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| | <!--Parts--> | | <!--Parts--> |
| − | <li class="item"> | + | <li class="item active"> |
| | <a href="https://2021.igem.org/Team:Shanghai_high_school/PartsCollection">Parts</a> | | <a href="https://2021.igem.org/Team:Shanghai_high_school/PartsCollection">Parts</a> |
| | <div class="child-nav"> | | <div class="child-nav"> |
| | <ul class="child-list"> | | <ul class="child-list"> |
| − | <li class="child-item"> | + | <li class="child-item active"> |
| | <a href="https://2021.igem.org/Team:Shanghai_high_school/Parts_Collection">Parts | | <a href="https://2021.igem.org/Team:Shanghai_high_school/Parts_Collection">Parts |
| | Collection</a> | | Collection</a> |
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| | <!--Project--> | | <!--Project--> |
| − | <li class="item active"> | + | <li class="item"> |
| | <a href="https://2021.igem.org/Team:Shanghai_high_school/Description">Project</a> | | <a href="https://2021.igem.org/Team:Shanghai_high_school/Description">Project</a> |
| | <div class="child-nav"> | | <div class="child-nav"> |
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| | <a href="https://2021.igem.org/Team:Shanghai_high_school/Experiments">Experiments</a> | | <a href="https://2021.igem.org/Team:Shanghai_high_school/Experiments">Experiments</a> |
| | </li> | | </li> |
| − | <li class="child-item active"> | + | <li class="child-item"> |
| | <a href="https://2021.igem.org/Team:Shanghai_high_school/Results">Result</a> | | <a href="https://2021.igem.org/Team:Shanghai_high_school/Results">Result</a> |
| | </li> | | </li> |
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| | <div class="page-header"> | | <div class="page-header"> |
| | <img src="https://static.igem.org/mediawiki/2021/f/fd/T--Shanghai_high_school--bg_1.png" alt=""> | | <img src="https://static.igem.org/mediawiki/2021/f/fd/T--Shanghai_high_school--bg_1.png" alt=""> |
| − | <h1>Result</h1> | + | <h1>Parts |
| | + | Collection</h1> |
| | </div> | | </div> |
| | | | |
| | <!--内容--> | | <!--内容--> |
| | <div class="content-small"> | | <div class="content-small"> |
| − | <section class="article p-t-54 p-b-54"> | + | <section class="article p-t-72 p-b-54"> |
| − | <section> | + | <table> |
| − | <h1 class="title">Plasmid Construction</h1> | + | <tr> |
| − | | + | <th>Part Number</th> |
| − | <section>
| + | <th>Part Name</th> |
| − | <h2 class="title2">Polymerase Chain Reaction</h2> | + | <th>Type</th> |
| − | | + | </tr> |
| − | <p>A PCR verification was performed to confirm whether the synthesized plasmid pET28a-VP7-LTB contains | + | <tr> |
| − | the correct gene sequences. According to Snap Gene data (Fig 1&2), gene for VP7 has 846 nucleotides
| + | < td>< u><a href=" http:// parts.igem.org/ Part:BBa_K3992000" style=" color: #0000FF"> BBa_K3992000</ a></ u></ td> |
| − | (nt) and VP7-LTB has 1242 nt. Agarose gel electrophoresis was used to assess the PCR’s result.
