Difference between revisions of "Part:BBa K3098015"

Latest revision as of 03:22, 22 October 2019

sfGFP+Avi-tag+His-tag

iGEM19_WHU-China added Avi-tag to C-terminal of sfGFP to confirm the function of BirA enzyme(BBa_K2485001). sfGFP is codon optimized from BBa_I746916. Avi-tag can perform normal function whether added to C-terminal or N-terminal of the peptide.Avi-tag can be specifically recognized by BirA enzyme in vitro/vivo to attach a biotin. Furthermore, this part can be used to produce biotinylated sfGFP for its purification and other signal detection/amplification possibilities in future versatile platform.

We made a comparison of fluorescence intensity between BBa_K3098015 and BBa_I746916.

Figure 1 Visible difference between BBa_I746916 (left in the picture) and BBa_K3098015 (right in the picture).

We constructed a recombined plasmid composed of pR (BBa_R0051)+RBS (BBa_B0030)+sfGFP (BBa_I746916)+6xHis-tag to express the sfGFP (BBa_I746916). By the way, there was no repressor CI of pR in the expression system.

We also constructed a recombined pet28a plasmid composed of T7 promoter and sfGFP BBa_K3098015 to express the sfGFP with avi-tag.

After purification by Ni resin, we made it a series of gradient dilutions of BBa_K3098015 and BBa_I746916 respectively. Then we measured the sfGFP protein concentration by Bradford method and the fluorescence intensity under 475nm as emission wavelength and 545nm as excitation wavelength.

Then we fit data into a straight line: y=kx+b. Due to the fluorescence intensity of blank group without any sfGFP is 113 A.U., the ordinate at the origin should be 113. So the b should be 113. It is easy to understand that the higher the value of k is, the stronger the sfGFP fluorescence is.

Therefore, we draw following conclusions:
(1) sfGFP (BBa_I746916) is available in constructs driven by the pR(BBa_R0051);
(2)According to the k of relationship(y=kx+113) between protein concentration and fluorescence intensity through our measurements, the fluorescence of sfGFP-Avitag (BBa_K3098015) is stronger than sfGFP (BBa_I746916).

Sequence and Features