Part:BBa_K4202006
This part is a fusion protein of Hag and SpyTag , and it`s used for the bio-scafford.
In this protein, SpyTag is inserted at a specific site for presentation of the epitope on the flagellar surface after assembly. In addition, we replaced amino acid at 222 with Cys ,which can be reacted with maleimide. In our experiments , we will use the SpyCatcher-mRFP , maleimide and other fluorescent dye to characterize this protein by fluorescence fluorescence microscope.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 568
Result
We tranformed the plasmid PHY-P43-HagT209C::SpyTag588-DT into Bacillus subtillis WB600 strain and inoculated into tetracycline resistant LB medium for overnight . Then we cultured the Bacillus subtillis WB600 containing PHY-HagT209C::SpyTag588 and PHY-EutM-SpyCatcher(BBa_K4202015) in the tetracycline resistance SMM medium. After culturing 36h, we added the 0.2 mg purified SpyCatcher-mRFP per 5 ml medium into the medium and cultured the bacteria for 12h.Besides, we settled WB600 group and calcium carbonate group for control. Then we utilized the confocal laser scanning microscope (FLUOVIEW FV3000) to detect the cultures treated by SYTO.
We can clearly observe the green fluorescence at 507 nm and red fluorescence at 610nm, while the group for control showed negative outcome. So we could conclude that the biological scafforld can be assembled reasonably.
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