Polymerase Chain Reaction
A PCR verification was performed to confirm whether the synthesized plasmid pET28a-VP7-LTB contains the correct gene sequences. According to Snap Gene data (Fig 1&2), gene for VP7 has 846 nucleotides (nt) and VP7-LTB has 1242 nt. 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. This verification was critical for our subsequent experiments—if the original plasmid was defective, every other plasmid constructed based on its DNA would have no chance of being right.
Restriction enzyme digestion
At this point we had two empty plasmid vectors (pET28a and pHT43-his) and two DNA fragments (vp7 and Ltb) awaiting to be inserted. To that end, we used 5 μL BamHI (a restriction enzyme) to digest and linearize the plasmid, making a specific site for fragment insertion . When BamHI was mixed with the plasmid vectors in a 10.5 μL Cusmart solution that created the chemical environment for the enzyme to function, it targeted its unique recognition sequence 5’-GGATCC-3’, cut this site open, and linearized the plasmid (Fig 4).
Once digestion completed, we set up gel electrophoresis again to assess the result. According to the gel (Fig 5), the first three lanes was DNA marker ladder, pET28a after digestion, and the control, respectively. The control was the uncut plasmid pET28a and showed two DNA bands in nicked DNA and supercoiled DNA confirmation, respectively. Super coiled was the native conformation of a plasmid extracted from a bacterium that is highly compacted and is able to migrate faster through a gel. Therefore, it presented smaller band than that of the equivalently sized within the ladder. Nicked DNA was also commonly found in uncut plasmid, which migrates even slower than linear DNA. Now, on the second lane was our linear pET28a with 5639 nt. The place of band in the gel was consistent with its real size.Figure 5 The digestion of pET28a.
We did the same digestion of pHT43-His. On the following image (Fig 6), 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. Unlike the one clear band of linear DNA shown in figure 5, many additional vague but distinguishable smaller bands can be found in figure 6. One probable explanation to this might be the plasmid in each sample were digested incompletely, leading to additional bands. Those stayed in supercoiled or circular conformation and had a larger migration speed.Figure 6 The digestion of pHT43-His.
The two gel images demonstrated that we had successfully digested the plasmid vectors pET28a and pHT43-his. According to the standard ladder, linearized DNAs are consistent with their size and distinctively different to the controls.
NEBuider HiFi DNA Assembly Reaction
NEBuider HiFi DNA Assembly is an effective method for the high-fidelity assembly of multiple DNA fragments. In our project, we used this technique to insert VP7 and VP&-LTB’s DNA to the linearized plasmid vectors through assembling both 5’ and 3’ restriction enzyme mismatches. In the first step (Fig 7), a 5’ to 3’ exonuclease activity created single stranded 3’ overhangs. These complementary sequences then annealed, creating the double stranded DNA of interest. A high-fidelity DNA polymerase then extended the 3’ ends, filling in the gaps, and a DNA ligase sealed the remining nicks. The end products were four complete circular plasmids-pET28a-VP7, pET28a-VP7-LTB, pHT43-His-VP7 and pHT43-His-VP7-LTB—that could be used for direct transformation.
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. We then performed a colony PCR to verify if the two types of bacteria contain the target plasmids.
pET28a-VP7 & pET28a-VP7-LTB
To verify plasmid transformation, we extracted 1 μL of DNA template from both the BL21 culture containing pET28a-VP7 and the other containingpET28a-VP7-LTB, and mixed the solution with primers, DNA polymerase, and ddH2O. According to the agarose gel electrophoresis image (Fig 8), using with a 2000bp DNA ladder, we were able to identify that the target genes of the correct sizes have been successfully amplified. Sequence VP7 had 846 nt, which was parallel to the data, and same was for VP7-LTB that had 1242 nt.Figure 8 PCR verification of E. coli BL21 containing pET28a-VP7 and pET28a-VP7-LTB.
pHT43-His-VP7 & pHT43-His-VP7-LTB
We expected these two plasmids to be inserted into WB800N at the end of the project. However, considering that WB800N is Gram-positive, we suspect whether they are able to be entered at all. For this reason, 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 using 1 μL of the solution containing the cell as DNA template. As a result, the gel image (Fig 9&10) clearly demonstrates that our target genes with correct size have been amplified from the cell’s DNA.Figure 9 PCR verification of E. coli BL21 containing pHT43-His-VP7.Figure 10 PCR verification of E. coli BL21 containing pHT43-His-VP7-LTB.
