Difference between revisions of "Team:SDU-Denmark/Tour50"
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<p> <i>"I've always believed that if you put in the work, the results will come."</i> - <b> Michael Jordan </b></p> | <p> <i>"I've always believed that if you put in the work, the results will come."</i> - <b> Michael Jordan </b></p> | ||
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<h1 align="center"> Results</h1> | <h1 align="center"> Results</h1> | ||
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− | <a class="popupImg alignRight" style="width: | + | <a class="popupImg alignRight" style="width:150px" target="_blank" href="https://static.igem.org/mediawiki/2015/c/c6/PcstASDU-denmark.png" title="Transcriptional activity of PcstA during growth by measurering RNA. Negative control of MG1655Δ<i>cyaA</i> in LB, WT in LB+0.2% glucose, and WT in LB. Samples collected at different OD<sub>600</sub> measurements. Graph shows intensities of mRNA GFP normalized according to intensity of 5S."> |
<img src="https://static.igem.org/mediawiki/2015/c/c6/PcstASDU-denmark.png" style="width:155px"/> </a> | <img src="https://static.igem.org/mediawiki/2015/c/c6/PcstASDU-denmark.png" style="width:155px"/> </a> | ||
− | <div class="thumbcaption" | + | <div class="thumbcaption">Figure 1: Transcriptional activity of PcstA during growth measured by RNA levels. |
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<p> | <p> | ||
− | + | <span class="intro">In the following</span> chapter you will see all our results from the lab. | |
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− | <a class="popupImg alignRight" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2015/d/de/Two-hybrid-system1-SDU_Denmark.png" title=" | + | <a class="popupImg alignRight" style="width:180px;" target="_blank" href="https://static.igem.org/mediawiki/2015/d/de/Two-hybrid-system1-SDU_Denmark.png" title="Competent BTH101 cells were co-transformed with the plasmids pSB1C3-T18 + pSB1K3-T25, pSB1C3-T18-Zip + pSB1K3-T25, pSB1C3-T18-Zip + pSB1K3-T25 and pSB1C3-T18-Zip + pSB1K3-T25-Zip. Transformations were plated out on LB plates containing 40 µg/ml x-gal, 30 µg/ml kan and 25 µg/ml cml and incubated at 37°C overnight. Only cells that were co-transformed with pSB1C3-T18-Zip + pSB1K3-T25-Zip got blue, while the others remained white. This indicates that β-Galactosidase was expressed in these cells and that the adenylate cyclase activity was reconstituted."> |
− | <img src="https://static.igem.org/mediawiki/2015/d/de/Two-hybrid-system1-SDU_Denmark.png" style="width:180px"/> | + | |
− | + | <img src="https://static.igem.org/mediawiki/2015/d/de/Two-hybrid-system1-SDU_Denmark.png" style="width:180px"/>ipsum | |
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+ | <div class="thumbcaption">Figure 2: Streaks of BTH101 on LB/X-gal plates, containing pSB1C3-T18 + pSB1K3-T25, pSB1C3-T18-Zip + pSB1K3-T25, pSB1C3-T18-Zip + pSB1K3-T25 and pSB1C3-T18-Zip + pSB1K3-T25-Zip.</div> | ||
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− | < | + | <p> |
+ | <span class="intro">Our goal</span> was to screen for peptide aptamers using a bacteria-based system. This system can be used to study protein-protein interactions. We used the bacterial two-hybrid system which is based on the reconstitution of the adenylate cyclase. In order to detect the rise in intracellular cAMP we used a Red Fluorescence Protein (RFP) reporter system with a cAMP-activated promoter, PcstA. Validation of a functional two-hybrid system and characterization of the reporter system was needed, before we could screen for peptide aptamers. | ||
+ | </p> | ||
− | + | <p> | |
+ | <span class="intro">Characterization</span> of <a href="https://2015.igem.org/Team:SDU-Denmark/Tour51">promotor PcstA</a>, was done by meassuring levels of mRNA during growth. The promoter is sensitive to the glucose concentrations, which was tested in the experiment. | ||
+ | </p> | ||
− | < | + | <p><span class="intro">The bacterial two-hybrid system</span> |
+ | has never been introduced to iGEM before. For this reason we wanted to validate the function of the system. To examine if the bacterial two-hybrid system can be used to study protein-protein interactions, we made a control experimental setup, where the leucine zipper region from the GCN4 yeast (<i>Saccharomyces cerevisiae</i>) protein were fused to the T18 and T25 domains (T18-Zip+T25-Zip). As leucine zippers form homodimers, their interaction should lead to functional complementation between T18 and T25. | ||
