Difference between revisions of "Team:Shanghai Metro Utd/Results"

(Prototype team page)
 
 
(One intermediate revision by the same user not shown)
Line 1: Line 1:
−
{{IGEM_TopBar}}
+
<html lang="en">
−
{{Shanghai_Metro_Utd}}
+
−
<html>
+
−
 
+
−
<div class="column full_size">
+
−
<h1>Results</h1>
+
−
<p>You can describe the results of your project and your future plans here. </p>
+
−
</div>
+
−
 
+
−
 
+
−
<div class="column third_size" >
+
−
 
+
−
<h3>What should this page contain?</h3>
+
−
<ul>
+
−
<li> Clearly and objectively describe the results of your work.</li>
+
−
<li> Future plans for the project. </li>
+
−
<li> Considerations for replicating the experiments. </li>
+
−
</ul>
+
−
</div>
+
−
 
+
−
 
+
−
 
+
−
 
+
−
<div class="column two_thirds_size" >
+
−
<h3>Describe what your results mean </h3>
+
−
<ul>
+
−
<li> Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for. </li>
+
−
<li> Show data, but remember <b>all measurement and characterization data must also be on the Part's Main Page on the <a href="http://parts.igem.org/Main_Page">Registry</a>.</b> Otherwise these data will not be in consideration for any medals or part awards! </li>
+
−
<li> Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project. </li>
+
−
</ul>
+
−
</div>
+
−
 
+
−
 
+
−
<div class="clear extra_space"></div>
+
−
 
+
−
 
+
−
 
+
−
<div class="column two_thirds_size" >
+
−
<h3> Project Achievements </h3>
+
−
 
+
−
<p>You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.</p>
+
−
 
+
−
<ul>
+
−
<li>A list of linked bullet points of the successful results during your project</li>
+
−
<li>A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.</li>
+
−
</ul>
+
−
 
+
−
</div>
+
−
 
+
−
 
+
−
 
+
−
<div class="column third_size" >
+
−
<div class="highlight decoration_A_full">
+
−
<h3>Inspiration</h3>
+
−
<p>See how other teams presented their results.</p>
+
−
<ul>
+
−
<li><a href="https://2019.igem.org/Team:Newcastle/Results">2019 Newcastle</a></li>
+
−
<li><a href="https://2019.igem.org/Team:Munich/Results">2019 Munich </a></li>
+
−
<li><a href="https://2019.igem.org/Team:Tec-Chihuahua/Results">2019 Tec Chihuahua</a></li>
+
−
<li><a href="https://2020.igem.org/Team:Aalto-Helsinki/Results">2020 Aalto Helsinki</a></li>
+
−
<li><a href="https://2020.igem.org/Team:GreatBay_SCIE/Results">2020 GreatBay SCIE</a></li>
+
−
<li><a href="https://2020.igem.org/Team:Queens_Canada/Results">2020 Queens Canada</a></li>
+
−
 
+
−
</ul>
+
−
</div>
+
−
</div>
+
−
 
+
  
 +
<head>
 +
    <meta charset="UTF-8">
 +
    <meta http-equiv="X-UA-Compatible" content="IE=edge">
 +
    <meta name="viewport" content="width=device-width, initial-scale=1.0">
 +
    <title>Shanghai_Metro_Utd</title>
 +
    <link rel="stylesheet"
 +
        href="https://2021.igem.org/wiki/index.php?title=Template:Shanghai_Metro_Utd/Main_CSS&action=raw&ctype=text/css" />
  
