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:
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
- 10COMPATIBLE WITH RFC
- 12Illegal NheI site found at 131
- 21Illegal BamHI site found at 71
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000COMPATIBLE WITH RFC
This part was characterised using a fluorescent assay to investigate proper folding of the BFP
Unfortunately from this data we had to conclude that our device does not differ in blue fluorescence from the negative controls. This most likely means that the blue fluorescent protein is not properly folded and not functional or induction of the arabinose inducible promoter did not result in gene expression.
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.
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.