Difference between revisions of "Part:BBa K4161301"
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===Usage and Biology=== | ===Usage and Biology=== | ||
| + | The yeasts that were transformed with pYD1-ipaD, pYD1-20ipaD, and pYD1 were amplified in SDCAA liquid medium for 36h. The cells were harvested by centrifuge at 5000xg for 5 minutes and diluted in SGCAA liquid containing the induction agent galactose to obtain an OD600=0.5. Four milliliters of each diluted cells were transferred into new culture tubes and induced at 21°C for 24h. An additional prep of EBY100 cells without plasmids were also diluted to OD600=0.5 and induced in SGCAA for 24h. | ||
| + | The induced cells were harvested and washed with PBS. The yeasts containing pYD1-ipaD were inoculated with rabbit anti-HA tag antibodies, while the yeasts containing pYD1-20ipaD, pYD1, or no plasmids were inoculated with rabbit anti-FLAG tag antibodies and BSA. After washed with PBS, the cells were inoculated with FITC-conjugated goat anti-rabbit immunoglobin antibodies and BSA without light. The cells were washed twice with PBS before imaged under UV-light. As Fig5 shows, the yeasts containing pYD1-ipaD and pYD1-20ipaD emitted green fluorescence, whereas the yeasts containing empty plasmids, or no plasmids did not. This fluorescence pattern proved the yeasts indeed expressed and displayed the nanobody and the antigen on their surfaces, which announced the success of engineering. | ||
| + | https://static.igem.wiki/teams/4161/wiki/y-fig5.png | ||
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===Design details=== | ===Design details=== | ||
| + | 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. | ||
| + | |||
The yeasts that were transformed with pYD1-ipaD, pYD1-20ipaD, and pYD1 were amplified in SDCAA liquid medium for 36h. The cells were harvested by centrifuge at 5000xg for 5 minutes and diluted in SGCAA liquid containing the induction agent galactose to obtain an OD600=0.5. Four milliliters of each diluted cells were transferred into new culture tubes and induced at 21°C for 24h. An additional prep of EBY100 cells without plasmids were also diluted to OD600=0.5 and induced in SGCAA for 24h. | The yeasts that were transformed with pYD1-ipaD, pYD1-20ipaD, and pYD1 were amplified in SDCAA liquid medium for 36h. The cells were harvested by centrifuge at 5000xg for 5 minutes and diluted in SGCAA liquid containing the induction agent galactose to obtain an OD600=0.5. Four milliliters of each diluted cells were transferred into new culture tubes and induced at 21°C for 24h. An additional prep of EBY100 cells without plasmids were also diluted to OD600=0.5 and induced in SGCAA for 24h. | ||
The induced cells were harvested and washed with PBS. The yeasts containing pYD1-ipaD were inoculated with rabbit anti-HA tag antibodies, while the yeasts containing pYD1-20ipaD, pYD1, or no plasmids were inoculated with rabbit anti-FLAG tag antibodies and BSA. After washed with PBS, the cells were inoculated with FITC-conjugated goat anti-rabbit immunoglobin antibodies and BSA without light. The cells were washed twice with PBS before imaged under UV-light. As Fig5 shows, the yeasts containing pYD1-ipaD and pYD1-20ipaD emitted green fluorescence, whereas the yeasts containing empty plasmids, or no plasmids did not. This fluorescence pattern proved the yeasts indeed expressed and displayed the nanobody and the antigen on their surfaces, which announced the success of engineering. | The induced cells were harvested and washed with PBS. The yeasts containing pYD1-ipaD were inoculated with rabbit anti-HA tag antibodies, while the yeasts containing pYD1-20ipaD, pYD1, or no plasmids were inoculated with rabbit anti-FLAG tag antibodies and BSA. After washed with PBS, the cells were inoculated with FITC-conjugated goat anti-rabbit immunoglobin antibodies and BSA without light. The cells were washed twice with PBS before imaged under UV-light. As Fig5 shows, the yeasts containing pYD1-ipaD and pYD1-20ipaD emitted green fluorescence, whereas the yeasts containing empty plasmids, or no plasmids did not. This fluorescence pattern proved the yeasts indeed expressed and displayed the nanobody and the antigen on their surfaces, which announced the success of engineering. | ||
| + | https://static.igem.wiki/teams/4161/wiki/y-fig5.png | ||
Latest revision as of 15:55, 12 October 2022
pyD1 plasmid
adkfhasdslhakfhsalk
Sequence and Features
- 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
Source
https://kdw-lab.mit.edu/reagents/ Wittrup Lab at MIT
Design details
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.
The yeasts that were transformed with pYD1-ipaD, pYD1-20ipaD, and pYD1 were amplified in SDCAA liquid medium for 36h. The cells were harvested by centrifuge at 5000xg for 5 minutes and diluted in SGCAA liquid containing the induction agent galactose to obtain an OD600=0.5. Four milliliters of each diluted cells were transferred into new culture tubes and induced at 21°C for 24h. An additional prep of EBY100 cells without plasmids were also diluted to OD600=0.5 and induced in SGCAA for 24h.
The induced cells were harvested and washed with PBS. The yeasts containing pYD1-ipaD were inoculated with rabbit anti-HA tag antibodies, while the yeasts containing pYD1-20ipaD, pYD1, or no plasmids were inoculated with rabbit anti-FLAG tag antibodies and BSA. After washed with PBS, the cells were inoculated with FITC-conjugated goat anti-rabbit immunoglobin antibodies and BSA without light. The cells were washed twice with PBS before imaged under UV-light. As Fig5 shows, the yeasts containing pYD1-ipaD and pYD1-20ipaD emitted green fluorescence, whereas the yeasts containing empty plasmids, or no plasmids did not. This fluorescence pattern proved the yeasts indeed expressed and displayed the nanobody and the antigen on their surfaces, which announced the success of engineering.
