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<div class="sub-nav"> | <div class="sub-nav"> | ||
<ul> | <ul> | ||
− | <li><a href="https://2021.igem.org/Team:Shanghai_Metro_Utd/ | + | <li><a href="https://2021.igem.org/Team:Shanghai_Metro_Utd/Parts" class="sub-nav-74">Parts |
− | + | ||
Collection</a></li> | Collection</a></li> | ||
<li><a href="https://2021.igem.org/Team:Shanghai_Metro_Utd/Engineering" | <li><a href="https://2021.igem.org/Team:Shanghai_Metro_Utd/Engineering" | ||
class="sub-nav-74">Engineering</a></li> | 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> | </ul> | ||
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digestion was conducted and it was linked with digested pET-28a.</div> | digestion was conducted and it was linked with digested pET-28a.</div> | ||
<div class="img-wrap no-margin"> | <div class="img-wrap no-margin"> | ||
− | <img src="https://static.igem.org/mediawiki/2021/ | + | <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> | <span>Figure 6. E. coil having the desired pET28a-rANG</span> | ||
</div> | </div> | ||
<div class="article-content"> | <div class="article-content"> | ||
− | The pET28a-rANG | + | The plates in figure 6 (1) showed monoclonals of pET28a-rANG constructs.<br /> |
− | The | + | The pET28a-rANG was constructed successfully which has been proved by sequencing as shown in figure 6 (2). |
</div> | </div> | ||
<div class="article-content"><b>Recombination E. coli</b></div> | <div class="article-content"><b>Recombination E. coli</b></div> | ||
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<div class="article-content"><b>Protein Purification</b></div> | <div class="article-content"><b>Protein Purification</b></div> | ||
<div class="img-wrap no-margin"> | <div class="img-wrap no-margin"> | ||
− | <img src="https://static.igem.org/mediawiki/2021/1/ | + | <img src="https://static.igem.org/mediawiki/2021/1/1d/T--Shanghai_Metro_Utd--chg4.jpg" alt="" /> |
− | <span>Figure 9. | + | <span>Figure 9. SDS-PAGE and stained with Coomassie Brilliant Blue after protein purifying: (1) first |
− | + | attempt; (2) second attempt</span> | |
</div> | </div> | ||
− | <div class="article-content">Based on figure 9, there was a band at 15 KDa before passing through the column | + | <div class="article-content">Based on figure 9 (1), there was a band at 15 KDa before passing through the column |
− | disappeared after passing through the column, indicating that the protein had hung the column. But we | + | but disappeared after passing through the column, indicating that the protein had hung the column. But we |
− | that there is a miscellaneous band with a small amount, | + | 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"> | <div class="img-wrap no-margin"> | ||
<img src="https://static.igem.org/mediawiki/2021/f/fd/T--Shanghai_Metro_Utd--result10.jpg" alt="" /> | <img src="https://static.igem.org/mediawiki/2021/f/fd/T--Shanghai_Metro_Utd--result10.jpg" alt="" /> |
Latest revision as of 18:56, 19 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/2021/5/5d/T--Shanghai_Metro_Utd--chg2.jpg)
![](https://static.igem.org/mediawiki/2021/3/3e/T--Shanghai_Metro_Utd--chg3.jpg)
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).
The pET28a-rANG was constructed successfully which has been proved by sequencing as shown in figure 6 (2).
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/2021/1/1d/T--Shanghai_Metro_Utd--chg4.jpg)
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.
![](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.