Difference between revisions of "Part:BBa K2201320"

Latest revision as of 21:53, 25 October 2017

functional GFP-streptavidin fusion protein with medium gly-gly-ser linker and T7-promoter and RBS

This fusion protein containing GFP and streptavidin connected by a Gly-Gly-Ser-linker is used to show the properties and usage of the non-canonical amino acid 2-nitrophenylalanine. 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.

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 K2201320 (Figure 1) and the transformation into E.coli strain BL21(DE3), the fusion protein is functional.

Figure 1: History of the plasmid construction of K2201320.

The protein will be expressed in high amount if induced with 0.5 mM IPTG. For that, the strain BL21(DE3) has a IPTG-inducible T7-Polymerases. The strain will also produce a small amount of the fusion protein without induction.

Figure 2: Plasmid map of K2201320 coding for a fusion protein containing GFP (extracted from E0040), a linker, and a streptavidin tag (extracted from J36848), assembled with a T7 promoter and RBS (K525998)

To proof the fluorescence property of the Biobrick, we compared the fluorescence of two cultures of E.coli BL21(DE3) transformed with K2201320. Both were cultures of 5 mL LB Media with 1.25 µL of 100 mg/mL chloramphenicol and inoculated with 10 µL preculture. After six hours at 37 °C, one was treated with IPTG to induce the synthesis of the T7 polymerases and thus the expression of the fusion protein. Both were incubated over night at 37 °C for 12 hours. In a plate reader, the OD600 and the fluorescence (excitation: 485 nm, emission: 520 nm) of seven technical replicates of pure LB-media and twenty technical replications of both cultures were measured. The absolute fluorescence of the induced culture was 4-times higher than the absolute fluorescence of the culture not induced with IPTG. After subtraction of the fluorescence signal of the LB-media, the induced cultures corrected fluorescence value was 20-times higher, although the OD600 was 0.1 lower than from the other culture. When the corrected fluorescence signal was normalized to the absorbance at 600 nm, we got the relative fluorescence per OD600, which was 30-timer higher in the induced culture.

Figure 3: Comparison of the fluorescence signal per OD600 of E. coli BL21(DE3) culture containing K2201320 with and without treatment with IPTG and incubation for 12 hours at 37 °C to induce the expression of the fusion protein.

The fluorescence property of this part and the functionality of the IPTG-induced production of the fusion protein were demonstrated. Because of the basic activity of the T7-promoter, as shown in Figure 3, the culture will always express some amount of the protein. This allows identifying positive colonies when streaked out because of their fluorescence (Figure 4). Also, a IPTG-induced culture with this Biobrick will have a bright fluorescence when cultivated for 8 hours at 37 °C or 24 hours at 18 °C. There was no visible difference in brightness between both cultivation conditions.

Figure 4: Visible fluorescence of streaked out positive clones (P) and mRFP-colony on LB-Agar and in a 100 mL culture 8 hours after induction with IPTG and incubation at 37 °C in LB-media.

To determine if the whole fusion protein is expressed, and not only the GFP part, a western blot with GFP-antibodies was performed on a lysate of the cultivated cells transformed with K2201321 (Figure 5). The blot shows a band at approximately 40 kDa, which correlates with the expected size of the whole fusion protein. GFP alone has a mass of approximately 27 kDa.

Figure 5: Western blot with GFP-antibodies of the expressed fusion protein of K2201321 with a mass of 40.9 kDa.

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