 +
</head>
  
 +
<body>
 +
    <nav class="head-nav clearfix">
 +
        <div class="top-block"></div>
 +
        <div class="top-nav-bar">
 +
            <ul class="clearfix">
 +
                <span class="small-logo"></span>
 +
                <li>
 +
                    <a href="https://2021.igem.org/Team:Shanghai_Metro_Utd">Home</a>
 +
                </li>
 +
                <li class="active">
 +
                    <a href="">Project</a>
 +
                    <div class="sub-nav">
 +
                        <ul>
 +
                            <li><a href="https://2021.igem.org/Team:Shanghai_Metro_Utd/Description"
 +
                                    class="sub-nav-74">Description</a></li>
 +
                            <li><a href="https://2021.igem.org/Team:Shanghai_Metro_Utd/Experiments"
 +
                                    class="sub-nav-74">Experiments</a></li>
 +
                            <li class="current-sub-nav"><a href="#" class="sub-nav-74">Results</a></li>
 +
                            <li><a href="https://2021.igem.org/Team:Shanghai_Metro_Utd/Proof_Of_Concept"
 +
                                    class="sub-nav-52">Proof Of Concept</a></li>
 +
                            <li><a href="https://2021.igem.org/Team:Shanghai_Metro_Utd/Notebook"
 +
                                    class="sub-nav-52">Notebook</a></li>
 +
                            <li><a href="https://2021.igem.org/Team:Shanghai_Metro_Utd/Safety">Safety</a></li>
 +
                        </ul>
 +
                    </div>
 +
                </li>
 +
                <li>
 +
                    <a href="">Parts</a>
 +
                    <div class="sub-nav">
 +
                        <ul>
 +
                            <li><a href="https://2021.igem.org/Team:Shanghai_Metro_Utd/Parts" class="sub-nav-74">Parts
 +
                                    Collection</a></li>
 +
                            <li><a href="https://2021.igem.org/Team:Shanghai_Metro_Utd/Engineering"
 +
                                    class="sub-nav-74">Engineering</a></li>
 +
                            <li><a href="https://2021.igem.org/Team:Shanghai_Metro_Utd/Contribution"
 +
                                    class="sub-nav-74">Contribution</a></li>
 +
                        </ul>
 +
                    </div>
 +
                </li>
 +
                <li>
 +
                    <a href="">Human Practices</a>
 +
                    <div class="sub-nav">
 +
                        <ul>
 +
                            <li><a href="https://2021.igem.org/Team:Shanghai_Metro_Utd/Human_Practices"
 +
                                    class="sub-nav-74">Integrated Human Practice</a></li>
 +
                            <li><a href="https://2021.igem.org/Team:Shanghai_Metro_Utd/Communication"
 +
                                    class="sub-nav-74">Communication</a></li>
 +
                            <li><a href="https://2021.igem.org/Team:Shanghai_Metro_Utd/Fundraising"
 +
                                    class="sub-nav-74">Fundraising</a></li>
 +
                        </ul>
 +
                    </div>
 +
                </li>
 +
                <li>
 +
                    <a href="https://2021.igem.org/Team:Shanghai_Metro_Utd/Implementation">Implementation</a>
 +
                </li>
 +
                <li>
 +
                    <a href="https://2021.igem.org/Team:Shanghai_Metro_Utd/Entrepreneurship">Entrepreneurship</a>
 +
                </li>
 +
                <li>
 +
                    <a href="https://2021.igem.org/Team:Shanghai_Metro_Utd/Model">Model</a>
 +
                </li>
 +
                <li>
 +
                    <a href="">Team</a>
 +
                    <div class="sub-nav">
 +
                        <ul>
 +
                            <li><a href="https://2021.igem.org/Team:Shanghai_Metro_Utd/Members" class="sub-nav-74">Team
 +
                                    Members</a></li>
 +