In the previous experiments, pHT43-His had been transferred into WB800N by electroporation, which opened up temporary pores on host’s membrane to facilitate the entry of the plasmid. Again, a colony PCR was used to verify the transformation using 1 μL of DNA template from the host. The gel image with a 2000 bp DNA ladder (Fig 11) identified that our pHT43-His sequence had been successfully amplified from the solution. Unlike BL21’s plasmid transformation gel results, WB800N’s showed additional vague gene bands whose bp values deviate from the standard. This was because, during PCR, not all DNA from the template was paired with the primers; the remaining DNA may paired with random gene sequences, which lead to various sizes.Figure 11 PCR verification of WB800N containing pHT43.
PHT43-His-VP7 & PHT43-His-VP7-LTB (WB800N)
A colony PCR was conducted to verity the plasmid transformation of PHT43-His-VP7 & PHT43-His-VP7-LTB into bacteria WB800N. According to the gel electrophoresis image, we collected 2 samples of WB800N containing PHT43-His-VP7 and 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 12 PCR verification of WB800N containing PHT43-His-VP7 & PHT43-His-VP7-LTB
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. Furthermore, It can be identified that some lanes have gene bands substantially thicker than the others. This tells us that the amount of DNA amplified in each sample is different. This is caused by the difference between cell cultures from which the DNA template is extracted from. Whether the template is exactly 1 μL can also influence the result.
OD600 growth curve
The Curve of OD600 Concentration
In general, our experiments went very orderly. At the beginning of the experiment, we prepared the IPTG. Then, after adding the IPTG into the PET28a-VP7-LTB sample, we started to wait for the strain to grow. The strain grew well and the experiment was very successful. You can see that here is a picture of the OD600 growth curve, in which the bacteria firstly slow growth, ushered in rapid growth after reaching about 3, growth again becomes slow. Thus, bacterial culture is very successful.
Figure 13 shows the protein expression of E. coli with SDS-PAGE. SDS-PAGE is a gel electrophoresis technique used for protein separation based on their molecular weight. Our purpose was to identify the presence of new proteins and confirm whether they are our proteins of interest --- VP7 and VP7-LTB.Figure 13(a), SDS-PAGE of BL21 protein expression
There are supernatants and precipitates. The precipitates are usually debris of the cell such as organelle. The supernatants are usually protein, carbohydrates, nucleic acids of the bacteria. Usually, the soluble protein should appear in the supernatant. However, it appeared in the 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. If there are extra bands, it is highly possible that they are our target proteins. Due to the presence of IPTG, the amount of proteins could be limited. 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. Because IPTG may make the bacteria less active, 1nM is a suitable concentration.
Compared the right figure with the other one, we could find that the adding of LTB stimulates the expression of VP7.Figure 13(b), SDS-PAGE of WB800n protein expression
Unfortunately, after IPTG induced expression, WB800n did not find the target protein from SDS-PAGE, and there were many hetero-proteins. In conclusion, the protein expression of Bacillus subtilis failed. The picture above is the SDS-PAGE of WB800n.
These graphs show the relationship between time and protein expression of BL21. The horizontal axis represents to the duration time of induced protein expression, and the vertical axis represents to the Gray value which means the amount of protein expression. The higher the gray value is, the higher the protein expressed.
The gray value are calculated by “ImageJ” which is a software used to show protein expression the by calculating the amounts of black pixels in the pictures.
Figure 14 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 15 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 m mole IPTG for 6 h induction.
Figure 16 VP7 1mM
Figure 17 VP7 expression with 2 mM IPTG.
Conclusion: VP7 was present in both the supernatant and sediment, but predominantly as inclusion bodies in the sediment.
The researchers believed that the two phenomena on the above tables could be explained as bellow.
The appearance of heteroprotein
For the presence of a hybrid protein, we believe that there are two factors. First, the nonspecific binding of the His amino acid tag leads to the presence of some non-target proteins. Second, there may be some enzymes that degrade a portion of the protein, leading to the appearance of some target protein fragments of lower mass.
The production of the target protein without IPTG induction in the 0 h sample
The reason for this is the background expression of VP7. It means that BL21, the bacteria itself, have the possibility to express target protein spontaneously even without IPTG induction.
The protein expression of WB800n failed. SDS-PAGE did not find the target protein. Western blot was performed, and there was no target protein. Besides, a large number of hetero-proteins appeared in the Western blot of wb800n.
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.