Difference between revisions of "Team:NUDT CHINA/Collaborations"

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<span><span style="color:#7f1015">Mentoring TMMU_China </span></span><hr  
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<span><span style="color:#7f1015">Collaboration with NJU-China</span></span><hr />
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<span style="line-height:2;font-family:Perpetua;font-size:18px;">It was
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<span style="line-height:2;font-family:Perpetua;font-size:18px;">NJU-China performed a Cas9-digest assay to help us validating the effectiveness of their sgRNAs, since our team met some trouble in assessing the efficiency of the single guide RNAs we designed for our further experiments. Also, they helped us to optimize our own protocol for in vitro expression of sgRNAs, so that our project could be performed more smoothly. </span>
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</p>
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the first time for TMMU_China  to take part in iGEM, so they had something
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<p style="text-indent:22pt;">
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unfamiliar about it. We provided a vigorous support for their project from
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<span style="line-height:2;font-family:Perpetua;font-size:18px;">In return, we performed TEM imaging of exosomes carrying KRAS siRNA and expressing iRGD peptide on their membrane for NJU-China, as well as nanoparticle tracking analysis (NTA) to help them to ascertain the relationship between protein concentration and exosomes quantity.</span>  
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beginning to end.</span>  
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</p>
 
</p>
 
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<h3>
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<span><span style="color:#7f1015">Collaboration with NJU-China</span></span><hr />
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</h3>
  
 
<!--正文-->
 
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<p style="text-indent:22pt;">
 
<p style="text-indent:22pt;">
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<span style="line-height:2;font-family:Perpetua;font-size:18px;">In
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<span style="line-height:2;font-family:Perpetua;font-size:18px;">After co-transfection of the two plasmids mentioned above, we performed a transmission electron microscopy (TEM) to characterize the iRGD-modified siRNA-loaded exosomes. The TEM image showed that the exosomes presented normal morphological characteristics after outside modification and siRNA loading, with a diameter of approximately 200 nanometer and a double-layer membrane. These characteristics indicate that the exosome properties won’t be affected by our modifications. </span>
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March of 2016, we attended a meet-up hosted by The Third Military Medical
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[[File:T--NUDT_CHINA--collaborationsfig1.jpg|700px|center]]
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University (TMMU) to discuss on some issues about iGEM.</span>  
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<span style="line-height:2;font-family:Perpetua;font-size:16px;text-align:center;font-weight:bold">Figure1.TEM image of iRGD-modified exosomes packaging KRAS siRNA</span>  
 
</p>
 
</p>
 
</br>
 
</br>
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<h3>
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<span><span style="color:#7f1015">Nanoparticle tracking analysis to ascertain the relationship between protein concentration and exosomes quantity</span></span><hr />
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</h3>
  
 
<!--正文-->
 
<!--正文-->
 
<p style="text-indent:22pt;">
 
<p style="text-indent:22pt;">
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<span style="line-height:2;font-family:Perpetua;font-size:18px;">We  
+
<span style="line-height:2;font-family:Perpetua;font-size:18px;">We then performed nanoparticle tracking analysis (NTA) for a further evaluation of the quantity and size of secreted exosomes. The use of Nanosight enabled quantification and size determination of the extracellular vesicles, as nanoparticles can be automatically tracked and sized based on Brownian motion and the diffusion coefficient. The size of exosomes attained ranged around 270nm. Basing the particle size and relative intensity, we also created a 3D plot for a visual explanation. Under measurement condition listed, the exosomes extracted from HEK293 cells were assayed for 2.95 E8 particles each milliliter. Then the relationship between particle number and protein was determined that exosomes in 1 ng protein were equivalent to 6277.95 particles, according to the dilution multiple (24) and protein concentration (1127.756 ng/ul) we have tested. All the data collected help us decide the transfection dosage of siRNA and dosing of treatment prepared for animal experiment.</span>
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</p>
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</br>
  
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introduced the concepts and rules for iGEM in detail, and we also talked about
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[[File:T--NUDT_CHINA--collaborationsfig2.jpg|700px|center]]
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some crucial points that should be paid attention to. Having had two years’
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<!--图注-->
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<p>
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<span style="line-height:2;font-family:Perpetua;font-size:16px;font-weight:bold">Figure 2. Nanoparticle tracking analysis (NTA) for characterization of secreted exosomes.</span>
  
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experience of taking part in the iGEM, we also shared our own lessons that we
 