                            <li><a href="https://2021.igem.org/Team:Shanghai_Metro_Utd/Attributions"
 +
                                    class="sub-nav-74">Attributions</a></li>
 +
                            <li><a href="https://2021.igem.org/Team:Shanghai_Metro_Utd/Collaborations"
 +
                                    class="sub-nav-74">Collaborations</a></li>
 +
                        </ul>
 +
                    </div>
 +
                </li>
 +
            </ul>
 +
        </div>
 +
    </nav>
 +
    <div class="sub-content">
 +
        <div class="sub-title">Results</div>
 +
        <div class="article-title">Overview</div>
 +
        <div class="article-content">As previously mentioned, we are interested in the property of angiogenin, and we
 +
            hope to get its prokaryotic expression. </div>
 +
        <div class="article-content">Section 1 is made up of Plasmid Extraction, Restriction Enzyme Digestion,
 +
            Transformation, and Recombination. Section 2 is made up of Protein Inducible Expression, Preparation and
 +
            Dissolution of Inclusion Body, Renaturation, Purification, and Activity Validation. </div>
 +
        <div class="article-title">Results</div>
 +
        <div class="article-content"><b>Plasmid Construction</b></div>
 +
        <div class="img-wrap no-margin">
 +
            <img src="https://static.igem.org/mediawiki/2021/2/22/T--Shanghai_Metro_Utd--result01.jpg" alt="" />
 +
            <span>Figure 1: the extracted plasmid which has been used in the later part of Restriction Enzyme Digestion
 +
                and Transformation.</span>
 +
        </div>
 +
        <div class="article-content">We extracted pET-28a plasmid for the use in the later part of restriction enzyme
 +
            digestion and transformation.</div>
 +
        <div class="img-wrap no-margin">
 +
            <img src="https://static.igem.org/mediawiki/2021/7/71/T--Shanghai_Metro_Utd--result02.jpg" alt="" />
 +
            <span>Figure 2: the resulting concentration of the extracted plasmid in Figure 1</span>
 +
        </div>
 +
        <div class="article-content">We measured the concentration of the extracted pET-28a plasmid. According to the
 +
            concentration, we took a
 +
            certain volume for enzyme digestion.</div>
 +
        <div class="img-wrap no-margin">
 +
            <img src="https://static.igem.org/mediawiki/2021/1/19/T--Shanghai_Metro_Utd--result03.jpg" alt="" />
 +
            <span>Figure 3: Construction flowchart of the target plasmid pET-28a-rANG (by Snapgene)</span>
 +
        </div>
 +
        <div class="img-wrap no-margin">
 +
            <img src="https://static.igem.org/mediawiki/2021/f/fc/T--Shanghai_Metro_Utd--result04.jpg" alt="" />
 +
            <span>Figure 4: the scanning gel electrophoresis map of the plasmid vector after enzyme digestion</span>
 +
        </div>
 +
        <div class="article-content">As seen in figure 4, lane 1 is the pET-28a without enzyme digestion and lane 2 to 9
 +
            is the result for pET-28a after enzyme digestion. The bands of our plasmid vectors after enzyme
 +
            digestion(5311bp) showed at 5000 bp around which are correct and the bands at 300 bp around were the SUMO
 +
            fragments that were cut by enzyme digestion. After that, we could further conduct the E. coli
 +
            transformation.</div>
 +
        <div class="img-wrap no-margin">
 +
            <img src="https://static.igem.org/mediawiki/2021/3/3f/T--Shanghai_Metro_Utd--result05.jpg" alt="" />
 +
            <span>Figure 5: The gel electrophoresis map of PCR</span>
 +
        </div>
 +
        <div class="article-content">Lane 1 to 6 is the result of PCR. We got rANG band at around 400bp (369bp). Enzyme
 +
            digestion was conducted and it was linked with digested pET-28a.</div>
 +
        <div class="img-wrap no-margin">
 +
            <img src="https://static.igem.org/mediawiki/2021/5/5d/T--Shanghai_Metro_Utd--chg2.jpg" alt="" />
