Part:BBa_K1955000

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.

In order to prove the HA(H1N1) sequence which we designed can express protein correctly, we use BL21 competent cell to express the HA sequence and detect the protein by Western blot analysis.

(1) Transform pSB1C3-J04500-HA into BL21 to express desired gene and validate the normal function of J04500 by GFP.

J04500 is a LacI inducible promoter, so it can be induced by IPTG to activate the promoter for transcription. We picked two colonies of pSB1C3-J04500-HA and pSB1A2-J04500-GFP and incubated in 4ml LB broth containing antibiotics at 37℃, 250rpm for overnight. Inoculate 4ml fresh LB supplemented with antibiotics with 20μl of overnight cultures. Shake cultures at 37℃, 250rpm for 3hr. Split cultures into two (2ml each) and add 2μl 1M IPTG to one of the tube to reach 1mM IPTG for induction. The other tube will be the uninduced control. Shake both tubes for another 3hrs at 37℃, 250rpm.

For the pSB1C3-J04500-HA, their induction can be checked by Coomassie blue or Western blot analysis. Transfer cultures from both tubes to two Eppendorf tubes and centrifuge bacteria at max speed for 3min. Discard the supernatant, and resuspend each pellet in 100μl 1x SDS sample buffer by pipetting. Fit cap-guards onto Eppendorf tubes and boil the samples at 100℃ for 10min. Run SDS-PAGE to check the expression of desired protein.

Conduct the Western blot analysis, we successfully recognize the HA protein (approximately 62.3kd) in our two colonies (JH1 and JH2) when induced by IPTG (Fig. 1A).

To check the feasibility of J04500, we also built up a construct containing J04500 and GFP. After induction of 3hrs, the induced bacteria turn fluorescent green. In contrast, the uninduced bacteria did not appear to have any color change. (Fig. 1B)



Fig. 1. Protein expression of pSB1C3-HA and pSB1A2-GFP.

The day before induction, two different colonies were picked up from each construct into 4ml LB broth at 37℃, 250rpm for overnight. Inoculated 4ml fresh LB broth with 20μl of overnight cultured at 37℃, 250rpm for 3hrs. Splited into two tubes(each 2ml), and added 2μl 1M IPTG to one tube as the induced group, the other tube without IPTG was the uninduced group. IPTG induction for 3~4hrs.

(A) After IPTG induction, centrifuged bacteria at max speed. Discarded the supernatant, lysed the cell with 100μl 1x SDS sample buffer. Western blotting to check the desired gene expression. In fig. 5A, His-tag HA serves as positive control to validate that the antibody can work normally. We also transform pUC-19 into BL21 and follow the induction protocol as the negative control.

(B) IPTG induction of pSB1A2-J04500-GFP to verify the LacI inducible promoter. The GFP fluorescence can be observed easily by bare eye.

(2) 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.

Contribution

• Group: (CSMU_Taiwan2019)
• Author: (YU-RU,LIN)
• Summary: (Fusion with LysRS, linker design, and codon optimization for improved solubility and efficiency production. )