Part:BBa_K4161900:Design
Single Domain Antibody 20ipaD Conjugated Aga2 Expression Module
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 857
Illegal NotI site found at 961 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 920
Illegal XhoI site found at 968 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 70
Illegal AgeI site found at 1559 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1541
Design Notes
One of the most important proteins in the 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 the 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 a 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 the GAL1 promoter that can be induced by galactose to control the expression of the proteins of interest. We used the pYD1 vector as the backbone which contains an 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.