Difference between revisions of "Team:BostonU/Experiments"

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<p class="body-type mainwrap"><img src="https://static.igem.org/mediawiki/2017/4/46/T--BostonU--BxbIRecombFig1.png" width=30%></imgFor this set of experiments, we used a BxbI recombinase in the pBEST plasmid. We exchanged the deGFP with the BxbI sequence, achieving a constitutive BxbI plasmid. The reporter we used was a promoter inversion promoter. In the presence of BxbI, the formerly inverted promoter should be moved into the proper orientation, allowing for deGFP expression. </p>
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<p class="body-type mainwrap"><img src="https://static.igem.org/mediawiki/2017/4/46/T--BostonU--BxbIRecombFig1.png" width=30%></img>For this set of experiments, we used a BxbI recombinase in the pBEST plasmid. We exchanged the deGFP with the BxbI sequence, achieving a constitutive BxbI plasmid. The reporter we used was a promoter inversion promoter. In the presence of BxbI, the formerly inverted promoter should be moved into the proper orientation, allowing for deGFP expression. </p>
 
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<p class="body-type mainwrap"><img src="https://static.igem.org/mediawiki/2017/8/83/T--BostonU--BxbI.png" width=30%></img>We ran a cell free reaction with the constitutive BxbI plasmid added to the reporter plasmid. Again, this only showed background expression as compared to a reaction containing no DNA. A reaction containing the constitutive BxbI plasmid added to the constitutive deGFP showed fluorescence at about 66% compared to a reaction with just the constitutive deGFP. View these results <a href="#">here</a>.  We believe that this is indicative that both the deGFP and BxbI recombinase are being transcribed and translated. Because of limited machinery, the deGFP expression would be decreased to allow for BxbI expression. Future work will be aimed at proving this hypothesis. In addition, a literature search revealed that only a small subset of previously tested recombinases were shown to have functionality in cell free [3]. Future work will also aim at discovering which recombinase show the best functionality in our cell free system.</p>
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<p class="body-type mainwrap"><https://static.igem.org/mediawiki/2017/7/77/T--BostonU--Cre.png" width=30%></img>We ran a cell free reaction with the constitutive BxbI plasmid added to the reporter plasmid. Again, this only showed background expression as compared to a reaction containing no DNA. A reaction containing the constitutive BxbI plasmid added to the constitutive deGFP showed fluorescence at about 66% compared to a reaction with just the constitutive deGFP. View these results <a href="#">here</a>.  We believe that this is indicative that both the deGFP and BxbI recombinase are being transcribed and translated. Because of limited machinery, the deGFP expression would be decreased to allow for BxbI expression. Future work will be aimed at proving this hypothesis. In addition, a literature search revealed that only a small subset of previously tested recombinases were shown to have functionality in cell free [3]. Future work will also aim at discovering which recombinase show the best functionality in our cell free system.</p>
  
  

Revision as of 06:23, 31 October 2017

EXPERIMENTS