Difference between revisions of "Part:BBa K2201321"

Revision as of 14:10, 25 October 2017

functional 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 containing an amber codon is used to show the properties and usages of the ncAA 2-NPA. 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. After the radiation, the GFP can be removed by simply washing it off the surface, so that the following loss of fluorescence shows the usage of this ncAA.

To get the fusion proteins encoded in the parts K2201220 and K2201221, a BioBrick assembly with a suitable promoter is needed to achieve sufficient protein expression (e.g. T7 promoter and RBS of K525998). This alone worked for the fusion protein without the amber codon of the part K2201320. To get the fusion protein containing the 2-NPA (K2201321) a co-transformation of the fusion protein and the RS-Part needed.

After the assembly of K525998 and K2201321 (Figure 1) and transformation in the E.coli strain BL21(DE3), the fusion protein is partially functional.

Figure 1: Construction history of K2201321.

The GFP will be expressed after treatment with IPTG, but the amber codon in the linker will be read as stop codon so that the fusion protein will deliver a fluorescence signal but no binding properties due to the absence of the streptavidin part.

Figure 2: Plasmid map of K2201321 encoding a fusion protein consisting of GFP (extracted from E0040), a linker containing an amber codon, and a streptavidin tag (extracted from J36848). This part was placed under the control of a T7 promoter and RBS (K525998).

Through the basic expression of the GFP-unit of the fusion protein or by picking and streaking out on LB-plates with IPTG, the positive colonies can be easily detected by their fluorescence (Figure 3A). If cultivated at 37°C over night and IPTG-induction on an OD600 of 0,6-0,8, the culture will show a bright fluorescence (Figure 3B).

Figure 3: A) LB-plate with 0.5 mM IPTG with control GFP-culture and five positive clones (P) and twenty-five negative clones after transformation and streaking. B) Culture of one of the positive clones in a 500 mL shaker on a blue light LED-Transilluminator.

To proof that only the GFP-unit of the fusion protein is expressed and not the whole fusion protein, a western blot with GFP-antibodies was performed (Figure 4). The GFP with a part of the linker has a calculated mass of 27,2 kDa and the whole fusion protein would have a mass of 40,9 kDa. The blot only shows a band near the 25,0 kDa marker and so proofs the functionality of the amber-codon.

Figure 4: Western blot of the expressed partial fusion protein of K2201321. The only band at approximately 25,0 kDa shows the expression GFP-unit of the fusion protein.

Unfortunately, we had some issues with proofing the binding activity to biotinylated surfaces. We recommend to replace the streptavidin with another tag or express the fusion protein in a biotin-deficient strain.

Sequence and Features