Part:BBa_K1074005

LTB

This eltB gene encodes for the Heat-labile enterotox(LT) in certain virulent strains of E.coli.However,in vitro,like Chinese Hamster Ovarylera toxin B subunit (CTB), it is an efficient mucosal adjuvant and carrier molecule for the generation of mucosal antibody responses and induction of systemic T-cell tolerance to linked antigens. So we use it to enhance the immune response to our transdermal vaccine.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 235
  

Characterization by 2021iGEM_Shanghai_high_school

Improvement of an existing part

Compared to the old part BBa_K1074005, we design a new part BBa_K3992004, which contains the and VP7-LTB fusion protein. The VP7 protein is on the outermost layer of virus particles, is the first choice for the development of genetic engineering vaccines.

The Group iGEM13_USTC_CHINA aimed to prove the secretion and expression possibility of TD1-antigen, TD1-adjuvant, and the antigenicity of TD1-antigen after transdermal process.

Based on the group’ contribution, our team design the new composite part BBa_K3992004 to express VP7-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_K3992004 and the old parts BBa_K1074005.

First of all, we constructed a composite part BBa_K3992004 and transformed it into E.coli. Furthermore, VP7 and VP7-LTB were successfully expression in E. coli predominantly as inclusion bodies. As a mucosal immune adjuvant, LTB enhances the antiviral ability of vp7.

In addition, we are trying to develop a new oral vaccine for hand, foot and mouth disease due to its advertisement for infants and young children. our project has a huge potential commercial market.

Vp7-GS linker-LTB

Usage and Biology

Otavirus (RV) is the main viral pathogen that causes severe acute diarrhea in infants and young children. Almost all children under five weeks of age have been infected with the virus, causing nearly 130,000 deaths worldwide each year. Social conditions in developing countries have led to reduced effectiveness of oral rehydration solutions and vaccines, as well as a lack of approved antiviral drugs, making rotavirus infection a global health problem. RV structural protein vp7, on the outermost layer of virus particles, is the first choice for the development of genetic engineering vaccines. We are trying to develop a new oral vaccine for hand, foot and mouth disease due to its advertisement for infants and young children. The B subunit LTB in the heat-labile enterotoxin (LT) of Escherichia coli heat-labile enterotoxin (LT) has strong immunogenicity and adjuvant activity, and will not cause harm to the human body. LTB and a variety of non-related proteins and their non-protein antigens can increase the mucosal IgA and humoral immune IgG response levels of the antigen through different immunization pathways. Currently, there are three main types of vaccines, including inactivated vaccines/attenuated vaccines, mRNA vaccines/DNA vaccines, and neutralizing antibody/non-neutralizing antibody vaccines. Human vaccination methods include injection (hepatitis B vaccine, BCG vaccine, flu vaccine, etc.) and oral administration (poliomyelitis, cholera vaccine, and rotavirus vaccine).

Construct design

VP7-LTB had been linked by P2A linker. VP7-LTB is inserted into plasmid (Figure 1). The sequence of pHT43-VP7-LBT and pET28a-VP7-LBT are shown in Figure 2.

Figure 1. Genetic design of the plasmid.

Figure 2. Schematic map of plasmids.

The profiles of every basic part are as follows:

Usage and Biology

BBa_K3992000 is a coding sequence of from E. coli. RV structural protein vp7 is on the outermost layer of virus particles.

Profile

Usage and Biology

BBa_K3992001 is a coding sequence of E. coli, which has strong immunogenicity and adjuvant activity, and will not cause harm to the human body.

BBa_K3992002

Usage and Biology

BBa_K3992002 is a part of GS linker. It can link different proteins to make them become a fusion protein. In our group, we use this part to link VP7 protein and LTB protein.

Plasmid Construction

Polymerase Chain Reaction

A PCR verification was performed to confirm whether the sequence of synthesized VP7-LTB is correct. Agarose gel electrophoresis was used to assess the PCR’s result. According to the 15000 bp DNA marker, the PCR amplified DNA fragments possess the desired right size.

Figure 3 PCR verification of VP7 and VP7-LTB.

Restriction enzyme digestion

At this point we had two empty plasmid vectors and two DNA fragments (vp7 and Ltb) awaiting to be inserted. To that end, we used BamHI to digest and linearize the plasmid, making a specific site for fragment insertion.

pET28a

Once digestion completed, we set up gel electrophoresis again to assess the result. The first three lanes were DNA marker ladder, pET28a after digestion, and the control, respectively. The second lane was our linear pET28a with 5639 nt. The place of band in the gel was consistent with its real size.

Figure 4 The result of enzyme digestion of pET28a.

pHT43-His

We did the same digestion of pHT43-His. As shown in Figure 5, lanes 2 to 7 were the linearized plasmids and the last was the original supercoiled and nicked DNA. It can be identified that the DNA was around 8000 bp, which was consistent to pHT43-His’s standard size-8101 bp.

