Part:BBa_K1583113

pAra + fusion protein BFP_Spycatcher_His

This device expresses the fusion protein BFP_SpyCatcher_His under control of an arabinose inducible promoter.

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 (Sun et al. 2014).

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.

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]

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.

Fig. 2: Sample overview of the fluorescence assay.

Data interpretation:
Unfortunately from this data we had to conclude that our device does not differ in blue fluorescence from the negative controls meaning: the blue fluorescent protein is not properly folded and not functional.

However, a publication (Botyanszki et al, 2015) successfully used the SpyTag/SpyCatcher system to link a fusion protein of amylase with SpyCatcher to the nanowires. We are thus hopeful, that later iGEM teams pick up our idea and further develop our devices to promote this idea.

References

Botyanszki, Z. et al., 2015. Engineered catalytic biofilms: Site-specific enzyme immobilization onto E. coli curli nanofibers. Biotechnology and Bioengineering, 112(10), pp.2016–2024.

Sun, F. et al., 2014. Synthesis of bioactive protein hydrogels by genetically encoded SpyTag-SpyCatcher chemistry. Proceedings of the National Academy of Sciences of the United States of America, 111(31), pp.11269–74.