Difference between revisions of "Part:BBa K4004004"

Revision as of 03:50, 20 October 2021

pGEX-vp1

promoter

Profile

Name: pGEX-vp1

Base Pairs: 5860bp

Origin: E. coli, synthetic

Properties: Hand-foot-mouth disease Drinkable EV71 Vaccine

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. 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. Generally, the vaccinated population, especially infants and young children, are more compliant with oral vaccines, so we are trying to develop oral HFMD vaccines. Probiotics bacteria Bifidobacteria, as the natural host of the intestinal tract, can adhere to intestinal epithelial cells and are ideal oral live vaccine expression vectors, and related studies have found that their preventive effects on gastrointestinal pathogens are more significant. Therefore, we can use the bifidobacterium in lactic acid bacteria as an expression system to express EV71 vp1.

Figure 1. Concept map of the EV71 oral vaccine...

Construct design

Figure 2. The expression system of EV71 vp1 in plasmid pGEX...

Figure 3. Schematic map of expression system of pGEX-VP1 plasmids...

The profiles of every basic part are as follows:

BBa_K4004001

Name: vp1

Base Pairs: 891bp

Origin: E. coli

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

Usage and Biology

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.

BBa_K4004003

Name: pGEX vector

Base Pairs: 4969bp

Origin: Addgene

Properties: A plasmid that allows cloning gene.

Usage and Biology

BBa_K4004003 is a plasmid backbone. pGEX plasmid allows cloning of gene of interest into bacterial expression vector with PreScission Protease cleavable N-terminal GST tag.

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 4. Gel electrophoresis of VP1 fragments after PCRs...

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.

·Clonexpress Ligation reaction for pGEX-VP1

We first needed to use the same restriction enzymes, SalⅠ and BamHⅠ, to digest pGEX-6P-1 and make the plasmids available for ligation. We then run the gel electrophoresis of digested pGEX-6P-1, identified the fragments we wanted, and extracted them from the gel. After that, we conducted ClonExpress ligation reaction to ligate VP1 fragments with pGEX-6P-1.

Figure 5. gel electrophoresis of pGEX-6P-1 after double digestion...

Conclusion: Theoretically, pGEX-6P-1 after double digestion of SalI and BamHI is 4975bp in length. Compared with the markers, the strong band fit in the right range, so we can continue to conduct ClonExpress ligation reaction for pGEX-6P-1-VP1.

Plasmid transformation

In this part, we transformed the plasmids we constructed into E.coli to replicate them, then extracted and verified the plasmids, and transformed them into E.coli.

Verification of transformed plasmids

·Electrophoresis of pGEX-VP1

After transforming the plasmids into E.coli DH5α to replicate them, we picked 6 individual colonies from each petri dish and extracted the plasmids from them. To confirm whether the extracted plasmids were the ones we required, we ran a DNA gel electrophoresis of the transformed plasmids; we also run gel electrophoresis of pGEX-6P-1 after single digestion on the same gel as a negative group. We then scanned the gel, compared the brightest DNA bands with the markers, and identifies pGEX-6P-1-VP1 on the gel.

Figure 6. gel electrophoresis of pGEX-6P-1-VP1...

Conclusion: Theoretically, pGEX-6P-1 is 4984 bp long in linear shape; pGEX-6P-1-VP1 is 5853 bp long in linear shape. Accordingly, pGEX-6P-1, which became linear-shaped after single digestion, should be around 5000bp in length, while pGEX-6P-1-VP1 was in the form of cccDNA, should be around 2500bp in length. Compared with the markers, the plasmids that we extracted from E.coli DH5α all fit in the right range. Therefore, we reached a preliminary conclusion that we succeeded in the experiments of plasmid transformation and obtained the plasmids we wanted.

·Restriction enzyme double digestion of pGEX-VP1 and

We had tested the plasmids we extracted from E.coli DH5α in terms of their length. To further confirm whether the plasmids carried the fragments we wanted, we used SalⅠ and BamHⅠ to cut off the fragments between their cutting sites from pGEX-6P-1-VP1; simultaneously, we conducted single digestion on each group as the negative control group. Then we run the gel electrophoresis of the digested plasmids to separate and identify the fragments cut off from the plasmids.

Figure 7. gel electrophoresis of pGEX-6P-1-VP1 and pGEX-6P-1-VP1-LTB after double digestion...

Conclusion: Theoretically, VP1 fragment is 891bp in length; pGEX-6P-1 after double digestion by SalⅠ and BamHⅠis 4975 in length. Compared with the markers, samples 3 and 4 of pGEX-6P-1-VP1 were both correct. We discarded this sample and sent the samples that were proved right after enzyme digestion to a company for sequencing.

Sequencing for VP1 and VP1-LTB fragments on transformed plasmids

After enzyme digestion and gel electrophoresis of pGEX-6P-1-VP1, we tentatively confirmed that the experiment of plasmid transformation into E.coli DH5α was successful, and we obtained the plasmids we wanted. To further verify whether there was any mutation in VP1 fragments on the plasmids, we needed to obtain and compare the upstream and downstream sequences of the two fragments.

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.

Sequence and Features