Difference between revisions of "Team:Lethbridge/Results"
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<h2>Full-Length Arc</h2> | <h2>Full-Length Arc</h2> | ||
| − | <center><img src="https://static.igem.org/mediawiki/2018/e/e9/T--Lethbridge--vincentFace.png" alt="TEM | + | <center><img src="https://static.igem.org/mediawiki/2018/e/e9/T--Lethbridge--vincentFace.png" alt="TEM Full" style="width: 200px"></center> |
<p>TEM image (60,000X magnification) of negatively-stained self-assembled full-length Arc PNCs. Scale bar = 200um.</p> | <p>TEM image (60,000X magnification) of negatively-stained self-assembled full-length Arc PNCs. Scale bar = 200um.</p> | ||
</div> | </div> | ||
<div class="twoText-Text"> | <div class="twoText-Text"> | ||
<h2>Minimal Arc Gag</h2> | <h2>Minimal Arc Gag</h2> | ||
| − | <center><img src="https://static.igem.org/mediawiki/2018/e/e9/T--Lethbridge--vincentFace.png" alt="TEM Arc | + | <center><img src="https://static.igem.org/mediawiki/2018/e/e9/T--Lethbridge--vincentFace.png" alt="TEM Arc Min" style="width:200px"></center> |
<p>TEM image (100,000X magnification) of negatively-stained self-assembled minimal Arc Gag PNCs. Scale bar = 100um.</p> | <p>TEM image (100,000X magnification) of negatively-stained self-assembled minimal Arc Gag PNCs. Scale bar = 100um.</p> | ||
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| + | <p class="f14">TEM also confirmed the presence of spherical nanoparticles in the P22 protein sample (without added scaffolding proteins; SPs) and P22 + SP-Cas9 protein samples. In the absence of the scaffolding protein, P22 capsid proteins were capable of self-assembly but with a significant variation in size and stability (many collapsed structures were evident in the sample). The P22 + SP-Cas9 PNCs had a much more consistent architecture but had a diameter of 120 +/- 5nm, which is approximately twice that of typical P22 virus-like particles. The 1:1 capsid to cargo ratio may have caused overloading of the PNCs, which caused an increase in overall size.</p> | ||
| + | </div> | ||
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| + | <h2>P22 coat proteins</h2> | ||
| + | <center><img src="https://static.igem.org/mediawiki/2018/e/e9/T--Lethbridge--vincentFace.png" alt="TEM P22" style="width: 200px"></center> | ||
| + | <p>TEM image (40,000X magnification) of negatively-stained self-assembled P22 capsids in the absence of scaffolding proteins. Scale bar = 200um.</p> | ||
| + | </div> | ||
| + | <div class="twoText-Text"> | ||
| + | <h2>P22-SP-Cas9</h2> | ||
| + | <center><img src="https://static.igem.org/mediawiki/2018/e/e9/T--Lethbridge--vincentFace.png" alt="TEM P22-SP-Cas9" style="width:200px"></center> | ||
| + | <p>TEM image (40,000X magnification) of negatively-stained self-assembled P22 capsids with the SP-Cas9 cargo. Scale bar = 100um.</p> | ||
</div> | </div> | ||
</div> | </div> | ||
Revision as of 02:31, 16 October 2018
Purification of Arc Min and Arc Full
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Transmission Electron Microscopy
Transmission electron microscopy (TEM) confirmed the presence of spherical nanoparticles in both the full-length M. musculus Arc protein and minimal Arc Gag protein samples. Even in the absence of RNA cargo, the Arc-derived PNCs were capable of self-assembly in vitro. In both samples, the average PNC diameter was approximately 30nm +/- 10nm.
Full-Length Arc
TEM image (60,000X magnification) of negatively-stained self-assembled full-length Arc PNCs. Scale bar = 200um.
Minimal Arc Gag
TEM image (100,000X magnification) of negatively-stained self-assembled minimal Arc Gag PNCs. Scale bar = 100um.
TEM also confirmed the presence of spherical nanoparticles in the P22 protein sample (without added scaffolding proteins; SPs) and P22 + SP-Cas9 protein samples. In the absence of the scaffolding protein, P22 capsid proteins were capable of self-assembly but with a significant variation in size and stability (many collapsed structures were evident in the sample). The P22 + SP-Cas9 PNCs had a much more consistent architecture but had a diameter of 120 +/- 5nm, which is approximately twice that of typical P22 virus-like particles. The 1:1 capsid to cargo ratio may have caused overloading of the PNCs, which caused an increase in overall size.
P22 coat proteins
TEM image (40,000X magnification) of negatively-stained self-assembled P22 capsids in the absence of scaffolding proteins. Scale bar = 200um.
P22-SP-Cas9
TEM image (40,000X magnification) of negatively-stained self-assembled P22 capsids with the SP-Cas9 cargo. Scale bar = 100um.
Mass Spectrometry
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Arc Min
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P22 Capsid
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Analytical Ultracentrifugation of Arc Min
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Transfection of Arc Min
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Purification of P22 and spCas9
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Collaboration
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Improved Parts
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