Part:BBa_K2201221

CDS of GFP-streptavidin fusion protein with medium gly-gly-ser linker containing an amber-codon

This fusion protein containing GFP and streptavidin connected by a Gly-Gly-Ser-linker (Figure 1) containing an amber codon is used to show the properties and usages of the non-canonical amino acid 2-nirophenylalanine (incorporated by BBa_K2201200). The streptavidin of this fusion protein can bind to biotin, such that the protein could be immobilized on any biotinylated surface. The GFP is fluorescent, such that the binding of the protein can easily be detected. The 2-NPA in the linker induces a cleavage of the peptide backbone when radiated with light at ʎ = 365 nm.

Figure 1: Plasmid map of the BioBrick K2201221 coding for a fusion protein containing GFP (extracted from E0040), a linker with an amber codon, and a streptavidin tag (extracted from J36848).

It was constructed by combining the GFP-part E0040 and the part J36848 containing streptavidin. Both protein coding sequences were extracted with primers and combined using Gibson assembly, as both parts had the linker-sequence (GFP at 2’side and streptavidin at the 5’side) as overlap. For this reason, the stop-codon at the 3’-end of the GFP-Protein was removed and replaced by the sequence of the linker. At the streptavidin protein, the start-codon at the 5’-end was replaced by the linker sequence and a His(6)-tag was removed at the 3’-end and replaced by a stop-codon. The two fragments were then assembled with a linearized pSB1C3 vector (Figure 2).

Figure 2: History of the plasmid construction of K2201221.

Important: This part contains only the coding sequence of a fusion protein of GFP and streptavidin with an amber-codon and will not be expressed if transformed. It will only be fully expressed when assembled with a matching promotor and RBS like in part K2201321 and co-transformed with an ncAA tRNA synthetase like in part K2201200. The GFP unit will be expressed only when assembled with a matching promotor and RBS like in part K2201321 without cotransformation.

Sequence and Features

Assembly Compatibility:

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

  Illegal BamHI site found at 787
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 829
  Illegal AgeI site found at 880
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 644