| + | <td>VP7</td> |
| − | According to the 15000 bp DNA marker, the PCR amplified DNA fragments possess the desired right
| + | <td>Basic</td> |
| − | size. This verification was critical for our subsequent experiments—if the original plasmid was
| + | </tr> |
| − | defective, every other plasmid constructed based on its DNA would have no chance of being right.</p>
| + | <tr> |
| − | | + | < td>< u><a href=" http:// parts.igem.org/ Part:BBa_K3992001" style=" color: #0000FF"> BBa_K3992001</ a></ u></ td> |
| − | <div class="img-container m-t-18"> | + | <td>LTB</td> |
| − | <img src="https://static.igem.org/mediawiki/2021/0/06/T--Shanghai_high_school--img_results_1.jpg" alt="" style="width: 55%">
| + | <td>Basic</td> |
| − | <span class="figure">Figure 1 The genetic information of vp7.</span>
| + | </tr> |
| − | </div>
| + | <tr> |
| − | | + | < td>< u><a href=" http:// parts.igem.org/ Part:BBa_K3992002" style=" color: #0000FF"> BBa_K3992002</ a></ u></ td> |
| − | <div class="img-container">
| + | <td>GS linker</td> |
| − | <img src="https://static.igem.org/mediawiki/2021/5/5a/T--Shanghai_high_school--img_results_2.jpg" alt="" style="width: 75%">
| + | <td>Basic</td> |
| − | <span class="figure">Figure 2 The genetic information for fusion protein of Vp7 and Ltb.</span>
| + | </tr> |
| − | </div>
| + | <tr> |
| − | | + | < td>< u><a href=" http:// parts.igem.org/ Part: BBa_K3992003" style=" color: #0000FF"> BBa_K3992003</a></ u></ td> |
| − | <div class="img-container m-t-12">
| + | <td>PHT43</td> |
| − | <img src="https://static.igem.org/mediawiki/2021/4/42/T--Shanghai_high_school--img_results_3.jpg" alt="" style="width: 65%">
| + | <td>Basic</td> |
| − | <span class="figure">Figure 3 PCR verification of pET28a-VP7-LTB.</span>
| + | </tr> |
| − | </div>
| + | <tr> |
| − | </section> | + | <td><u><a href="http://parts.igem.org/Part:BBa_K3992004" style="color: #0000FF">BBa_K3992004</a></u></td> |
| − | | + | <td>Vp7-GS linker-LTB</td> |
| − | <section class="p-t-30"> | + | <td>Composite</td> |
| − | <h2 class="title2">Restriction enzyme digestion</h2> | + | </tr> |
| − | | + | <tr> |
| − | <p>At this point we had two empty plasmid vectors (pET28a and pHT43-his) and two DNA fragments
| + | < td>< u><a href=" http:// parts.igem.org/ Part: BBa_K3992005" style=" color: #0000FF"> BBa_K3992005</ a></ u></ td> |
| − | (<i>vp7</i> and <i>Ltb</i>) awaiting to be inserted. To that end, we used 5 μL BamHI (a restriction
| + | <td>PHT43-HIS- VP7-LTB</td> |
| − | enzyme) to digest and linearize the plasmid, making a specific site for fragment insertion . When
| + | <td>Composite</td> |
| − | BamHI was mixed with the plasmid vectors in a 10.5 μL Cusmart solution that created the chemical
| + | </tr> |
| − | environment for the enzyme to function, it targeted its unique recognition sequence 5’-GGATCC-3’,
| + | </table> |
| − | cut this site open, and linearized the plasmid (Fig 4). </p>
| + | |
| − | | + | |
| − | <div class="half m-t-18">
| + | |
| − | <div class="left">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2021/f/f7/T--Shanghai_high_school--img_results_4.jpg" alt="" style="width: 90%">
| + | |
| − | </div>
| + | |
| − | <div class="right">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2021/1/1e/T--Shanghai_high_school--img_results_5.jpg" alt="" style="width: 90%">
| + | |
| − | </div>
| + | |
| − | </div> | + | |
| − | <span class="figure">Figure 4 Restriction enzyme digestion sites of BamHI for cloning.</span>
| + | |
| − | | + | |
| − | <ul class="normal m-t-18"> | + | |
| − | <li>
| + | |
| − | <p>pET28a</p>
| + | |
| − | <p>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. </p>
| + | |
| − | | + | |
| − | <div class="img-container">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2021/a/a6/T--Shanghai_high_school--img_results_27.jpg" alt="" style="width: 80%">
| + | |
| − | | + | |
| − | <span class="figure">Figure 5 The digestion of pET28a.</span>
| + | |
| − | </div>
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | <p>pHT43-His</p>
| + | |
| − | <p>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. </p>
| + | |
| − | | + | |
| − | <div class="img-container m-t-18">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2021/c/c8/T--Shanghai_high_school--img_results_8.jpg" alt="" style="width: 80%">
| + | |
| − | | + | |
| − | <span class="figure">Figure 6 The digestion of pHT43-His.</span>
| + | |
| − | </div>
| + | |
| − | </li>
| + | |
| − | </ul> | + | |
| − | </section>
| + | |
| − | | + | |
| − | <section>
| + | |
| − | <h2 class="title2">Conclusion</h2> | + | |
| − | | + | |