+ | </p> | ||
− | <span class="intro"> | + | <p> |
− | + | <span class="intro">We used</span> the <i>E. coli</i> K12 BTH101-strain, which is deficient in the gene encoding adenylate cyclase, <i>cyaA</i>. Our results showed that only when leucine zippers was fused to both T18 and T25, complementation was observed. We wanted to carry out a screening for peptide aptamers, but due to delayed deliverance of our nucleotide library and problems inserting it in our scaffold, we did not accomplish this. | |
+ | </p> | ||
+ | <p> | ||
− | < | + | <span class="intro">17 parts</span> were sent to the Parts Registry. In <a href="https://2015.igem.org/Team:SDU-Denmark/Tour53">Submitted Parts</a> you can browse through our parts and be redirected to the Parts Registry. Here you will find more information on our parts, e.g. function, sequencing and characterization. |
+ | </p> | ||
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− | < | + | <span class="intro">This year</span> |
+ | we participated in iGEM's <a href="https://2015.igem.org/Team:SDU-Denmark/Tour54">Second International InterLab Measurement Study</a>. The goal of this study was to measure fluorescence from three different devices expressing GFP. We were able to measure the fluorescence of these devices using FACS. The data we acquired showed that there was a relative difference in promoter activity in the mutated promoters compared to the wild type. | ||
+ | </p> | ||
<b>Dig deeper</b> to get a more detailed description of our results. | <b>Dig deeper</b> to get a more detailed description of our results. | ||
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{{:Team:SDU-Denmark/core/footer}} | {{:Team:SDU-Denmark/core/footer}} |
Latest revision as of 16:23, 4 October 2015
"I've always believed that if you put in the work, the results will come." - Michael Jordan
Results
In the following chapter you will see all our results from the lab.
Our goal was to screen for peptide aptamers using a bacteria-based system. This system can be used to study protein-protein interactions. We used the bacterial two-hybrid system which is based on the reconstitution of the adenylate cyclase. In order to detect the rise in intracellular cAMP we used a Red Fluorescence Protein (RFP) reporter system with a cAMP-activated promoter, PcstA. Validation of a functional two-hybrid system and characterization of the reporter system was needed, before we could screen for peptide aptamers.
Characterization of promotor PcstA, was done by meassuring levels of mRNA during growth. The promoter is sensitive to the glucose concentrations, which was tested in the experiment.
The bacterial two-hybrid system has never been introduced to iGEM before. For this reason we wanted to validate the function of the system. To examine if the bacterial two-hybrid system can be used to study protein-protein interactions, we made a control experimental setup, where the leucine zipper region from the GCN4 yeast (Saccharomyces cerevisiae) protein were fused to the T18 and T25 domains (T18-Zip+T25-Zip). As leucine zippers form homodimers, their interaction should lead to functional complementation between T18 and T25.
We used the E. coli K12 BTH101-strain, which is deficient in the gene encoding adenylate cyclase, cyaA. Our results showed that only when leucine zippers was fused to both T18 and T25, complementation was observed. We wanted to carry out a screening for peptide aptamers, but due to delayed deliverance of our nucleotide library and problems inserting it in our scaffold, we did not accomplish this.
17 parts were sent to the Parts Registry. In Submitted Parts you can browse through our parts and be redirected to the Parts Registry. Here you will find more information on our parts, e.g. function, sequencing and characterization.
This year we participated in iGEM's Second International InterLab Measurement Study. The goal of this study was to measure fluorescence from three different devices expressing GFP. We were able to measure the fluorescence of these devices using FACS. The data we acquired showed that there was a relative difference in promoter activity in the mutated promoters compared to the wild type.
Dig deeper to get a more detailed description of our results.