Part:BBa_K4161301
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
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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