| − | <p>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. </p>
| + | |
| − | </section> | + | |
| − | | + | |
| − | <section class="p-t-30"> | + | |
| − | <h2 class="title2">NEBuider HiFi DNA Assembly Reaction</h2> | + | |
| − | | + | |
| − | <p>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. </p>
| + | |
| − | | + | |
| − | <div class="img-container m-t-18">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2021/f/f6/T--Shanghai_high_school--img_results_9.jpg" alt="" style="width: 70%">
| + | |
| − | | + | |
| − | <span class="figure">Figure 7 The depiction of NEBuider HiFi DNA Assembly Reaction.</span>
| + | |
| − | </div>
| + | |
| − | </section>
| + | |
| − | | + | |
| − | <section class="p-t-30">
| + | |
| − | <h2 class="title2">Colony PCR</h2> | + | |
| − | | + | |
| − | <p>After completing restriction enzyme digestion and ligation, we had transformed the new plasmids into | + | |
| − | different cultures of <i>E. coli</i> 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. </p>
| + | |
| − | | + | |
| − | <ul class="normal m-t-18">
| + | |
| − | <li>
| + | |
| − | <p>pET28a-VP7 & pET28a-VP7-LTB</p>
| + | |
| − | <p>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 ddH<sub>2</sub>O. 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. </p>
| + | |
| − | | + | |
| − | <div class="img-container m-t-18">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2021/4/49/T--Shanghai_high_school--img_results_10.jpg" alt="" style="width: 35%">
| + | |
| − | | + | |
| − | <span class="figure">Figure 8 PCR verification of <i>E. coli</i> BL21 containing pET28a-VP7 and pET28a-VP7-LTB.</span>
| + | |
| − | </div>
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | <p>pHT43-His-VP7 & pHT43-His-VP7-LTB</p>
| + | |
| − | <p>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 <i>E. coli</i> 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. </p>
| + | |
| − | | + | |
| − | <div class="img-container m-t-18">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2021/a/aa/T--Shanghai_high_school--img_results_11.jpg" alt="" style="width: 35%">
| + | |
| − | | + | |
| − | <span class="figure">Figure 9 PCR verification of <i>E. coli</i> BL21 containing pHT43-His-VP7.</span>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <div class="img-container m-t-18">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2021/6/6c/T--Shanghai_high_school--img_results_12.jpg" alt="" style="width: 35%">
| + | |
| − | | + | |
| − | <span class="figure">Figure 10 PCR verification of <i>E. coli</i> BL21 containing pHT43-His-VP7-LTB.</span>
| + | |
| − | </div>
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | <p>pHT43-His</p>
| + | |
| − | <p>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. </p>
| + | |
| − | | + | |
| − | <div class="img-container m-t-18">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2021/e/e8/T--Shanghai_high_school--img_results_13.jpg" alt="" style="width: 35%">
| + | |
| − | | + | |
| − | <span class="figure">Figure 11 PCR verification of WB800N containing pHT43.</span>
| + | |
| − | </div>
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | <p>PHT43-His-VP7 & PHT43-His-VP7-LTB (WB800N)</p>
| + | |
| − | <p>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. </p>
| + | |
| − | | + | |
| − | <div class="img-container m-t-18">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2021/e/e4/T--Shanghai_high_school--img_results_14.jpg" alt="" style="width: 45%">
| + | |
| − | | + | |
| − | <span class="figure">Figure 12 PCR verification of WB800N containing PHT43-His-VP7 & PHT43-His-VP7-LTB</span>
| + | |
| − | </div>
| + | |
| − | </li>
| + | |
| − | </ul> | + | |
| − | </section>
| + | |
| − | | + | |
| − | <section> | + | |
| − | <h2 class="title2">Conclusion</h2>
| + | |
| − | | + | |
| − | <p>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.</p>
| + | |
| − | </section> | + | |
| − | | + | |
| − | <section class="p-t-30">
| + | |
| − | <h2 class="title2">Inducible expression</h2> | + | |
| − | | + | |
| − | <ul class="normal">
| + | |
| − | <li>
| + | |
| − | <h3 class="title3">OD<sub>600</sub> growth curve</h3>
| + | |
| − | <img src="https://static.igem.org/mediawiki/2021/f/f1/T--Shanghai_high_school--img_results_15.jpg" alt="" style="width: 70%">
| + | |
| − | </li>
| + | |
| − | <li class="m-t-18">
| + | |
| − | <h3 class="title3">The Curve of OD<sub>600</sub> Concentration</h3>
| + | |
| − | <img src="https://static.igem.org/mediawiki/2021/6/66/T--Shanghai_high_school--img_results_28.jpg" alt="" style="width: 90%">
| + | |
| − | | + | |
