Difference between revisions of "Team:SDU-Denmark/Tour51"
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| − | <a class="popupImg alignRight" style="width:250px" target="_blank" href="https://static.igem.org/mediawiki/2015/c/c7/SDU2015_ReporterSystem.png" title="The experimental setup to measure promotor activity of PcstA by measurering levels of <i>gfp</i>-mRNA with bacteria transformed with BBa_K1135002. MG1655Δ<i>cyaA</i>, LB was used as a negative control. Samples were collected at different OD<sub>600</sub>-measurements. A single sample from the negative control was collected at OD<sub>600</sub> = 0.3. The RNA from the samples was purified and a Northern Blot with <i>gfp</i> and | + | <a class="popupImg alignRight" style="width:250px" target="_blank" href="https://static.igem.org/mediawiki/2015/c/c7/SDU2015_ReporterSystem.png" title="The experimental setup to measure promotor activity of PcstA by measurering levels of <i>gfp</i>-mRNA with bacteria transformed with BBa_K1135002. MG1655Δ<i>cyaA</i>, LB was used as a negative control. Samples were collected at different OD<sub>600</sub>-measurements. A single sample from the negative control was collected at OD<sub>600</sub> = 0.3. The RNA from the samples was purified and a Northern Blot with <i>gfp</i> and 5S probes was performed."> |
<img src="https://static.igem.org/mediawiki/2015/a/a4/SDU2015_ReporterSystem_thumbnail.png" style="width:255px"/> </a> | <img src="https://static.igem.org/mediawiki/2015/a/a4/SDU2015_ReporterSystem_thumbnail.png" style="width:255px"/> </a> | ||
<div class="thumbcaption">Figure 1: The experimental setup | <div class="thumbcaption">Figure 1: The experimental setup | ||
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| − | <a class="popupImg alignRight" style="width:250px" target="_blank" href="https://static.igem.org/mediawiki/2015/c/c6/PcstASDU-denmark.png" title="<b>Transcriptional activity of PcstA during growth by measurering RNA.</b> <p>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 <i>gfp</i>-mRNA normalized according to intensity of | + | <a class="popupImg alignRight" style="width:250px" target="_blank" href="https://static.igem.org/mediawiki/2015/c/c6/PcstASDU-denmark.png" title="<b>Transcriptional activity of PcstA during growth by measurering RNA.</b> <p>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 <i>gfp</i>-mRNA normalized according to intensity of 5S.</p> |
<p>Both from the graph and northern blot, it is quite clear that very little <i>gfp</i>-mRNA is present in the single sample from the negative control Δ<i>cyaA</i>. This strain lacks the ability to synthesize cAMP, thus little transcription is initiated, eventhough the bacteria is grown without excess glucose. Samples collected from WT, LB+0.2% glucose show a small increase in mRNA levels of <i>gfp</i> during exponential growth. However, samples from WT, LB show a even greater increase in amount of <i>gfp</i>-mRNA. There is a single sample from this collection that stands out (OD<sub>600</sub>=0.8). This might simply be due to some error in the execution of the experiment. However, the tendencies correlates with the expected. The inverse relationship between glucose and cAMP means that in high-energy states (high glucose), intracellular concentration of cAMP will be low. Therefore we expect little transcription to be initated when extra glucose is added. In low-energy states concentrations of cAMP rises, which will induce transcription of the PcstA-controlled gene <i>gfp</i>. </p> "> | <p>Both from the graph and northern blot, it is quite clear that very little <i>gfp</i>-mRNA is present in the single sample from the negative control Δ<i>cyaA</i>. This strain lacks the ability to synthesize cAMP, thus little transcription is initiated, eventhough the bacteria is grown without excess glucose. Samples collected from WT, LB+0.2% glucose show a small increase in mRNA levels of <i>gfp</i> during exponential growth. However, samples from WT, LB show a even greater increase in amount of <i>gfp</i>-mRNA. There is a single sample from this collection that stands out (OD<sub>600</sub>=0.8). This might simply be due to some error in the execution of the experiment. However, the tendencies correlates with the expected. The inverse relationship between glucose and cAMP means that in high-energy states (high glucose), intracellular concentration of cAMP will be low. Therefore we expect little transcription to be initated when extra glucose is added. In low-energy states concentrations of cAMP rises, which will induce transcription of the PcstA-controlled gene <i>gfp</i>. </p> "> | ||
<img src="https://static.igem.org/mediawiki/2015/c/c6/PcstASDU-denmark.png" style="width:255px"/> </a> | <img src="https://static.igem.org/mediawiki/2015/c/c6/PcstASDU-denmark.png" style="width:255px"/> </a> | ||
Revision as of 02:16, 19 September 2015
"It is beyond a doubt that all our knowledge begins with experience." - Immanuel Kant
Reporter System
We sat out to improve the original reporter system – the PcstA-induced transcription of the gene encoding Red Fluorescent Protein (RFP). We wanted to further characterize the PcstA promoter PcstA PromoterPcstA is a carbon stress induced promotor. In absence of glucose the enzyme adenylate cyclase will synthesize cAMP from ATP. cAMP will bind CAP, catabolite activator protein, encoded by crp, and the complex recruit RNA polymerase to the promotor. There is an inverse relationship between cAMP and glucose, and this means that glucose can repress promotor activity., part: BBa_K118011, by measuring the levels of mRNA through a Northern Blot.
Originally our reporter system was based on the part BBa_K861173 in which the promotor controls transcription of rfp. However, it is challenging to work with RFP, partly due to its relatively long folding time. Therefore we decided to perform the experiments with another brick, which also contained PcstA; BBa_K1135002. In this brick PcstA will initiate transcription of the gene encoding GFP.
Generally during the exponential phase of the bacteria, they have a high level of transcriptional activity. However, levels of 5S rRNA are relatively constant at all times. The transcription of gfp increases as the cells enter exponential phase between the two OD600 measurements 0.1 and 0.3. As expected, very low levels of gfp can be detected in the negative control. This strain lacks the ability to generate cAMP, and thus very little transcription is induced. The small amounts of gfp could be explained by leakiness of PcstA or that CAP alone initiates some transcription.
In the setup with WT, LB it is quite clear that the amount of gfp rises, compared to WT, LB+0.2% glucose. Transcription is clearly affected by the presence of glucose. One measurement WT, LB OD600 = 0.8 stands out. The result is not readily explained, but is probably due to some error. But the tendency of the results correlates with the knowledge of the invert relationship between glucose and cAMP. Glucose signaling will repress adenylate cyclase-activity, thus intracellular levels of cAMP will be low in high-energy states, and little transcription of gfp will be initiated.
We also tried to measure RNA levels induced by cAMP. This experiment was performed in two adenylate cyclase deficient strains; MG1655ΔcyaA and BTH101. In this setup, 1 mM cAMP was added to the medium of exponential cells and samples were subsequently collected at specific times. However, levels of RNA did not seem to be affected by cAMP. But this is probably not due to insensitivity of PcstA to cAMP. The most reasonable explanation is that cAMP failed to activate transcription of gfp>/i>, because the molecule couldn’t pass the cell membrane. However, RNA levels were at best slightly increased after approximately 8 minutes (data)