Coding
Part:BBa_K1955000
Designed by: Justine Hsu Group: iGEM16_CGU_Taiwan (2016-10-14)
pSB1C3-Hemagglutinin
The coding sequence of Hemagglutinin (HA) from H1N1 acquired from NCBI. HA is a glycoprotein found on the surface of influenza viruses and can be used in immunological experiments as the target antigen.
(1) Insert 5’HYG, 3’UTR, HA and OVA gBlocks into pSB1C3 vector:
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.
(Fig. 2) The basic part checked by PCR
We used pSB1C3-5’HYG, pSB1C3-3’UTR, pSB1C3-HA, pSB1C3-OVA as template, to check the length of the inserts. The PCR reaction was performed with Taq polymerase, and screened in 0.8％ agarose gel by electrophoresis.
(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.
(Fig. 2) The basic part checked by PCR
We used pSB1C3-5’HYG, pSB1C3-3’UTR, pSB1C3-HA, pSB1C3-OVA as template, to check the length of the inserts. The PCR reaction was performed with Taq polymerase, and screened in 0.8％ agarose gel by electrophoresis.
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