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| + | <img src="https://static.igem.org/mediawiki/2021/f/fd/T--Shanghai_high_school--bg_1.png" alt=""> |
| + | <h1>Proof of Concept</h1> |
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| + | <!--内容--> |
| + | <div class="content-small"> |
| + | <section class="article p-t-54 p-b-54"> |
| + | <section> |
| + | <h1 class="title">Overview</h1> |
| + | |
| + | <img class="m-b-18" src="https://static.igem.org/mediawiki/2021/a/a8/T--Shanghai_high_school--img_proof_of_concept_1.jpg" alt="" |
| + | style="width: 60%"> |
| + | |
| + | <p>Our goal is to produce a vaccine that includes VP7, a surface protein of rotavirus, and adjuvant LTB, a |
| + | subunit of Heat Labile Enterotoxin B. VP7, being the most abundant surface protein of rotavirus, is the |
| + | primary antigen detected by antibodies when rotavirus enters the body. This makes it the optimal protein |
| + | candidate for the vaccine. </p> |
| + | |
| + | <p>In order to obtain the most successful protein expression, we resulted in using the popular E. Coli as |
| + | the bacteria for this experiment. This is because E.Coli is known to be the most practical bacteria due |
| + | to its rapid expression, inexpensive cultivation, and high yield. </p> |
| + | |
| + | <br> |
| + | <p>Ensuring that our proposed target gene, VP7 has been successfully expressed by testing through SDS PAGE |
| + | and Western Blot. </p> |
| + | </section> |
| + | |
| + | <section> |
| + | <h1 class="title">SDS PAGE</h1> |
| + | |
| + | <p>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> |
| + | |
| + | <br> |
| + | <div class="half"> |
| + | <div class="left"> |
| + | <p>First, we made a Polyacrylamide gel, which consists of the pink stacking gel and the transparent |
| + | separating gel. </p> |
| + | |
| + | <p>We inserted the gel into a chamber and filled in some running buffer, and then we pipetted |
| + | protein |
| + | samples into the wells, except for the first and last wells, we filled in with marker proteins, |
| + | as a |
| + | reference to determine the molecular weight of the proteins in the sample. Lastly, we connected |
| + | the |
| + | electrodes to the power supply and start running the gel. </p> |
| + | </div> |
| + | <div class="right" style="text-align: right"> |
| + | <img class="m-b-18" src="https://static.igem.org/mediawiki/2021/a/a7/T--Shanghai_high_school--img_proof_of_concept_4.jpg" alt="" |
| + | style="width: 95%"> |
| + | </div> |
| + | </div> |
| + | |
| + | <div class="half m-t-18"> |
| + | <div class="left" style="text-align: left"> |
| + | <br> |
| + | <img class="m-b-18" src="https://static.igem.org/mediawiki/2021/0/00/T--Shanghai_high_school--img_proof_of_concept_3.jpg" alt="" |
| + | style="width: 95%"> |
| + | </div> |
| + | <div class="right"> |
| + | <p>Since the proteins were all denatured by heating at 95 degrees Celsius and masked by negative |
| + | charge, they would run from negative to positive terminals. First, they went through the |
| + | stacking gel, which had large pore size, so both large and small proteins were able to move in |
| + | the same speed, eventually stacking on one “starting line” prior to entering the separating gel. |
| + | In the separating gel, the pore size was much smaller, which allowed small proteins to run |
| + | faster than the larger ones. As a result, the proteins got separated based on their molecular |
| + | weight. </p> |
| + | </div> |
| + | </div> |
| + | |
| + | <p>After running the gel, we stained it with Coomassie Brilliant Blue to visualize proteins. After the |
| + | excess stain was eluted, blue bands of new proteins were revealed. The next step would be to confirm |
| + | whether they were our proteins of interest. </p> |
| + | |
| + | <p>We have three groups of samples which are VP7-LTB induced by 1nm of IPTG, VP7-LTB induced by 2nm of IPTG, |
| + | and VP7 induced by 1nm of IPTG. We took OD600, which is an abbreviation indicating the optical density |
| + | of a sample measured at a wavelength of 600 nm, for each of the groups. The optimal density id |
| + | proportional to the concentration of the sample. Thus, it is used to measure the concentration of the |
| + | bacterium. After testing the samples which the OD600 of those is about 1, we collect five groups of each |
| + | at 0, 2, 4, 5, and 6 hours to run the SDS page. </p> |
| + | |
| + | <p>Below are the imgas of SDS PAGE results:</p> |
| + | |
| + | <img class="m-t-18 m-b-18" src="https://static.igem.org/mediawiki/2021/0/0f/T--Shanghai_high_school--img_proof_of_concept_5.jpg" alt="" |
| + | style="width: 95%"> |
| + | |
