to a frameshift in our construct CsgA_SpyTag we were not able to produce amyloid nanowires with this certain tag in time.

Characterization

This part was characterised using a fluorescent assay to investigate proper folding of the BFP

Fluorescence assay

Fig. 1: Fluorescence signal normalized by the number of cells (OD600) over the time of 18 hours.

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Unfortunately from this data we had to conclude that our device does not differ in blue fluorescence from the negative controls meaning thus: the Blue Fluorescent Protein is not properly folded and not functional.