Difference between revisions of "Part:BBa K2201220"

Latest revision as of 17:20, 25 October 2017

CDS of GFP-streptavidin fusion protein with medium gly-gly-ser linker

This fusion protein containing GFP and streptavidin connected by a Gly-Gly-Ser-linker (Figure 1) is used to show the properties and uses of the ncAA 2-NPA. The streptavidin of this fusion protein is able to bind 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. This version of the fusion protein is needed to validate the binding and detection properties without the incorporation of the ncAA 2-NPA compared to the part K2201221. Experiments with this protein can deliver the reference values of binding efficiency and fluorescence signal undisturbed by the ncAA. When compared to the values of the amber variant, it can be determined if there is any loss or change in the binding or in the fluorescence properties caused by the 2-NPA or the synthetase.

Figure 1: Plasmid map of the BioBrick K2201220 coding for a fusion protein containing GFP (extracted from E0040) a linker, and a streptavidin tag (extracted from J36848).

It was constructed through the combination of the GFP part E0040 and the part J36848 encoding a streptavidin. Both protein coding sequences were extracted with primers and combined by means of Gibson assembly, as both parts had the linker-sequence (GFP at 3’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 K2201220.

Important: This part only contains the coding sequence and will not be expressed if transformed. For any further results look up to BBa_K2201320.

Sequence and Features