Part:BBa_K3484000:Design
Intein-mediated T3(THRB) → sfGFP
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 174
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 661
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 217
Illegal SapI.rc site found at 1576
Design Notes
The Fh8 solubility tag is added to the N-terminal of the construct to avoid the formation of inclusion bodies when translating the sequence [1].
The FLAGx3 tag was added at the C-terminal of the construct for immunodetection purposes.
Superfolder GFP is split between 70th-71st residues to allow the C-terminal of the intein to be followed by a Cys residue and perform an optimal splicing [2].
From the Mtu RecA endonuclease PI-MtuI only the first 110 and the last 167 aminoacids were kept, substituting the middle by the thyroid hormone receptor β1 ligand-binding domain and the extremes by the split superfolder GFP [3].
The V67L post-translational sequence mutation on the endonuclease PI-MtuI was performed to enhance the functionality of the mini-intein, as stated by Wood et al. [4].
Once the whole part was constructed, the sequence was codon-optimized to have a better expression in E. coli bacteria.
Source
The ligand-binding domain of the THR-β; comes from the human thyroid receptor β-1. Sequence available at the supplementary material of Gierach et al. [3].
Superfolder GFP is a green fluorescent protein derived from Aequorea victoria, with the codon sequence available at the FPBase.
The intein splicing domains come from the Endonuclease PI-MtuI region of the Mycobacterium tuberculosis (Mtu) RecA protein, codon sequence available at Uniprot.
The Fh8 tag is available at Uniprot.
The FLAGx3 tag is available at the iGEM registry BBa_K823034.
References
[1] Costa, S., Almeida, A., Castro, A., & Domingues, L. (2014). Fusion tags for protein solubility, purification and immunogenicity in Escherichia coli: the novel Fh8 system. Frontiers in Microbiology, 5. DOI:10.3389/fmicb.2014.00063
[2] Tornabene, P., Trapani, I., Minopoli, R., Centrulo, M., Lupo, M., de Simone, S., Auricchio, A., et Al. (2019). Intein-mediated protein trans-splicing expands adeno-associated virus transfer capacity in the retina. Science Translational Medicine, 11(492), eaav4523. DOI:10.1126/scitranslmed.aav4523
[3] Gierach, I., Li, J., Wu, W.-Y., Grover, G. J., & Wood, D. W. (2012). Bacterial biosensors for screening isoform-selective ligands for human thyroid receptors α-1 and β-1. FEBS Open Bio, 2(1), 247–253. DOI:10.1016/j.fob.2012.08.002
[4] Wood, D. W., Wu, W., Belfort, G., Derbyshire, V., & Belfort, M. (1999). A genetic system yields self-cleaving inteins for bioseparations. Nature Biotechnology, 17(9), 889–892. DOI:10.1038/12879