Composite
Single Dom

Part:BBa_K4161900:Design

Designed by: Yuxin Wang   Group: iGEM22_DKU   (2022-10-13)
Revision as of 16:31, 13 October 2022 by Yy1130 (Talk | contribs) (added the map)

No part name specified with partinfo tag.

No part name specified with partinfo tag.


Design Notes

One of the most important proteins in type III secretion system (T3SS) of pathogenic Shigella called ipaD is our attack target8. Because ipaD is the primary trigger to form translocon pores within the host cell membrane, the inhibition of it would probably inhibit the invasion of Shigella and thus treat the infection9,10. Several single domain antibodies (nanobodies) have been previously reported to target ipaD, the most efficient of which is 20ipaD11. Therefore, 20ipaD was chosen for further engineering. To have the yeasts displayed 20ipaD on surface, we adopted the strategy of Aga2 yeast surface display system. Aga2 is a membrane protein of S. cerevisiae, which would be sealed on membrane by the interaction with Aga16. By fusing the Aga2 protein with 20ipaD, the nanobody could thus be brought to the membrane, and that was what we designed as shown in the figure. Additionally, we introduced 3xFLAG tag behind 20ipaD for expression assays like immunofluorescent microscopic imaging, western blot, and FACS. The whole expression module was designed under the control of GAL1 promotor that can be induced by galactose to control the expression of the proteins of interest. We used pYD1 vector as the backbone which contains ampicillin resistance marker, E. coli replication origin, and tryptophan deficiency marker. These features enable us to store the recombinant plasmids in E. coli, and easily select the yeasts after transformation.