 +
            <img src="https://static.igem.org/mediawiki/2021/3/3e/T--Shanghai_Metro_Utd--chg3.jpg" />
 +
            <span>Figure 6. E. coil having the desired pET28a-rANG</span>
 +
        </div>
 +
        <div class="article-content">
 +
            The plates in figure 6 (1) showed monoclonals of pET28a-rANG constructs.<br />
 +
            The pET28a-rANG was constructed successfully which has been proved by sequencing as shown in figure 6 (2).
 +
        </div>
 +
        <div class="article-content"><b>Recombination E. coli</b></div>
 +
        <div class="img-wrap no-margin">
 +
            <img src="https://static.igem.org/mediawiki/2021/a/a1/T--Shanghai_Metro_Utd--result07.jpg" alt="" />
 +
            <span>Figure 7: the scanning gel electrophoresis map of Colony PCR (the top was before and the bottom was
 +
                after)</span>
 +
        </div>
 +
        <div class="article-content">Lane NC to XCN6 and lane XCN7 to N6 are the results of colony PCR. We get the band
 +
            531bp at 500bp around. It indicates that the obtained recombinant monoclonals were positive monoclonals
 +
            containing the pET-28a-rANG recombinant plasmid.</div>
 +
        <div class="article-title">SDS PAGE</div>
 +
        <div class="img-wrap no-margin">
 +
            <img src="https://static.igem.org/mediawiki/2021/7/7e/T--Shanghai_Metro_Utd--result08.jpg" alt="" />
 +
            <span>Figure 8. SDS-PAGE Analyzing the proteins before and after induction, within inclusion bodies, and in
 +
                supernatants. </span>
 +
        </div>
 +
        <div class="article-content">The results for SDS-PAGE shows that the induction was successful, and the target
 +
            protein (about 15KDa) was
 +
            mainly in the inclusion body.</div>
 +
        <div class="article-content"><b>Protein Purification</b></div>
 +
        <div class="img-wrap no-margin">
 +
            <img src="https://static.igem.org/mediawiki/2021/1/1d/T--Shanghai_Metro_Utd--chg4.jpg" alt="" />
 +
            <span>Figure 9. SDS-PAGE and stained with Coomassie Brilliant Blue after protein purifying: (1) first
 +
                attempt; (2) second attempt</span>
 +
        </div>
 +
        <div class="article-content">Based on figure 9 (1), there was a band at 15 KDa before passing through the column
 +
            but disappeared after passing through the column, indicating that the protein had hung the column. But we
 +
            notice that there is a miscellaneous band with a small amount. In order to obtain the purified protein, we
 +
            repeated the protein purification experiments and finally obtained the more purified protein as seen in
 +
            figure 9 (2) where you could only see one band at 15 KDa.</div>
 +
        <div class="img-wrap no-margin">
 +
            <img src="https://static.igem.org/mediawiki/2021/f/fd/T--Shanghai_Metro_Utd--result10.jpg" alt="" />
 +
            <span>Figure 10. First SDS-PAGE, Coomassie blue staining. Samples from left to right: marker; 1.before
 +
                column(unconcentrated); 2. after column 1(unconcentrated); 3. after column 2(unconcentrated); 4. buffer
 +
                A rinsed; 5 second elution; 6 third elution.</span>
 +
        </div>
 +
        <div class="img-wrap no-margin">
 +
            <img src="https://static.igem.org/mediawiki/2021/2/26/T--Shanghai_Metro_Utd--result11.jpg" alt="" />
 +
            <span>Figure 11. Second SDS-PAGE, Coomassie blue staining. Samples from left to right: marker; 1. second
 +
                elution; 2.third elution; 3.fourth elution; 4. fifth elution.</span>
 +
        </div>
 +
        <div class="article-content">It can be seen from Figure 10 and Figure 11 that there is actually not much protein
 +
            after the third
 +
            elution.</div>
 +
        <div class="img-wrap no-margin">
 +
