Difference between revisions of "Team:BostonU/Results"

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  <p class="inline-heading-type mainwrap"> Characterizing Recombinase Activity in Cell Free </p>
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  <p class="body-type mainwrap">Once toehold switches were functional in cell free, we then shifted to generating functional recombinases in our cell free system. First we set out to characterizing the performance of commercially available Cre recombinase in cell free. Using a terminator excision reporter plasmid (shown below), the Cre recombinase should excise the premature termination and allow for deGFP expression. When fluorescence from this reporter plasmid with added Cre recombinase is compared to fluorescence from a reaction with no DNA and a reaction with constitutively active deGFP, only background levels of fluorescence are seen from the reporter. In addition, to see how the Cre recombinase affects cell free activity, we set up a reaction with Cre recombinase and the constitutively active deGFP plasmid. In this case, we see that the fluorescence is only 25% of the fluorescence shown with the fluorescence. We believed that this may have been a result of the solution containing the Cre recombinase. Our next step was to determine if similar results occurred when testing with plasmid contained recombinases. </p>
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  <p class="inline-heading-type mainwrap"> FIGURE </p>
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  <p class="inline-heading-type mainwrap"> Characterizing Recombinase Activity in Cell Free </p>
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  <p class="body-type mainwrap">We finally tested the a plasmid bound BxbI recombinase and a reporter plasmid with an inverted promoter (shown below). The BxbI recombinase should put the promoter in the proper orientation, allowing for deGFP expression. Fluorescence from a reaction containing recombinase and reporter again shows only background levels of fluorescence as compared to a reaction with no DNA. A reaction containing the recombinase and the constitutively active deGFP showed expression at about 66% as compared to a reaction containing just a constitutively active deGFP plasmid. In this case, we believe that the decrease may be caused by transcription and translation machinery is being diverted to produce the recombinase. Future experiments will be aimed at testing additional types of recombinases in cell free and understanding in more detail the mechanisms leading to the decreased deGFP expression. </p>
 
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Revision as of 16:08, 29 October 2017

RESULTS