Team:CSL Pittsburgh/Notebook

Notebook

Protocols

• Part BBa_K112624 in psb1c3 was transformed into DH5 competent cells using pitts protocol called piGEM plasmids.



• Colony pcr of piGEM was performed to make sure insert was present.



• Digestion of IDT oligos and linearized psb1c3 plasmid was done and ligated overnight. It was transformed with pitts transformation protocol.



• Colony PCR was performed for the pCYP6AY1 was performed to make sure insert was present.



• pCYP6AY1 and piGEM were plasmid prepped to obtain purified plasmid. IBI kit.



• Plasmids were digested with BamHI and NdeI. No band was observed for piGEM that we were going to insert into pCYP6AY1.



• Single digests of piGEM were done with BamHI and NdeI to see if they digested. BamHI did not digest the plasmid.



• Returned to our transformants from step 3 for the other oligo inserted into pSB1C3. Resulted in pBee plasmid.



• Colony PCR on pBee colony transformants to see if any contained insert.



• Used the positive culture EpBee and WT dh5 alpha in imidacloprid to see if there was a significant difference in their growth.



• Used WT and EpBee cultures to extract his tagged proteins in the supernatant and quantified with nanodrop.



• Fed the WT and EpBee cultures to bees and tested their survival with imidacloprid treatment.