  
−
got from iGEM.</span>  
+
<!--图注-->
 +
 
 +
<span style="line-height:2;font-family:Perpetua;font-size:16px;">(a) Concentration of different particle sizes of exosomes. (b) 3D plot of particle size and relative intensity. (c) Experiment condition for our measurement. (d) Results attained after measurement of exosomes.</span>  
 
</p>
 
</p>
 
</br>
 
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</br>
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[[File:T--NUDT_CHINA--collaborationsteam1.jpg|700px|center]]
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<html>
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</br>
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<!--标题-->
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<h2>
 +
<span><span style="color:#7f1015">Mentoring TMMU-China</span></span><hr />
 +
</h2>
  
 
<!--正文-->
 
<!--正文-->
 
<p style="text-indent:22pt;">
 
<p style="text-indent:22pt;">
−
<span style="line-height:2;font-family:Perpetua;font-size:18px;">During
+
<span style="line-height:2;font-family:Perpetua;font-size:18px;">It was the first time for TMMU-China to take part in iGEM, so they had something unfamiliar about it. We provided a vigorous support for their project from beginning to end.</span>
 +
</p>
 +
</br>
  
−
the meet-up, both teams communicated and exchanged each other’s ideas through
+
<!--正文-->
 +
<p style="text-indent:22pt;">
 +
<span style="line-height:2;font-family:Perpetua;font-size:18px;">In March of 2016, we attended a meet-up hosted by The Third Military Medical University (TMMU) to discuss on some issues about iGEM.</span>
 +
</p>
 +
</br>
  
−
presentation, brainstorm and other methods. This discussion had brought us some
 
  
−
inspiration for our own project this year. </span>  
+
<!--正文-->
 +
<p style="text-indent:22pt;">
 +
<span style="line-height:2;font-family:Perpetua;font-size:18px;">We introduced the concepts and rules for iGEM in detail, and we also talked about some crucial points that should be paid attention to. Having had two years’ experience of taking part in the iGEM, we also shared our own lessons that we got from iGEM.</span>  
 
</p>
 
</p>
 
</br>
 
</br>
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<!--正文-->
 
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<p style="text-indent:22pt;">
 
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<span style="line-height:2;font-family:Perpetua;font-size:18px;">The
+
<span style="line-height:2;font-family:Perpetua;font-size:18px;">During the meet-up, both teams communicated and exchanged each other’s ideas through presentation, brainstorm and other methods. This discussion had brought us some inspiration for our own project this year.</span>
 +
</p>
 +
</br>
  
−
connection between us has accelerated the project's improvement. Meanwhile, it  
+
<!--正文-->
−
 
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<p style="text-indent:22pt;">
−
let us understand one of iGEM’s core value---collaboration, sharing and  
+
<span style="line-height:2;font-family:Perpetua;font-size:18px;">The connection between us has accelerated the project's improvement. Meanwhile, it let us understand one of iGEM’s core value---collaboration, sharing and community.</span>  
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community. </span>  
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</p>
 
</p>
 
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[[File:T--NUDT_CHINA--collaborationsteam2.jpg|700px|center]]
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[[File:T--NUDT_CHINA--collaborationsfig3.jpg|700px|center]]
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[[File:T--NUDT_CHINA--collaborationsfig4.jpg|700px|center]]
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<span><span style="color:#7f1015">Collaboration with NEFU_China</span></span><hr />
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<span style="line-height:2;font-family:Perpetua;font-size:18px;">In August of this year, we went to NEFU_China and carried out an issue communication. They took advantage of magnet tool to purify protein, which would improve the yield and purity of some protein. This idea may help us reduce the complexity of the protein cas9 purification, so as to focus more on the miRNA detection method developments. Besides, they are also very interested in our project and describe it open, close to the forefront and worth their studying. Briefly, we are pleasant to have the communication with NEFU_China.</span>  
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<td style = "padding: 0px 15px;"><img src="https://static.igem.org/mediawiki/2016/a/a4/T--NUDT_CHINA--collaborationfig3.jpg" style = "width: auto; height:200px;"></td>
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<td style = "padding: 0px 15px;"><img src="https://static.igem.org/mediawiki/2016/7/77/T--NUDT_CHINA--collaborationfig1.jpg" style = "width: auto; height:200px;"></td>
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<td>NUDT_CHINA</td>
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<!--正文-->
 
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<p style="text-indent:22pt;">
 
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<span style="line-height:2;font-family:Perpetua;font-size:18px;">In
 
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August of this year, we went to NEFU_China and carried out an issue
 
−
 
−
communication. They took advantage of magnet tool to purify protein, which would
 
−
 
−
improve the yield and purity of some protein. This idea may help us reduce the
 
−
 
−
complexity of the protein cas9 purification, so as to focus more on the miRNA
 
−
 
−
detection method developments. Besides, they are also very interested in our
 
−
 
−
project and describe it open, close to the forefront and worth their studying.
 
