Part:BBa_K1457000
mRFP_Spycatcher
This part is the coding sequence for a modified red fluorescent protein fused to 'Spycatcher' protein. Spycatcher specifically amide bonds to an orthogonal polypeptide sequence called 'Spytag'. This part can be used to bind and detect any engineered constructs displaying Spytag. In our specific system, it is engineered to bind csgA protein fused to spytag and is applied extracellularly to easily detect CsgA production.
Usage and Biology
The red chromoprotein in this part is conjugated to the SpyCatcher peptide tag. This tag has been demonstrated to spontaneously form a covalent bond to the corresponding SpyTag domain when the two come close to one another in a solution (Zakeri 2011). Therefore, this construct can be used to tag any protein or protein complex fused to a SpyTag domain with a red chromoprotein, allowing the complex to absorb light around the 490nm wavelength.
A gel shift assay was performed to confirm covalent linkage between Csga-Spytag and mRFP-Spycatcher. After 24 hours of incubation of mRFP-Spycatcher with CsgA-Spytag, a noticeable population of mRFP spycatcher shifted upwards to ~67kDa indicating a conjugation event.
Design Considerations
Included between the red chromoprotein and the SpyCatcher sequence is a short amino-acid linker sequence. This linker ensures that the SpyCatcher domain will be fully available to react with other SpyTag domains in the solution, and will be less likely to be unavailable to due the specific geometry of a direct chromoprotein-SpyCatcher fusion protein that lacks a linker sequence.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 555
Illegal AgeI site found at 667 - 1000COMPATIBLE WITH RFC[1000]
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