Difference between revisions of "Part:BBa K1955007"

Since we can’t detect the HA and OVA protein by western blotting after the pSB1C3-5’HYG-HA-3’UTR and pSB1C3-5’HYG-OVA-3’UTR plasmid were transfected into leishmania. We decided to construct pSB1C3-5’HYG-GFP-3’UTR in order to prove if our leishmania shuttle vector could express the second protein or not. The GFP sequence came from BBa_E0040 in the vector pSB1A2.

The pSB1C3-3’UTR was digested with EcoRI and XbaI, The pSB1A2-GFP and pSB1C3-5’HYG were digested with EcoRI and SpeI. After the purification, the pSB1C3-3’UTR was ligated with GFP and 5’HYG successively, then transformed into DH5a. The colonies were checked by colony PCR. The right length of GFP-3’UTR should be approximately 1.5 kb (720 bp +774 bp), while the 5’HYG-GFP-3’UTR should be about 3 kb (1446 bp +720 bp +774 bp). As the result, we knew that all the colonies contained the correct plasmid after the construction. The right colony of pSB1C3-5’HYG-GFP-3’UTR was picked and amplified in 200 ml LB broth, then the plasmid DNA was purified by midiprep.

pSB1C3-GFP-3’UTR and pSB1C3-5’HYG-GFP-3’UTR check by colony PCR

The PCR was performed with Taq polymerase, and screened in 0.8％ agarose gel by electrophoresis. The GFP-3’UTR was about 1.5 kb in length, and the 5’HYG-GFP-3’UTR was about 3 kb.