Difference between revisions of "Team:BostonU/Experiments"

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   <p class="inline-heading-type mainwrap">Characterizing Our Cell Free System</p>
 
   <p class="inline-heading-type mainwrap">Characterizing Our Cell Free System</p>
 
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   <p class="body-type mainwrap">
In order to characterize the fluorescence capabilities of our cell-free system, we measured the fluorescence from a plasmid coding for a  constitutively active deGFP at varying concentrations. Specifically, the plasmid was at added to cell-free at 0 nM, 10 nM, 20 nM, 30 nM, and 40 nM concentrations. Fluorescence was measured over eight hours. The data was then used to inform our <a href="https://2017.igem.org/Team:BostonU/Model">model</a>.</p>
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In order to characterize the fluorescence capabilities of our cell-free system, we measured the fluorescence from a plasmid coding for a  constitutively active deGFP at varying concentrations. Our plasmid of choice was pBEST, which comes from the Noireaux Lab, whose cell-free protocol we used to make our in house cell-free. pBEST was shown by the Noireaux lab to have high performance in cell-free [1]. The plasmid was designed modularly, so each part could be replaced using a simple digestion ligation reaction. </p>
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Tthe plasmid was at added to cell-free at 0 nM, 10 nM, 20 nM, 30 nM, and 40 nM concentrations. Fluorescence was measured over eight hours. The data was then used to inform our <a href="https://2017.igem.org/Team:BostonU/Model">model</a>.</p>
 
<p class="body-type mainwrap">&nbsp;</p>
 
<p class="body-type mainwrap">&nbsp;</p>
  
 
   <p class="inline-heading-type mainwrap">Characterizing Toehold Activity Our Cell Free System</p>
 
   <p class="inline-heading-type mainwrap">Characterizing Toehold Activity Our Cell Free System</p>
  
  <p class="body-type mainwrap"> Our source plasmid pBEST comes from the Noireaux Lab, whose cell-free protocol we used to make our in house cell-free. pBEST was shown by the Noireaux lab to have high performance in cell-free [1]. The plasmid was designed modularly, so each part could be replaced using a simple digestion ligation reaction. </p>
 
<p class="body-type mainwrap">&nbsp;</p>
 
 
<p class="body-type mainwrap">We began by adapting our source plasmid to contain a toehold as its RBS. We did this by using primers: one set of master primers, which add in XbaI sites used to clone our final product into our final plasmid. The other set of primers added in the toehold or trigger architecture. The toehold switch and trigger sequences came from the best performing forward engineered toehold switch designed by Green et al [2]. We combined the two PCR products into a single linear piece of DNA using overlap extension PCR. </p>
 
<p class="body-type mainwrap">We began by adapting our source plasmid to contain a toehold as its RBS. We did this by using primers: one set of master primers, which add in XbaI sites used to clone our final product into our final plasmid. The other set of primers added in the toehold or trigger architecture. The toehold switch and trigger sequences came from the best performing forward engineered toehold switch designed by Green et al [2]. We combined the two PCR products into a single linear piece of DNA using overlap extension PCR. </p>
 
<p class="body-type mainwrap">&nbsp;</p>
 
<p class="body-type mainwrap">&nbsp;</p>

Revision as of 17:26, 1 November 2017

EXPERIMENTS