Difference between revisions of "Team:Vilnius-Lithuania/Results"
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− | <h3 class="text-heading"></h3> | + | <h3 class="text-heading"><em>In vivo</em> cell experiments</h3> |
<p class="text-justify">We planned an experiment, during which we can see our system's killing profficiency. We prepared BL21-DE3 strain <em>E. coli</em> cells with our Cascade expression construct, along with a complement plasmid, carrying the Cas3 gene in a pCola-Duet vector.</p> | <p class="text-justify">We planned an experiment, during which we can see our system's killing profficiency. We prepared BL21-DE3 strain <em>E. coli</em> cells with our Cascade expression construct, along with a complement plasmid, carrying the Cas3 gene in a pCola-Duet vector.</p> | ||
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<p class="text-justify">However, when IPTG is present, we can see a dramatic cell count drop of almost 100x in bacteria, harbouring our crRNA's (SP1 and SP2), compared to the control (K). According to this data, we can say, that our Cascade complex biobrick (BBa_K1773022) is expressed and actively functions in degradation of cell DNA.</p> | <p class="text-justify">However, when IPTG is present, we can see a dramatic cell count drop of almost 100x in bacteria, harbouring our crRNA's (SP1 and SP2), compared to the control (K). According to this data, we can say, that our Cascade complex biobrick (BBa_K1773022) is expressed and actively functions in degradation of cell DNA.</p> | ||
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+ | <h3 class="text-heading">Promoter characterization</h3> | ||
+ | |||
+ | <p class="text-justify">We wanted to make a contribution to the ever growing iGEM parts registry by characterizing one of more frequently used promoters, that is called the standard (lambda cI regulated) promoter, biobrick code – BBa_R1051.</p> | ||
+ | |||
+ | <p class="text-justify">To do this, we employed a complex system that relies on GFP expression as a measurable identity that lets us to quantify levels of transcription from the cI regulated promoter. In order to achieve this, we cloned the cI regulated promoter in front of the GFP gene (BBa_I13504, the biobrick already contained appropriate RBS and terminator sequences).</p> | ||
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+ | <p class="text-justify">Though we proved that GFP expression from the cI regulated promoter is strong (measurement data), we also wanted to characterize the ability of the promoter to be repressed by producing the cI protein <em>in vivo</em>. Thus we constructed a regulatory unit of cI regulated promoter. The design is composed of pLac promoter sequence (BBa_R0010), which allows the induction of transcription by addition of IPTG, followed by cI coding gene (BBa_P0151, the biobrick also included the necessary RBS and terminator sequences).</p> | ||
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+ | <p class="text-justify">For the fluorescence measurement experiments we took advantage of protocol that was provided by the iGEM headquarters for the InterLab Measurement experiment. We transformed JM109 bacteria with our new constructs that were cloned into two separate compatible pasmid vectors. In theory, our regulatory unit upon induction with IPTG should produce cI repressor, which, in turn, should downregulate the expression of the GFP gene (under the control of cI regulated promoter). We seeked to quantify this by growing cell cultures overnight with different IPTG concentrations. We also used a negative and a positive control. </p> | ||
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Revision as of 22:15, 17 September 2015