Difference between revisions of "Team:Vilnius-Lithuania/Results"
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<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">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 | + | <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> |
− | <p class="text-justify">Though we | + | <p class="text-justify">Though we saw that GFP expression from the cI regulated promoter is strong. We also wanted to characterize the ability of the promoter to be repressed by producing the cI protein in vivo. So we emplyed a regulatory unit - BBa_K077039 which 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> |
<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> | <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> | ||
− | <p class="text-justify">We | + | <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 seperate 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 which is 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 control.</p> |
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+ | <p class="text-justify">Here are the results:</p> | ||
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<p style="border-left: 5px solid rgb(236,151,31); padding-left: 3px; border-bottom: none"><strong>Figure 1.</strong> Relative fluorescence data normalized to sample with highest fluorescence (0.5 mM IPTG). (-) – Negative control (only BBa_K077039); 0.1 IPTG – 10 mM IPTG – samples with different IPTG concentrations. Error bars represent standard deviations of two biological replicates for each type of IPTG concentration.</p> | <p style="border-left: 5px solid rgb(236,151,31); padding-left: 3px; border-bottom: none"><strong>Figure 1.</strong> Relative fluorescence data normalized to sample with highest fluorescence (0.5 mM IPTG). (-) – Negative control (only BBa_K077039); 0.1 IPTG – 10 mM IPTG – samples with different IPTG concentrations. Error bars represent standard deviations of two biological replicates for each type of IPTG concentration.</p> | ||
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+ | <h3 style="border-left: 5px solid rgb(236,151,31); padding-left: 5px; border-bottom: none">Discussion</h3> | ||
+ | <p class="text-justify">We determined that this device is sensitive to cI. It can be seen that upon induction of cI expression there is a significant drop in relative fluorescence, the general tendency is that d fluorescence signal depletes with increasing concentrations of IPTG, however we see great variation in our data. Similar data was obtained during replications of this experiment. We suggest that BBa_K195613 is indeed sensitive to cI and the effects of cI on transcription levels from this promoter are somewhat titratable, however we claim that this system (BBa_K077039 + BBa_K195613) is highly unpredictable. We suggest that this unpredictability might be due to the low translation levels of cI protein (weak RBS) combined with its instability (LVA tail) and its nature of a dominant repressor. These effects combined might lead to an effect in which changes in a small population of repressor might have great effects on the whole system.<p/> | ||
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Revision as of 03:36, 19 September 2015