Difference between revisions of "Team:BostonU/Experiments"

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<p class="body-type mainwrap">In order to determine how recombinases function in cell free, we obtained a commercially available Cre recombinase protein from New England Biolabs. We designed a reporter plasmid with the same design as pBEST, but with a premature terminator before the deGFP gene. This terminator was flanked with recombinase recognition sites, and in the presence of Cre should be excised, allowing for deGFP expression. The figure below shows the reporter architecture.</p>
 
<p class="body-type mainwrap">In order to determine how recombinases function in cell free, we obtained a commercially available Cre recombinase protein from New England Biolabs. We designed a reporter plasmid with the same design as pBEST, but with a premature terminator before the deGFP gene. This terminator was flanked with recombinase recognition sites, and in the presence of Cre should be excised, allowing for deGFP expression. The figure below shows the reporter architecture.</p>
 
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<center>
<img src-"https://static.igem.org/mediawiki/2017/e/e4/T--BostonU--CreRecombFig1.svg">  
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<img src="https://static.igem.org/mediawiki/2017/e/e4/T--BostonU--CreRecombFig1.svg" height = "600" width="600"></img>
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</center>
  
 
<p class="body-type mainwrap">We set up a cell free reaction in which we added one unit of the Cre recombinase to the reporter plasmid. We also added one unit of the Cre recombinase to a positive control reaction containing a constitutive deGFP plasmid. The reporter with Cre showed only background fluorescence as compared to a reaction with no DNA. The Cre with the constitutive deGFP plasmid showed only 25% fluorescence as compared to a constitutive deGFP reaction with no Cre. You can view these results <a href="#">here</a>. </p>
 
<p class="body-type mainwrap">We set up a cell free reaction in which we added one unit of the Cre recombinase to the reporter plasmid. We also added one unit of the Cre recombinase to a positive control reaction containing a constitutive deGFP plasmid. The reporter with Cre showed only background fluorescence as compared to a reaction with no DNA. The Cre with the constitutive deGFP plasmid showed only 25% fluorescence as compared to a constitutive deGFP reaction with no Cre. You can view these results <a href="#">here</a>. </p>

Revision as of 06:01, 31 October 2017

EXPERIMENTS