Difference between revisions of "Part:BBa K4004001"

Revision as of 09:39, 20 October 2021

vp1

promoter

Profile

Name: vp1

Base Pairs: 891bp

Origin: Enterovirus 71,genome

Properties: Vp1 is the main antigen gene of the EV71 virus

Usage and Biology

Hand-foot-mouth disease (HFMD) is an infectious disease caused by enterovirus 71 (EV71). The virus is an important pathogenic factor of hand, foot and mouth disease. BBa_K4004001 is a coding sequence of from E. coli. Vp1 protein is the viral capsid protein and promotes the infection of host cells by virus particles. Vp1 is also the main antigen gene of the EV71 virus.

Experimental approach

Firstly, to amplify VP1 fragments from pUC57-VP1, we added VP1-FP and VP1-RP into two tubes to amplify VP1 fragments.

To confirm whether we successfully amplified the fragment, we ran the electrophoresis of the fragment. We then scanned the gel, compared the strong bands with the markers, and identified VP1 fragment on the gel.

Figure 1. Gel electrophoresis of VP1 fragments after PCR...

Conclusion: Theoretically, VP1 fragment is 891bp in length. Compared with the markers, the strong bands all fit in the right range, so it proved that our PCR of fragment was successful, and we could continue our experiments. Then we put vp1 fragment into plasmid pGEX to get a recombinant plasmid pGEX-vp1 and we performed functional test on this recombinant plasmid.

Proof of function

SDS-PAGE and Coomassie Brilliant Blue staining for whole bacteria, supernate, and precipitation

We transformed pGEX-6P-1-VP1 into E.coli BL21 respectively and incubated them. Firstly, we ran a PAGE gel of the whole bacteria, supernate, and precipitation of E.coli BL21 and then stained the gel through Coomassie Brilliant Blue Staining.

Figure 2. PAGE gel of GST, GST-VP1 and GST-VP1-LTB after staining(W: whole bacteria; S: supernatant; P: precipitation)...

Conclusion: After Coomassie Brilliant Blue Staining, we found that the extent of the brightness of the band in the P group was comparable to that in the W group, while the band in the S group was nearly invisible. In other words, GST, GST-VP1 had all been successfully expressed by E.coli BL21, and they mainly existed in the precipitation in the form of inclusion body.

Due to the relatively low rate of growth and efficiency of electroporation of L. casei, our team first transformed E. coli BL21, which is commonly used in plasmids transformation, to verify the expression and antigencity of VP1 protein.

Figure 3. SDS-PAGE and Western Blot for expression of VP1 and VP1-LTB proteins...

Expression optimization

In order to find the optimum condition under which the proteins were expressed the most, we selected bacteria solution of different concentration (OD600=0.5/0.6/0.8/1), and inducted them with IPTG solution of different concentration (IPTG=1mM/10mM). Then we ran a PAGE gel of them and then marked the proteins with Coomassie Brilliant Blue Staining Solution. To visualize and compare the expression of proteins under different conditions, we used the software ImageJ to quantify specific bands on the gel, collected and arranged the data, and constructed a broken line graph with OD600 the x- axis and the gray value as the y-axis.

Figure 4. PAGE gel of GST, GST-VP1 and GST-VP1-LTB under different expression conditions...

Sequence and Features