Figure 5 The result of enzyme digestion of pHT43-His.

Conclusion

The two gel images demonstrated that we had successfully digested the plasmid vector and pHT43-his. According to the standard ladder, linearized DNAs are consistent with their size and distinctively different to the controls.

Colony PCR

After completing restriction enzyme digestion and ligation, we had transformed the new plasmids into different cultures of E. coli BL21 for cloning. Additionally, we also transformed the empty plasmid vector pHT43-HIS into WB800N through electroporation instead of normal processes.

pET28a-VP7-LTB

According to the agarose gel electrophoresis image, we were able to identify that the target genes of the correct sizes have been successfully amplified. Sequence VP7 had 846 nt, and same was for VP7-LTB that had 1242 nt.

Figure 6 PCR verification of E. coli BL21 containing pET28a-VP7 and pET28a-VP7-LTB.

pHT43-His-VP7-LTB

We expected these two plasmids to be inserted into WB800N at the end of the project. We first transformed the plasmids into E. coli BL21, a Gram-negative bacterium with substantially thinner cell wall, to test the feasibility, and performed a colony PCR. As a result, the gel image clearly demonstrates that our target genes with correct size have been amplified from the cell’s DNA.

Figure 7 PCR verification of E. coli BL21 containing pHT43-His-VP7-LTB.

PHT43-His-VP7 & PHT43-His-VP7-LTB (WB800N)

A colony PCR was conducted to verity the plasmid transformation of PHT43-His-VP7-LTB into bacteria WB800N. According to the gel electrophoresis image, we collected 1 sample of WB800N containing PHT43-His-VP7-LTB. Again, the band of VP7 is at 846 bp and that of VP7-LTB presents at 1242 bp. Therefore, the location of each gene bands on the following image is consistent with the values, which demonstrates successful transformations.

Figure 8 PCR verification of WB800N containing PHT43-His-VP7 & PHT43-His-VP7-LTB.

Conclusion

All gel images are the proofs that our target plasmids have been amplified from the DNA template, which indicates that they have been successfully connected to the cell’s DNA.

Proof of function

SDS PAGE

Figure 9 shows the protein expression of E. coli with SDS-PAGE. Our purpose was to identify the presence of new proteins and confirm whether they are our proteins of interest --- VP7 and VP7-LTB.

Figure 9 SDS-PAGE of BL21 protein expression.

There are supernatants and precipitates. We suggest that the virus-induced proteins existed in the form of insoluble inclusion body. Compared the samples of the precipitates and the supernatants at 0 h and other times of the figure on the left, we could see an extra band that located below 50 kD. We should see thinner bands in the samples, which are collected after adding IPTG. However, the extra bands are thicker than the parallel bands which are at the 0 h. This phenomenon demonstrates that the thicker bands could be our target proteins. The masses of the bands match pretty well with the theoretical molecular weight of VP7-LTB. Compared the left figure and the middle one, we could find that VP7-LTB induced by 1nM of IPTG expressed better than that induced by 2nM of IPTG. Compared the right figure with the other one, we could find that the adding of LTB stimulates the expression of VP7.

Figure 10 SDS-PAGE of WB800n protein expression.

Western Blot

BL21 These graphs show the relationship between time and protein expression

Figure 11 VP7-LTB expression with1mM IPTG.

Conclusion: VP7 LTB was successfully induced to express and was present as inclusion bodies, which increased gradually with increasing induction time.

Figure 12 VP7-LTB expression with 2mM IPTG.

Conclusion: VP7 LTB was successfully induced to express and was present as inclusion bodies, which gradually increased in expression with increasing induction time, but the heteroprotein appeared so the optimal induction conditions were: 1 mM IPTG for 6 h induction. In order to determine the optimum induction duration of our engineered BL21 strains for the antigen protein expression which is designed to induce neutralizing antibody immune response inside the human body, we conducted several Western Blot and collected the gray value which was calculated by ImageJ to quantify the protein expression against hours.

When IPTG concentration was given 1mM, VP7-LTB expresses the most fast during the third hour of the induction and it starts to flatten out after 4 hours. So we could imply that our engineered strain PET28a-VP7-LTB would start its maximum response efficiency after 2 hours and its optimum induction duration will be 4 hours around. When IPTG concentration was given 2 mM, the strain expresses the fast in the second hour of the induction but it is also earlier to get the steady phase which the peak of gray value is 120 around. In this case, the optimum induction duration of VP7-LTB shall be 2 hours.

Future plan

For BL21, in order to increase the expression of target proteins, we plan to optimize their expression conditions, such as temperature, time, pH value, IPTG concentration and so on.

For WB800n, we needed to analyze why it failed to express. After a successful protein expression, perhaps we would try to make it as through secretory protein expression.