Team:Wageningen UR/Notebook/toggleswitch
Week 1 (2nd-7th June 2016)
Some of important biobricks for constructing toggle switch were transformed for the iGEM 2016 kits, including repressors BBa_P0440, BBa_P0412, inducible promoters BBa_R0040, BBa_R0010 and reporter genes BBa_K515005, BBa_K1365020. Breed conversation of each biobrick had been made.
Week 2 (29th- 30th June , 2016)
Purified BBa_K1491009 plasmid (132 ng/μL) from breed conservation.
Week 3 (4th-8th July, 2016)
Firstly, purified plasmid of BBa_P0440, BBa_P0412, BBa_R0040, BBa_R0010 (60.7 ng/μL, 50.7 ng/μL, 66.3ng/μL, 26.3ng/μL), secondly, transformed parts from 2016 kits including, BBa_C0040, BBa_C0012, BBa_B0015, and BBa_K1365020. Picked up colony from the plates, cultured and purified plasmid of each part (207.5 ng/μL, 140.6 ng/μL, 96.1 ng/μL, 146.3 ng/μL). Last, tried to use PCR to amplify iGEM vectors of each resistance by using the linearized backbones as template, but it failed.
Week 3 (4th-8th July, 2016)
Firstly, purified plasmid of BBa_P0440, BBa_P0412, BBa_R0040, BBa_R0010 (60.7 ng/μL, 50.7 ng/μL, 66.3 ng/μL, 26.3 ng/μL), secondly, transformed parts from 2016 kits including, BBa_C0040, BBa_C0012, BBa_B0015, and BBa_K1365020. Picked up colony from the plates, cultured and purified plasmid of each part (207.5 ng/μL, 140.6 ng/μL, 96.1 ng/μL, 146.3 ng/μL). Last, tried to use PCR to amplify iGEM vectors of each resistance by using the linearized backbones as template, but it failed.
Week 4 (11th-16th July, 2016)
Firstly, I tried to make vectors of each resistance again. This time, I transformed 6 kind of backbones that contains RFP generator from 2016 kits and picked up colony from the plates, cultured and purified plasmid of each part. Then digested them with EcroRI and PstI overnight and did gel purification to recycle backbones fragments. Final concentration of each vector (pSB1A3 17.2 ng/μL, pSB1A5 21 ng/μL, pSB1C3 18.3 ng/μL, pSB1C5 45 ng/μL, pSB1K3 35.9 ng/μL, pSB1LK5 12.6 ng/μL). I got 4 parts from iGEM part registry BBa_K215108, BBa_K215107, BBa_K554007, BBa_K554102. I did plate streaking and selected signal colony from the plate, then purified plasmid of each part (BBa_K215108 46.3 ng/μL, BBa_K215107 51.5 ng/μL). (Fig.1)