            <img src="https://static.igem.org/mediawiki/2021/9/94/T--Shanghai_Metro_Utd--result12.jpg" alt="" />
 +
            <span>Figure 12 Lyophilized protein</span>
 +
        </div>
 +
        <div class="article-content">As seen in figure 12, the protein was successfully lyophilized into the form of
 +
            powder.</div>
 +
        <div class="article-content"><b>RNA Extraction</b></div>
 +
        <div class="img-wrap no-margin">
 +
            <img src="https://static.igem.org/mediawiki/2021/3/31/T--Shanghai_Metro_Utd--result13.jpg" alt="" />
 +
            <span>Figure 13. The concentration of the extracted RNA by nanodrop</span>
 +
        </div>
 +
        <div class="article-content"><b>RNA Degradation Experiment</b></div>
 +
        <div class="img-wrap no-margin">
 +
            <img src="https://static.igem.org/mediawiki/2021/d/d7/T--Shanghai_Metro_Utd--result14.jpg" alt="" />
 +
            <span>Figure 14. electrophoresis map after RNA degradation tests </span>
 +
        </div>
 +
        <div class="article-content">According to the electrophoresis map, the renatured rANG has ribonuclease activity
 +
            with the capability of RNA incomplete degradation and produced a band at about 100 bp, which is consistent
 +
            with the data in the literatures (Ref. 1, 2).</div>
 +
        <div class="img-wrap no-margin">
 +
            <img src="https://static.igem.org/mediawiki/2021/9/91/T--Shanghai_Metro_Utd--result15.jpg" alt="" />
 +
            <span>Figure 15. OD<sub>450</sub> curve of the system with heparin and rANG</span>
 +
        </div>
 +
        <div class="article-content">The OD<sub>450</sub> could indicate the interaction ability between heparin and
 +
            angiogenin which means that the higher the OD<sub>450</sub> value, the greater the interaction ability
 +
            between heparin and angiogenin. This curve shows the more angiogenin we added, the higher the OD450 value,
 +
            namely the interaction ability between heparin and angiogenin. Therefore, it can be concluded that the
 +
            interaction of heparin has dose-dependent with angiogenin.</div>
 +
        <div class="article-title">Future Plan</div>
 +
        <div class="article-content">As we have successfully obtained the eingineered E. coli to produce the angiogenin
 +
            with well activity. We would further focus more on the improvement of the purity and yield of our protein
 +
            and more tests should be conducted to ensure the safety and stability.</div>
 +
        <div class="article-title">Reference:</div>
 +
        <div class="article-content">[1]孙德森,盛静浩.重组人血管生成素制备和生物活性鉴定[J].中国生物化学与分子生物学报,2015,31(12):1315-1321.</div>
 +
        <div class="article-content">[2] Shapiro R, Riordan JF, Vallee BL. Characteristic ribonucleolytic activity of
 +
            human angiogenin. Biochemistry. 1986 Jun 17;25(12):3527-32. doi: 10.1021/bi00360a008. Erratum in:
 +
            Biochemistry 1986 Oct 21;25(21):6730. PMID: 2424496.</div>
 +
    </div>
 +
    <footer class="footer">
 +
        <section class="footer-wrap">
 +
            <p class="margin-bottom-14">Email us at:<i
 +
                    style="color:#0546ff; font-style: normal;">ashleyyang48@gmail.com;alexlin711@gmail.com</i></p>
 +
            <p>Follow us on Wechat: <i style="color:#650000; font-size: 18px;">血管马力Bloody
 +
                    Marry</i></p>
 +
        </section>
 +
    </footer>
 +
</body>
 +
<script>
 +
    let liTags = document.querySelectorAll(".top-nav-bar > ul > li");
 +
    let len = liTags.length;
 +
    for (let i = 0; i < len; i++) {
 +
        liTags[i].onclick = function (e) {
 +
            //先移除所有的点击样式
 +
            for (let j = 0; j < len; j++) {
 +
                liTags[j].classList.remove("active");
 +
            }
 +
            //再添加点击样式
 +
            let li = e.currentTarget;
 +
            li.classList.add("active");
 +
        }
 +
    }
 +
</script>
  
