Difference between revisions of "Part:BBa K1955002"

The biobrick parts, including 5’HYG, 3’UTR, HA and OVA, were synthesized directly by IDT. After receiving the synthesized parts, we used EcoRI and PstI to digest the parts and pSB1C3 backbone, then ligated and transformed the DNA samples into DH5a competent cells. According to the digestion and colony PCR results of the colony, all the parts were inserted into the pSB1C3 vector with the right length of DNA sequences, 5’HYG is 1446 bp, HA is 1700 bp, OVA is 2098 bp and 3’UTR is 774 bp.


(Fig. 1) pSB1C3-3’UTR, pSB1C3-5’HYG, pSB1C3-OVA checked by colony PCR and enzyme digestion

(A),(C) The pSB1C3-3’UTR and pSB1C3-OVA were transformed and the colonies were picked to perform colony PCR. The forward primer sequence was 5’- GAATTCGCGGCCGCTTCTAGAG-3’, which was in the prefix site. And the reverse primer sequence was 5’-CTGCAGCGGCCGCTACTAGTA-3’, which was in the suffix site. The PCR reaction was performed with Taq polymerase, and screened in 0.8％ agarose gel by electrophoresis. As the results, a 700~800 bp sequence was proliferated in pSB1C3-3’UTR, and a 2000~2500 bp sequence was proliferated from pSB1C3-OVA. (B) The pSB1C3-5’HYG was transformed and the colonies were picked and amplified in LB broth. pSB1C3-5’HYG plasmid was purified by miniprep, and digested with EcoRI and PstI for 4 hrs, then screened in 0.8％ agarose gel by electrophoresis. The results showed a 2000 bp band of pSB1C3 and the 1500 bp 5’HYG.

The pSB1C3-3’UTR was digested with EcoRI and XbaI, then the pSB1C3-HA and pSB1C3-OVA were digested with EcoRI and SpeI. After the purifying step, the pSB1C3-3’UTR was ligated with HA and OVA, then transformed after 16℃ overnight. The colony were checked with colony PCR, as the results, the HA-3’UTR would be about 2.6 kb (1774 bp +774 bp), and the OVA-3’UTR would be about 2.9 kb (2098 bp +774 bp).



pSB1C3-HA-3’UTR and pSB1C3-OVA-3’UTR checked by colony PCR

The pSB1C3-HA-3’UTR and pSB1C3-OVA-3’UTR were transformed and the colonies were picked to perform colony PCR. The PCR reaction was performed with Taq polymerase, and screened in 0.8％ agarose gel by electrophoresis. The 2600 bp HA-3’ UTR and 2900 bp OVA-3’UTR were proliferated from pSB1C3-HA-3’UTR and pSB1C3-OVA-3’UTR.

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.