| + | <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 protein should appear in the supernatant, however, it appears in the precipitates. We suggest that |
| + | the proteins from inclusion body since they are virus-induced proteins. </p> |
| + | |
| + | <p>Compared the samples of the precipitates and the supernatants at 0h and other times of the figure on the |
| + | left, we could see an extra band that is at a little 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 0h. 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 LTB stimulates the expression |
| + | of VP7.</p> |
| + | </section> |
| + | |
| + | <section> |
| + | <h1 class="title">Western Blot</h1> |
| + | |
| + | <p>Western blot technique uses three elements to achieve its task of separating a specific protein from a |
| + | complex: separation by size, transfer of protein to a solid support, and marking target protein using a |
| + | primary and secondary antibody to visualize. A synthetic or animal-derived antibody (known as the |
| + | primary antibody) is created that recognizes and binds to a specific target protein. The electrophoresis |
| + | membrane is washed in a solution containing the primary antibody, before excess antibody is washed off. |
| + | A secondary antibody is added which recognizes and binds to the primary antibody. The secondary antibody |
| + | is visualized through various methods such as staining, immunofluorescence, and radioactivity, allowing |
| + | indirect detection of the specific target protein.</p> |
| + | |
| + | <p>In our project, we used Anti-6×His rabbit polyclonal antibody as the primary antibody and the anti-GST |
| + | Tag rabbit polyclonal antibody as the secondary antibody to detect our protein.</p> |
| + | |
| + | <ul class="mark"> |
| + | <li class="m-t-24"> |
| + | <p>VP7-LTB inducible expression (1 mM IPTG): </p> |
| + | |
| + | <img class="m-t-18" src="https://static.igem.org/mediawiki/2021/9/95/T--Shanghai_high_school--img_proof_of_concept_6.jpg" alt="" |
| + | style="width: 90%"> |
| + | |
| + | <img src="https://static.igem.org/mediawiki/2021/1/15/T--Shanghai_high_school--img_proof_of_concept_11.jpg" alt="" |
| + | style="width: 90%"> |
| + | |
| + | <img src="https://static.igem.org/mediawiki/2021/6/68/T--Shanghai_high_school--img_proof_of_concept_12.jpg" alt="" |
| + | style="width: 500px"> |
| + | |
| + | <p>Through mathematical modeling, we see that, with higher gray values, there is an increase in |
| + | protein |
| + | expression. </p> |
| + | |
| + | <p>In summary, VP7-LTB was successfully induced and existed in the form of inclusion body. With the |
| + | increase of induction time, the expression increased gradually. </p> |
| + | </li> |
| + | <li class="m-t-24"> |
| + | <p>VP7-LTB inducible expression (2 mM IPTG): </p> |
| + | |
| + | <img class="m-t-18" src="https://static.igem.org/mediawiki/2021/f/f5/T--Shanghai_high_school--img_proof_of_concept_7.jpg" alt="" |
| + | style="width: 90%"> |
| + | |
| + | <img src="https://static.igem.org/mediawiki/2021/b/b5/T--Shanghai_high_school--img_proof_of_concept_8.jpg" alt="" |
| + | style="width: 90%"> |
| + | |
| + | <img src="https://static.igem.org/mediawiki/2021/0/01/T--Shanghai_high_school--img_proof_of_concept_13.jpg" alt="" |
| + | style="width: 500px"> |
| + | |
| + | <p>Summary: VP7 LTB was successfully induced and existed in the form of inclusion body. With the |
| + | increase of induction time, the expression increased gradually, but there were miscellaneous |
| + | proteins. Therefore, the best induction condition is induction with 1M IPTG for 6 hours.</p> |
| + | </li> |
| + | <li class="m-t-24"> |
| + | <p>VP7-LTB inducible expression (1 mM IPTG):</p> |
| + | |
| + | <img class="m-t-18" src="https://static.igem.org/mediawiki/2021/d/dd/T--Shanghai_high_school--img_proof_of_concept_9.jpg" alt="" |
| + | style="width: 90%"> |
| + | |
| + | <img src="https://static.igem.org/mediawiki/2021/1/14/T--Shanghai_high_school--img_proof_of_concept_14.jpg" alt="" |
| + | style="width: 500px"> |
| + | |
| + | <p>VP7 exists in both supernatant and precipitation, but mainly in the form of inclusion bodies in |
| + | precipitation.</p> |
| + | </li> |
| + | <li class="m-t-24"> |
| + | <p>VP7-LTB inducible expression (2 mM IPTG):</p> |
| + | |
| + | <img class="m-t-18" src="https://static.igem.org/mediawiki/2021/5/5d/T--Shanghai_high_school--img_proof_of_concept_10.jpg" alt="" |
| + | style="width: 90%"> |
| + | <img src="https://static.igem.org/mediawiki/2021/0/0b/T--Shanghai_high_school--img_proof_of_concept_15.jpg" alt="" |
| + | style="width: 500px"> |
| + | </li> |
| + | </ul> |
| + | </section> |
| + | </section> |
| + | </div> |
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