| − | <p>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 OD<sub>600</sub> 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.</p>
| + | |
| − | </li>
| + | |
| − | <li class="m-t-18">
| + | |
| − | <h3 class="title3">SDS PAGE</h3>
| + | |
| − | <p>Figure 13 shows the protein expression of <i>E. coli</i> 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. </p>
| + | |
| − | | + | |
| − | <div class="img-container m-t-18">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2021/9/9b/T--Shanghai_high_school--img_results_21.jpg" alt="" style="width: 80%">
| + | |
| − | | + | |
| − | <span class="figure">Figure 13(a), SDS-PAGE of BL21 protein expression</span>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <p>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. </p>
| + | |
| − | | + | |
| − | <p>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. </p>
| + | |
| − | | + | |
| − | <p>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. </p>
| + | |
| − | | + | |
| − | <p>Compared the right figure with the other one, we could find that the adding of LTB stimulates
| + | |
| − | the expression of VP7.</p>
| + | |
| − | | + | |
| − | <div class="img-container m-t-18">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2021/a/a4/T--Shanghai_high_school--img_results_22.jpg" alt="" style="width: 80%">
| + | |
| − | | + | |
| − | <span class="figure">Figure 13(b), SDS-PAGE of WB800n protein expression</span>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <p>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 <i>Bacillus
| + | |
| − | subtilis</i> failed. The picture above is the SDS-PAGE of WB800n.</p>
| + | |
| − | </li>
| + | |
| − | <li class="m-t-18">
| + | |
| − | <h3 class="title3">Western Blot</h3>
| + | |
| − | | + | |
| − | <section>
| + | |
| − | <h4 class="title4">BL21</h4>
| + | |
| − | <p>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.</p>
| + | |
| − | <p>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.</p>
| + | |
| − | | + | |
| − | <br>
| + | |
| − | <p>Figure 14 VP7-LTB expression with1mM IPTG.</p>
| + | |
| − | <img src="https://static.igem.org/mediawiki/2021/0/0e/T--Shanghai_high_school--img_results_23.jpg" alt="" style="width: 80%">
| + | |
| − | <p>Conclusion: VP7 LTB was successfully induced to express and was present as inclusion
| + | |
| − | bodies,
| + | |
| − | which increased gradually with increasing induction time.</p>
| + | |
| − | | + | |
| − | <br>
| + | |
| − | <p>Figure 15 VP7-LTB expression with 2mM IPTG.</p>
| + | |
| − | <img src="https://static.igem.org/mediawiki/2021/5/59/T--Shanghai_high_school--img_results_24.jpg" alt="" style="width: 80%">
| + | |
| − | <p>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.</p>
| + | |
| − | | + | |
| − | <br>
| + | |
| − | <p>Figure 16 VP7 1mM</p>
| + | |
| − | <img src="https://static.igem.org/mediawiki/2021/f/fd/T--Shanghai_high_school--img_results_25.jpg" alt="" style="width: 80%">
| + | |
| − | | + | |
| − | <br>
| + | |
| − | <p>Figure 17 VP7 expression with 2 mM IPTG. </p>
| + | |
| − | <img src="https://static.igem.org/mediawiki/2021/a/a9/T--Shanghai_high_school--img_results_26.jpg" alt="" style="width: 80%">
| + | |
| − | <p>Conclusion: VP7 was present in both the supernatant and sediment, but predominantly as
| + | |
| − | inclusion bodies in the sediment.</p>
| + | |
| − | | + | |
| − | <br>
| + | |
| − | <p>The researchers believed that the two phenomena on the above tables could be explained as
| + | |
| − | bellow.</p>
| + | |
| − | <ol>
| + | |
| − | <li>
| + | |
| − | <p>The appearance of heteroprotein</p>
| + | |
| − | <p>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.</p>
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | <p>The production of the target protein without IPTG induction in the 0 h sample</p>
| + | |
| − | <p>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.</p>
| + | |
| − | </li>
| + | |
| − | </ol>
| + | |
| − | </section>
| + | |
| − | | + | |
| − | <section class="m-t-18">
| + | |
| − | <h4 class="title4">WB800n</h4>
| + | |
| − | <p>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.</p>
| + | |
| − | </section>
| + | |
| − | | + | |
| − | <section class="m-t-18">
| + | |
| − | <h4 class="title4">Future plan</h4>
| + | |
| − | | + | |
| − | <p>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.</p>
| + | |
| − | | + | |
| − | <p>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.</p>
| + | |
| − | </section>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | </section> | + | |
| − | </section> | + | |
| | </section> | | </section> |
| | </div> | | </div> |
| − |
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