Difference between revisions of "Part:BBa K4229021"

Revision as of 12:22, 4 October 2022

sfGFP AMBER stop codon at the position of the 4th amino acid

Superfolder GFP (sfGFP) with an amber stop codon mutation at position 4, replacing phenylalanine

Short Description:

The G4 variant of sfGFP was created using site-directed mutagenesis, aiming to insert the amber stop codon at position 4. The sfGFP can only be completely synthesized upon successful amber stop codon suppression. Hence, sfGFP G4 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 G4, 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 G4 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 G4.

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.

Figure text: The median fluorescence of BL21 samples under different conditions is shown. On the left, the combinations of plasmids and the presence of the non-canonical amino acid pBpf is indicated. Each sample contains two plasmids: A pBAD33 backbone containing the pBpf synthetase (pBpf RS) and a pTrc99a backbone with sfGFP and its mutant.

The figure shows that fluorescence of the sfGFP mutant only occurs if the non-canonical amino acid and the corresponding synthetase are present. This suggests that the full sfGFP can only be synthesized upon the incorporation of pBpf into sfGFP G4. Compared to the control without an inserted amber stop codon, the mutant still exhibits less fluorescence intensity.

Sequence and Features BBa_K4229021