Part:BBa_K4229023

sfGFP with AMBER stop codon at the position of the 74th amino acid

The sequence for sfGFP (PDB: 2B3P) with an AMBER stop codon at the position of the 74th amino acid replacing tyrosine

Short Description: 

The Y74 variant of sfGFP was created using site-directed mutagenesis, aiming to insert the amber stop codon at position 74. The sfGFP can only be completely synthesized upon successful amber stop codon suppression. Hence, sfGFP Y74 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 Y74, is a model protein to test non-canonical amino acid incorporation. Without successful incorporation, a truncated and 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 Y74 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-Tyrnylalanine.  

E.coli BL21 (DE3) were co-transformed with a plasmid containing the pBpf synthetase, pEVOL-pBpF (Addgene 31190), and one plasmid with the sfGFP Y74 mutant, pTrc99a_sfGFP Y74. 

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 0.2 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 synthetase/pTrc99a-sfGFP Y74 to that of wild type sfGFP (pBpf RS/pTrc99a-sfGFP WT). 

Figure text:The 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 Y74 . Cells were induced in the presence (+) or in the absence (−) of 0.2 mM mM pBpF amino acid. Data are means ± STD of duplicates of samples from two experiment determinations.

As shown by a fluorescent signal, unnatural amino acid was successfully incorporated at Tyr74 of sfGFP only at the presence of p-BpF and the corresponding synthetase. In the absence of p-BpF or the synthetase no fluorescence signal was detected. 

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 421
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]