Difference between revisions of "Part:BBa K4229022"

Revision as of 09:31, 5 October 2022

sfGFP with AMBER stop codon at the position of the 8th amino acid

The sequence for sfGFP (PDB: 2B3P) with an AMBER stop codon at the position of the 8th amino acid

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 20 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 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 (DE3) were transformed with a plasmid containing the pBpf synthetase, pEVOL-pBpF (Addgene 31190), and one plasmid with the sfGFP mutant, pBAD33_sfGFP F8 (BBa...?).

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 additional 2.5 h.

Samples for the flow cytometry measurement were prepared by mixing 10 µL of the liquid bacterial cultures with 990 µL DPBS. The flourecent signal of those samples was analyzed with the CyAn ADP Analyzer from Beckman Coulter.

E.coli BL21 (DE3) were co-transformed with a plasmid containing the pBpf synthetase, pEVOL-pBpF (Addgene 31190), and one plasmid with the sfGFP F8 mutant, pTrc99a_sfGFP F8 (BBa...?).

A single colony from each co-transformation was grown overnight at 37°C in LB medium supplemented with ampicillin and chloramphenicol. Overnight cultures were diluted and grown to OD600 of 0.4 before induction. Samples were induced with 1 mM arabinose and XX mM unnatural amino acid, p-BpF (unless no unnatural amino acid is indicated). One hour after arabinose induction the samples were induced with 200 µM IPTG and growth was continued for additional 2.5 h at 37°C

GFP fluorescence was measured by flow cytometry with the CyAn ADP Analyzer from Beckman Coulter. Therefore, samples were prepared by mixing 10 µL of the liquid bacterial cultures with 990 µL DPBS.

The incorporation of unnatural amino acid was measured by comparing the fluorescence of E. coli cells co-transformed with pBpf RS/pTrc99a-sfGFP F8 to that of wild type sfGFP (pBpf RS/pTrc99a-sfGFPWT). (and normalized to culture density?)

Figure text: TThe Median Fluorescence Intensity (MFI) of bacterial samples at different conditions were measured by flow cytometry. The fluorescent GFP signal serves as a consequence of unnatural amino acid, p-BpF incorporation. E.coli BL21 (DE3) were co-transformed with pBpf RS/pTrc99a-sfGFP F8 . Cells were induced in the presence (+) or in the absence (−) of XX mM pBpF amino acid. Data are means ± SEM of triplicate samples from two experiment determinations.

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 F8. Compared to the control without an inserted amber stop codon, the mutant still exhibits less fluorescence intensity.

Sequence and Features