−
 
−
Briefly, we are pleasant to have the communication with NEFU_China.</span>
 
−
</p>
 
−
</br>
 
  
  
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<td style = "padding: 0px 15px;"><img src="https://static.igem.org/mediawiki/2016/c/ce/T--NUDT_CHINA--Collaborationfig2.jpg" style = "width: auto; height:200px;"></td>
 
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Revision as of 13:45, 19 October 2016

NUDT_CHINA 2016

HomePage • TEAM • Collaborations

Collaboration with NJU-China

NJU-China performed a Cas9-digest assay to help us validating the effectiveness of their sgRNAs, since our team met some trouble in assessing the efficiency of the single guide RNAs we designed for our further experiments. Also, they helped us to optimize our own protocol for in vitro expression of sgRNAs, so that our project could be performed more smoothly.


In return, we performed TEM imaging of exosomes carrying KRAS siRNA and expressing iRGD peptide on their membrane for NJU-China, as well as nanoparticle tracking analysis (NTA) to help them to ascertain the relationship between protein concentration and exosomes quantity.


Collaboration with NJU-China

After co-transfection of the two plasmids mentioned above, we performed a transmission electron microscopy (TEM) to characterize the iRGD-modified siRNA-loaded exosomes. The TEM image showed that the exosomes presented normal morphological characteristics after outside modification and siRNA loading, with a diameter of approximately 200 nanometer and a double-layer membrane. These characteristics indicate that the exosome properties won’t be affected by our modifications.



T--NUDT CHINA--collaborationsfig1.jpg


Figure1.TEM image of iRGD-modified exosomes packaging KRAS siRNA


Nanoparticle tracking analysis to ascertain the relationship between protein concentration and exosomes quantity

We then performed nanoparticle tracking analysis (NTA) for a further evaluation of the quantity and size of secreted exosomes. The use of Nanosight enabled quantification and size determination of the extracellular vesicles, as nanoparticles can be automatically tracked and sized based on Brownian motion and the diffusion coefficient. The size of exosomes attained ranged around 270nm. Basing the particle size and relative intensity, we also created a 3D plot for a visual explanation. Under measurement condition listed, the exosomes extracted from HEK293 cells were assayed for 2.95 E8 particles each milliliter. Then the relationship between particle number and protein was determined that exosomes in 1 ng protein were equivalent to 6277.95 particles, according to the dilution multiple (24) and protein concentration (1127.756 ng/ul) we have tested. All the data collected help us decide the transfection dosage of siRNA and dosing of treatment prepared for animal experiment.



T--NUDT CHINA--collaborationsfig2.jpg


Figure 2. Nanoparticle tracking analysis (NTA) for characterization of secreted exosomes. (a) Concentration of different particle sizes of exosomes. (b) 3D plot of particle size and relative intensity. (c) Experiment condition for our measurement. (d) Results attained after measurement of exosomes.



T--NUDT CHINA--collaborationsteam1.jpg


Mentoring TMMU-China

It was the first time for TMMU-China to take part in iGEM, so they had something unfamiliar about it. We provided a vigorous support for their project from beginning to end.


In March of 2016, we attended a meet-up hosted by The Third Military Medical University (TMMU) to discuss on some issues about iGEM.


We introduced the concepts and rules for iGEM in detail, and we also talked about some crucial points that should be paid attention to. Having had two years’ experience of taking part in the iGEM, we also shared our own lessons that we got from iGEM.


During the meet-up, both teams communicated and exchanged each other’s ideas through presentation, brainstorm and other methods. This discussion had brought us some inspiration for our own project this year.


The connection between us has accelerated the project's improvement. Meanwhile, it let us understand one of iGEM’s core value---collaboration, sharing and community.



T--NUDT CHINA--collaborationsteam2.jpg



T--NUDT CHINA--collaborationsfig3.jpg



T--NUDT CHINA--collaborationsfig4.jpg


Collaboration with NEFU_China

In August of this year, we went to NEFU_China and carried out an issue communication. They took advantage of magnet tool to purify protein, which would improve the yield and purity of some protein. This idea may help us reduce the complexity of the protein cas9 purification, so as to focus more on the miRNA detection method developments. Besides, they are also very interested in our project and describe it open, close to the forefront and worth their studying. Briefly, we are pleasant to have the communication with NEFU_China.



T--NUDT CHINA--collaborationsteam3.jpg



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