Part:BBa_K4004007

pGEX-vp1-LTB

promoter

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

We link vp1 and LTB with a linker. The vp1-LTB is inserted into plasmid pGEX, respectively (Figure 2 and 3).

Figure 2. The expression system of EV71 vp1...

Figure 3. Schematic map of expression system of pGEX-vp1-LTB plasmid...

The profiles of every basic part are as follows:

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.

Usage and Biology

BBa_K4004005 is a coding sequence of E. coli, which has strong immunogenicity and adjuvant activity, and will not cause harm to the human body.

BBa_K4004003

Name: pGEX vector

==== Base Pairs: 4969bp ==== Origin: Addgene

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

PCR for VP1, VP1-linker and LTB fragments

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

To confirm whether we successfully amplified the fragments we wanted from the plasmids, we ran the electrophoresis of the fragments in the six tubes. We then scanned the gel, compared the strong bands with the markers, and identified VP1, VP1-linker and LTB fragments on the gel. If we got the expected results, we can extract the three types of fragments from the gel and continue our experiments: digestion of VP1 fragments and OE PCR of VP1-LTB fragments.

Figure 4. Gel electrophoresis of VP1, VP1-linker and LTB fragments after PCR...

Conclusion: Theoretically, VP1 fragment is 891bp in length; VP1-linker fragment is 948bp in length; LTB fragment is 604bp in length. Compared with the markers, the strong bands in the six tubes all fit in the right range, so it proved that our PCR for the three types of fragments was successful, and we could continue our experiments.

·OE PCR for VP1-LTB fragments

After obtaining VP1-linker and LTB fragments from PCR, we overlapped them through OE PCR. We added the two types of fragments, VP1-FP, and LTB-RP into one tube and waited for them to overlap. Then we conducted double digestion on the newly ligated fragments.

To confirm whether we successfully overlapped the two fragments, we ran the electrophoresis of the fragments in the tubes. We then scanned the gel, compared the strong bands with the markers and identified VP1-LTB fragments on the gel. If we got the expected results, we can extract the fragments from the gel and insert them into the vectors.

Figure 5. Gel electrophoresis of VP1-LTB fragments after OE PCR and enzyme digestion...

Conclusion: Theoretically, digested VP1-LTB fragment is 1552bp in length. Compared with the markers, the strong band fit in the right range, so it proved that our OE PCR for VP1-LTB was successful, and we could continue our experiments.

·Clonexpress Ligation reaction for pGEX-VP1-LTB

We had already obtained digested VP1-LTB fragment after PCR and OE PCR. In order to insert them into the vectors pGEX-6P-1 respectively, 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-LTB fragment with pGEX-6P-1.

Figure 6. 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-LTB.

Simultaneously, we used Snapgene to construct the profiles of the plasmids we constructed, pGEX-6P-1-VP1-LTB. We demonstrated and understood the parts of the plasmids: restriction gene, target gene, and multiple cloning site.

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 and L.casei again. Finally, we verified the expression of VP1-LTB proteins in the two types of bacteria.

Verification of transformed plasmids

·Electrophoresis of pGEX-VP1-LTB

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-LTB on the gel.

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

Conclusion: Theoretically, pGEX-6P-1 is 4984 bp long in linear shape; pGEX-6P-1-VP1-LTB is 6511 bp long in linear shape. pGEX-6P-1, which became linear-shaped after single digestion, should be around 5000bp in length, Compared with the markers, the plasmids in the twelve groups 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-LTB

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-LTB; 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 plasmid.

Figure 8. gel electrophoresis of pGEX-6P-1-VP1 and pGEX-6P-1-VP1-LTB after double digestion...

Conclusion: Theoretically, VP1 fragment is 891bp in length; LTB fragment is 604bp in length；pGEX-6P-1 after double digestion by SalⅠ and BamHⅠis 4975 in length. Compared with the markers, sample 3 of pGEX-6P-1-VP1-LTB was correct as well. However, in sample 4 B+S (pGEX-6P-1-VP1-LTB after double digestion by SalⅠ and BamHⅠ), the weaker bands, which should represent the VP1-LTB fragments, did not appear on the gel, so we recognized the plasmids transformation in sample 4 of pGEX-6P-1-VP1-LTB as a failure. 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-LTB, 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-LTB 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 and pGEX-6P-1-VP1-LTB 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 9. 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 and GST-VP1-LTB 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 and VP1-LTB proteins.

Figure 10. 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 11. PAGE gel of GST, GST-VP1 and GST-VP1-LTB under different expression conditions...

Figure 12.Model...

Conclusion: Via this graph of model, we determined the optimum expression conditions of VP1 and VP1-LTB respectively in E.coli BL21: VP1 was expressed the most under OD600=0.5, IPTG=10mM; VP1-LTB was expressed the most under OD600=0.8, IPTG=1mM. Besides, we could see that when OD600 was over 0.8, the expression of VP1-LTB proteins was significantly higher than that of VP1 proteins in E.coli BL21. This meant that LTB could promote the expression of VP1 in E.coli BL21 during its late growth period.

Improvement of an existing part

Compared to the old part BBa_K3487013, we design a new part BBa_K4004007, which contains the and VP1-LTB fusion protein. The 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.

The Group iGEM20_SZPT-CHINA aimed to devlope a chewing gum that can prevent dental caries with expressed AMP or synthetic AMP probiotics.

Based on the group’ contribution, our team design the new composite part BBa_K4004007 to express VP1-LTB fusion protein. After the composite part was inserted in a particular plasmid vector and entered an industrial bacteria.

Figure 13. The blast results about the DNA sequence of our new part BBa_K4004007 and the old parts BBa_K3487013...

First of all, we constructed a composite part BBa_K4004007 and transformed it into E.coli.

Furthermore, VP1 and VP1-LTB were successfully expression in E. coli predominantly as inclusion bodies. As a mucosal immune adjuvant, LTB enhances the antiviral ability of vp1.

In addition, we are trying to develop a new oral vaccine for Hand-foot-mouth disease. our project has a huge potential commercial market.

Future plan

We combined VP1 and LTB to construct the recombinant expression system and used E.coli to express the proteins. We detected the protein expression with IPTG solution of different concentration at different growth stage, constructed related models, and determined the optimum expression conditions. All these laid a solid foundation for the development of HFMD oral vaccine.

1.Currently, we just verified the viability of our concept and determined the optimized condition of protein expression in E.coli. However, because of the difficulty in protein expression in L.casei, we would need to optimize the incubation and electroporation of L.casei, or improve and reconstruct the plasmids, to realize the expression of GST-VP1-LTB in L.casei.

2.If L.casei did express the proteins after the improvement of our design, the next part of our project would be that we let the mice take the transformed L.casei via gavage, and then measured the level of anti-VP1-LTB IgG in the serum of the mice and the level of IgA in their feces. Finally, we could verify immunogenicity of our oral vaccine.

References

6. Orlando, A.; Refolo, M. G.; Messa, C.; Amati, L.; Lavermicocca, P.; Guerra, V.; Russo, F. (October 2012). "Antiproliferative and Proapoptotic Effects of Viable or Heat-Killed IMPC2.1 and GG in HGC-27 Gastric and DLD-1 Colon Cell Lines". Nutrition and Cancer. 64 (7): 1103–1111. doi:10.1080/01635581.2012.717676

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 930
  Illegal XhoI site found at 954
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 5807
  Illegal AgeI site found at 5079
  Illegal AgeI site found at 5828
  
• 1000
  COMPATIBLE WITH RFC[1000]