 
</html>
 
</html>

Latest revision as of 18:56, 19 October 2021

Shanghai_Metro_Utd

Results
Overview
As previously mentioned, we are interested in the property of angiogenin, and we hope to get its prokaryotic expression.
Section 1 is made up of Plasmid Extraction, Restriction Enzyme Digestion, Transformation, and Recombination. Section 2 is made up of Protein Inducible Expression, Preparation and Dissolution of Inclusion Body, Renaturation, Purification, and Activity Validation.
Results
Plasmid Construction
Figure 1: the extracted plasmid which has been used in the later part of Restriction Enzyme Digestion and Transformation.
We extracted pET-28a plasmid for the use in the later part of restriction enzyme digestion and transformation.
Figure 2: the resulting concentration of the extracted plasmid in Figure 1
We measured the concentration of the extracted pET-28a plasmid. According to the concentration, we took a certain volume for enzyme digestion.
Figure 3: Construction flowchart of the target plasmid pET-28a-rANG (by Snapgene)
Figure 4: the scanning gel electrophoresis map of the plasmid vector after enzyme digestion
As seen in figure 4, lane 1 is the pET-28a without enzyme digestion and lane 2 to 9 is the result for pET-28a after enzyme digestion. The bands of our plasmid vectors after enzyme digestion(5311bp) showed at 5000 bp around which are correct and the bands at 300 bp around were the SUMO fragments that were cut by enzyme digestion. After that, we could further conduct the E. coli transformation.
Figure 5: The gel electrophoresis map of PCR
Lane 1 to 6 is the result of PCR. We got rANG band at around 400bp (369bp). Enzyme digestion was conducted and it was linked with digested pET-28a.
Figure 6. E. coil having the desired pET28a-rANG
The plates in figure 6 (1) showed monoclonals of pET28a-rANG constructs.
The pET28a-rANG was constructed successfully which has been proved by sequencing as shown in figure 6 (2).
Recombination E. coli
Figure 7: the scanning gel electrophoresis map of Colony PCR (the top was before and the bottom was after)
Lane NC to XCN6 and lane XCN7 to N6 are the results of colony PCR. We get the band 531bp at 500bp around. It indicates that the obtained recombinant monoclonals were positive monoclonals containing the pET-28a-rANG recombinant plasmid.
SDS PAGE
Figure 8. SDS-PAGE Analyzing the proteins before and after induction, within inclusion bodies, and in supernatants.
The results for SDS-PAGE shows that the induction was successful, and the target protein (about 15KDa) was mainly in the inclusion body.
Protein Purification
Figure 9. SDS-PAGE and stained with Coomassie Brilliant Blue after protein purifying: (1) first attempt; (2) second attempt
Based on figure 9 (1), there was a band at 15 KDa before passing through the column but disappeared after passing through the column, indicating that the protein had hung the column. But we notice that there is a miscellaneous band with a small amount. In order to obtain the purified protein, we repeated the protein purification experiments and finally obtained the more purified protein as seen in figure 9 (2) where you could only see one band at 15 KDa.
Figure 10. First SDS-PAGE, Coomassie blue staining. Samples from left to right: marker; 1.before column(unconcentrated); 2. after column 1(unconcentrated); 3. after column 2(unconcentrated); 4. buffer A rinsed; 5 second elution; 6 third elution.
Figure 11. Second SDS-PAGE, Coomassie blue staining. Samples from left to right: marker; 1. second elution; 2.third elution; 3.fourth elution; 4. fifth elution.
It can be seen from Figure 10 and Figure 11 that there is actually not much protein after the third elution.
Figure 12 Lyophilized protein
As seen in figure 12, the protein was successfully lyophilized into the form of powder.
RNA Extraction
Figure 13. The concentration of the extracted RNA by nanodrop
RNA Degradation Experiment
Figure 14. electrophoresis map after RNA degradation tests
According to the electrophoresis map, the renatured rANG has ribonuclease activity with the capability of RNA incomplete degradation and produced a band at about 100 bp, which is consistent with the data in the literatures (Ref. 1, 2).
Figure 15. OD450 curve of the system with heparin and rANG
The OD450 could indicate the interaction ability between heparin and angiogenin which means that the higher the OD450 value, the greater the interaction ability between heparin and angiogenin. This curve shows the more angiogenin we added, the higher the OD450 value, namely the interaction ability between heparin and angiogenin. Therefore, it can be concluded that the interaction of heparin has dose-dependent with angiogenin.
Future Plan
As we have successfully obtained the eingineered E. coli to produce the angiogenin with well activity. We would further focus more on the improvement of the purity and yield of our protein and more tests should be conducted to ensure the safety and stability.
Reference:
[1]孙德森,盛静浩.重组人血管生成素制备和生物活性鉴定[J].中国生物化学与分子生物学报,2015,31(12):1315-1321.
[2] Shapiro R, Riordan JF, Vallee BL. Characteristic ribonucleolytic activity of human angiogenin. Biochemistry. 1986 Jun 17;25(12):3527-32. doi: 10.1021/bi00360a008. Erratum in: Biochemistry 1986 Oct 21;25(21):6730. PMID: 2424496.

Email us at:ashleyyang48@gmail.com;alexlin711@gmail.com

Follow us on Wechat: 血管马力Bloody Marry