Part:BBa_K4229021
sfGFP AMBER stop codon at the position of the 4th amino acid
Superfolder GFP (sfGFP) with an amber stop codon mutation at position 8, replacing phenylalanine
Short Description:
The F8 variant of sfGFP was created using site-directed mutagenesis, aiming to insert the amber stop codon at position 8. The sfGFP can only be completely synthesized upon successful amber stop codon suppression. Hence, sfGFP F8 can be used as a model to test the efficiency of incorporation of non-canonical amino acids via amber stop codon suppression technology.
Usage:
Besides the well-known 21 canonical amino acids, there is a variety of non-canonical amino acids which can be used to further modify proteins. One way of incorporating non-canonical amino acids is via the amber stop codon suppression technology. This part, sfGFP F8, is a model protein to test non-canonical amino acid incorporation. Without successful incorporation, a non-functional version of sfGFP will be translated, and therefore not give a fluorescence signal. However, after successful incorporation of the non-canonical amino acid, fluorescence will be restored providing an immediate readout of the efficiency of amber stop codon suppression via orthogonal translation systems.
Characterization:
The incorporation of non-canonical amino acids into sfGFP F8 was characterized using flow cytometry. As an orthogonal translation system, an additional aminoacyl-tRNA synthetase and its corresponding tRNA are used, which in this case correspond to the pBpf synthetase, which incorporates 4-Benzoyl-l-phenylalanine.
First E.coli BL21 were transformed with a plasmid containing the pBpf synthetase, pEVOL-pBpF (Addgene 31190), and one plasmid with the sfGFP mutant, pBAD33_sfGFP F8.
The cells were grown in LB medium at 37°C. At an OD600 of 0.4, the samples were induced with 1 mM arabinose and then further incubated at 37°C. One hour after arabinose induction the samples were induced with 200 µM IPTG and, again, incubated for 2.5 h.
Samples for the flow cytometry measurement were prepared by mixing 10 µL of the liquid bacterial cultures with 990 µL DPBS. These samples were analyzed with the CyAn ADP Analyzer from Beckman Coulter.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 421
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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