Part:BBa_K4161301:Experience

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Applications of BBa_K4161301

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.

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