Difference between revisions of "Team:Northwestern/experiments"

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<p style="padding-top:2%; padding-right: 15%; padding-left:15%; font-size:14px;" class="big">In order to successfully package Cas9 into Outer Membrane Vesicles, we needed a source of OMVs.  We transformed our saCas9 encoding plasmids into a hyper-vesiculating strain of E. coli known as JC8031.  This strain is genetically engineered to create a large amount of OMVs and as a result, any substance whose concentration is high in the periplasm of the cell would also be expected to exist within the generated OMVs. The Cas9 itself also has a His6 tag attached for identification purposes, as well as a signal sequence meant to direct and secrete the protein into the periplasm.</font></p>
 
<p style="padding-top:2%; padding-right: 15%; padding-left:15%; font-size:14px;" class="big">In order to successfully package Cas9 into Outer Membrane Vesicles, we needed a source of OMVs.  We transformed our saCas9 encoding plasmids into a hyper-vesiculating strain of E. coli known as JC8031.  This strain is genetically engineered to create a large amount of OMVs and as a result, any substance whose concentration is high in the periplasm of the cell would also be expected to exist within the generated OMVs. The Cas9 itself also has a His6 tag attached for identification purposes, as well as a signal sequence meant to direct and secrete the protein into the periplasm.</font></p>
  
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<h3><center>Packaging Cas9</h3></center></h3>
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<h3><center>Signaling Sequences</h3></center></h3>
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<p style="padding-top:2%; padding-right: 15%; padding-left:15%; font-size:14px;" class="big">In order to successfully package Cas9 into Outer Membrane Vesicles, we needed a source of OMVs.  We transformed our saCas9 encoding plasmids into a hyper-vesiculating strain of E. coli known as JC8031.  This strain is genetically engineered to create a large amount of OMVs and as a result, any substance whose concentration is high in the periplasm of the cell would also be expected to exist within the generated OMVs. The Cas9 itself also has a His6 tag attached for identification purposes, as well as a signal sequence meant to direct and secrete the protein into the periplasm.</font></p>
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<p style="padding-top:2%; padding-right: 15%; padding-left:15%; font-size:14px;" class="big">The following table includes the signalling sequences that we used and tested over the course of the summer.  They are divided into the secretion pathway taken by the sequences, where an ambiguous sequence simply has a less defined pathway or may use multiple pathways.</font></p>
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<center><img src="https://static.igem.org/mediawiki/2017/4/41/T-Northwestern--table.png" width="700px" border="0">
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</center></figure>
  
  

Revision as of 01:07, 2 November 2017

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Packaging Cas9

In order to successfully package Cas9 into Outer Membrane Vesicles, we needed a source of OMVs. We transformed our saCas9 encoding plasmids into a hyper-vesiculating strain of E. coli known as JC8031. This strain is genetically engineered to create a large amount of OMVs and as a result, any substance whose concentration is high in the periplasm of the cell would also be expected to exist within the generated OMVs. The Cas9 itself also has a His6 tag attached for identification purposes, as well as a signal sequence meant to direct and secrete the protein into the periplasm.

Signaling Sequences

The following table includes the signalling sequences that we used and tested over the course of the summer. They are divided into the secretion pathway taken by the sequences, where an ambiguous sequence simply has a less defined pathway or may use multiple pathways.