(Prototype team page) |
|||
Line 1: | Line 1: | ||
− | + | <html lang="en"> | |
− | + | ||
− | <html | + | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
+ | <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/Collection" | ||
+ | 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> | ||
+ | </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/2021/4/4d/T--Shanghai_Metro_Utd--result06.jpg" alt="" /> | ||
+ | <span>Figure 6. E. coil having the desired pET28a-rANG</span> | ||
+ | </div> | ||
+ | <div class="article-content"> | ||
+ | The pET28a-rANG was constructed.<br /> | ||
+ | The plates showed monoclonals of pET28a-rANG constructs. | ||
+ | </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/2021/1/10/T--Shanghai_Metro_Utd--result09.jpg" alt="" /> | ||
+ | <span>Figure 9. The solution before and after the column chromatography and the corresponding concentrated | ||
+ | solution run SDS-PAGE and stained with Coomassie Brilliant Blue.</span> | ||
+ | </div> | ||
+ | <div class="article-content">Based on figure 9, 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, so we concentrated the solution before and after the | ||
+ | chromatography columns and SDS-PAGE was performed simultaneously.</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> |
Revision as of 16:48, 13 October 2021
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
![](https://static.igem.org/mediawiki/2021/2/22/T--Shanghai_Metro_Utd--result01.jpg)
We extracted pET-28a plasmid for the use in the later part of restriction enzyme
digestion and transformation.
![](https://static.igem.org/mediawiki/2021/7/71/T--Shanghai_Metro_Utd--result02.jpg)
We measured the concentration of the extracted pET-28a plasmid. According to the
concentration, we took a
certain volume for enzyme digestion.
![](https://static.igem.org/mediawiki/2021/1/19/T--Shanghai_Metro_Utd--result03.jpg)
![](https://static.igem.org/mediawiki/2021/f/fc/T--Shanghai_Metro_Utd--result04.jpg)
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.
![](https://static.igem.org/mediawiki/2021/3/3f/T--Shanghai_Metro_Utd--result05.jpg)
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.
![](https://static.igem.org/mediawiki/2021/4/4d/T--Shanghai_Metro_Utd--result06.jpg)
The pET28a-rANG was constructed.
The plates showed monoclonals of pET28a-rANG constructs.
The plates showed monoclonals of pET28a-rANG constructs.
Recombination E. coli
![](https://static.igem.org/mediawiki/2021/a/a1/T--Shanghai_Metro_Utd--result07.jpg)
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
![](https://static.igem.org/mediawiki/2021/7/7e/T--Shanghai_Metro_Utd--result08.jpg)
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
![](https://static.igem.org/mediawiki/2021/1/10/T--Shanghai_Metro_Utd--result09.jpg)
Based on figure 9, 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, so we concentrated the solution before and after the
chromatography columns and SDS-PAGE was performed simultaneously.
![](https://static.igem.org/mediawiki/2021/f/fd/T--Shanghai_Metro_Utd--result10.jpg)
![](https://static.igem.org/mediawiki/2021/2/26/T--Shanghai_Metro_Utd--result11.jpg)
It can be seen from Figure 10 and Figure 11 that there is actually not much protein
after the third
elution.
![](https://static.igem.org/mediawiki/2021/9/94/T--Shanghai_Metro_Utd--result12.jpg)
As seen in figure 12, the protein was successfully lyophilized into the form of
powder.
RNA Extraction
![](https://static.igem.org/mediawiki/2021/3/31/T--Shanghai_Metro_Utd--result13.jpg)
RNA Degradation Experiment
![](https://static.igem.org/mediawiki/2021/d/d7/T--Shanghai_Metro_Utd--result14.jpg)
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).
![](https://static.igem.org/mediawiki/2021/9/91/T--Shanghai_Metro_Utd--result15.jpg)
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.