Difference between revisions of "Team:BostonU/Experiments"

 
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<p class="body-type mainwrap"><img src="https://static.igem.org/mediawiki/2017/a/ab/T--BostonU--Experiments1.png" height="300" style="float:right;"></img>After this step, our plan was to test these linear PCR products in the cell-free transcription translation system. However, we observed basal levels of expression from linearized deGFP compared to the plasmid deGFP. After performing a literature search, we hypothesized that either our linear fragments were being degraded by exonucleases present in our cell-free system or RNAse present in our cell-free system degrading our RNA. In order to prevent exonuclease activity, we added purified gamS protein to our cell-free, which inhibits exonuclease activity. We also added commercial RNase inhibitor. However, after running cell-free tests with gamS, RNAse inhibitor, and linear DNA, we still saw only basal levels of expression. From this point onward we tested all our constructs in cell-free as plasmids. </p>
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<p class="body-type mainwrap"><img src="https://static.igem.org/mediawiki/2017/a/ab/T--BostonU--Experiments1.png" height="300" style="float:right;"></img>After this step, our plan was to test these linear PCR products in the cell-free transcription translation system. We wanted to test linear fragments beacuse it would drastically cut down on time and cost, and thus improve throughput. However, we observed basal levels of expression from linearized deGFP compared to the plasmid deGFP. After performing a literature search, we hypothesized that either our linear fragments were being degraded by exonucleases present in our cell-free system or RNAse present in our cell-free system degrading our RNA. In order to prevent exonuclease activity, we added purified gamS protein to our cell-free, which inhibits exonuclease activity. We also added commercial RNase inhibitor. However, after running cell-free tests with gamS, RNAse inhibitor, and linear DNA, we still saw only basal levels of expression. From this point onward we tested all our constructs in cell-free as plasmids. </p>
 
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<p class="body-type mainwrap">We moved on to adding our triggers as plasmid DNA. This resulted in expression higher than basal levels, however fluorescence still failed to reach the level of the constitutively active deGFP gene and was actually quite low.<img src="https://static.igem.org/mediawiki/2017/3/37/T--BostonU--Experiments3.png" height="300" style="float:left;">We hypothesized that too much of the cell-free's transcriptional machinery was being allocated to transcribing the trigger DNA and not enough was being allocated towards transcribing the toehold. We then decided to add the trigger as RNA instead as DNA. <img src="https://static.igem.org/mediawiki/2017/0/0b/T--BostonU--Experiments2.png" height="300" style="float:right;">We transcribed our trigger DNA into RNA using the Ampliscribe T7 Flash Transcription Kit before adding it into the cell-free system. Adding our trigger as RNA showed a more significant increase in fluorescence. Results from this experiment can be seen on the <a href="#">results</a> page.</p>
 
<p class="body-type mainwrap">We moved on to adding our triggers as plasmid DNA. This resulted in expression higher than basal levels, however fluorescence still failed to reach the level of the constitutively active deGFP gene and was actually quite low.<img src="https://static.igem.org/mediawiki/2017/3/37/T--BostonU--Experiments3.png" height="300" style="float:left;">We hypothesized that too much of the cell-free's transcriptional machinery was being allocated to transcribing the trigger DNA and not enough was being allocated towards transcribing the toehold. We then decided to add the trigger as RNA instead as DNA. <img src="https://static.igem.org/mediawiki/2017/0/0b/T--BostonU--Experiments2.png" height="300" style="float:right;">We transcribed our trigger DNA into RNA using the Ampliscribe T7 Flash Transcription Kit before adding it into the cell-free system. Adding our trigger as RNA showed a more significant increase in fluorescence. Results from this experiment can be seen on the <a href="#">results</a> page.</p>

Latest revision as of 23:12, 1 November 2017

EXPERIMENTS