Difference between revisions of "Team:DTU-Denmark/Demonstrate"
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<h2>Demonstrate</h2> | <h2>Demonstrate</h2> | ||
| − | <p><div> | + | <p><div>The goal for our project was to create a piece of software that could predict synthetic promoters that could be utilized by both future iGEM teams and by industry.</div></p> |
</div> | </div> | ||
</div><!-- /end col-md-4 --> | </div><!-- /end col-md-4 --> | ||
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<div class="sm-no-float col-md-8"> | <div class="sm-no-float col-md-8"> | ||
| − | <h2> | + | <h2>Introduction</h2> |
| − | <p> | + | <p>After deciding on an organism and consulting key stakeholders, it became apparent that one of the most important criteria for industry-relevant <i>Aspergilli</i> promoters was that they were functional and reliable in multiple scales. |
| + | To show the scalability of our promoters, we wanted to demonstrate that the synthetic promoters from our library not only worked under many different conditions but also had the same relative gene expression from each other, regardless of which scale we tested them in. We therefore performed experiments in four different scales to test the strength, and more importantly, the consistency of our promoters. | ||
| + | </a>. | ||
</p> | </p> | ||
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| − | <h2> | + | <h2>The experiments</h2> |
| − | <p> | + | <p>For these experiments, the two synthetic promoters PLEAPglaA_2 and PLEAPsonB_1, were inserted in a USER plasmid in front of the fluorescent reporter gene mCherry. The plasmids were then transformed into a protease deficient variation of the <i>Aspergillus niger</i> ATCC 1015 strain, which was used to test the promoter activity at multiple scales. |
| + | The model hypothesizes that both promoters are the most active in the exponential growth phase. Additionally, the model predicted PLEAPsonB_1 to be a, relatively speaking, weak-medium strength promoter. The PLEAPglaA_2 promoter is a weakened variation (as described in the <a target="_blank" href="https://2019.igem.org/Team:DTU-Denmark/Model">Model page</a>) of the PLEAPglaA_1 promoter. This weakened promoter was included to validate the noise injection procedure of the model. For verification of the promoter activity and measurement of fluorescence, we used an engineered <i>A. nidulans</i> strain producing mCherry as a positive control, and an <i>A. niger</i> ATCC 1015 without mCherry production, as a negative control. | ||
| + | The details regarding the choice of this specific <i>A. niger</i> strain, and fluorescent marker can be found on the <a target="_blank" href="https://2019.igem.org/Team:DTU-Denmark/Design_Measurement">Design page</a>. | ||
| + | It should be noted that since the positive control is an mCherry producing <i>A. nidulans</i> strain, and not an <i>A. niger</i> strain, the positive control cannot be compared quantitatively to our other data, as we are working with a different organism entirely. However, the strain is still useful in comparing mCherry production to no mCherry production. | ||
| + | |||
| + | The production of mCherry from the two promoters was evaluated in a confocal microscope based on fungi grown on agar plates and although we were unable to determine the relative promoter strength quantitatively, it was evident that both promoters produced mCherry. | ||
</p> | </p> | ||
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<div class=" sm-no-float col-md-4 col-sm-12"> | <div class=" sm-no-float col-md-4 col-sm-12"> | ||
| − | <img src="https:// | + | <figure> |
| + | <img src="https://2019.igem.org/File:T--DTU-Denmark--NotebookConfocal.png" alt="Conidiaphore with red conidia" class="safetysecondimg"/> | ||
| + | <figcaption>The figure above displays RFP fluorescence from a strain expressing mCherry under control of the PLEAPglaA_2 promoter as observed under a confocal microscope</figcaption> | ||
| + | </figure> | ||
</div> | </div> | ||
Revision as of 18:58, 21 October 2019
Demonstrate
Introduction
After deciding on an organism and consulting key stakeholders, it became apparent that one of the most important criteria for industry-relevant Aspergilli promoters was that they were functional and reliable in multiple scales. To show the scalability of our promoters, we wanted to demonstrate that the synthetic promoters from our library not only worked under many different conditions but also had the same relative gene expression from each other, regardless of which scale we tested them in. We therefore performed experiments in four different scales to test the strength, and more importantly, the consistency of our promoters. .
The experiments
For these experiments, the two synthetic promoters PLEAPglaA_2 and PLEAPsonB_1, were inserted in a USER plasmid in front of the fluorescent reporter gene mCherry. The plasmids were then transformed into a protease deficient variation of the Aspergillus niger ATCC 1015 strain, which was used to test the promoter activity at multiple scales. The model hypothesizes that both promoters are the most active in the exponential growth phase. Additionally, the model predicted PLEAPsonB_1 to be a, relatively speaking, weak-medium strength promoter. The PLEAPglaA_2 promoter is a weakened variation (as described in the Model page) of the PLEAPglaA_1 promoter. This weakened promoter was included to validate the noise injection procedure of the model. For verification of the promoter activity and measurement of fluorescence, we used an engineered A. nidulans strain producing mCherry as a positive control, and an A. niger ATCC 1015 without mCherry production, as a negative control. The details regarding the choice of this specific A. niger strain, and fluorescent marker can be found on the Design page. It should be noted that since the positive control is an mCherry producing A. nidulans strain, and not an A. niger strain, the positive control cannot be compared quantitatively to our other data, as we are working with a different organism entirely. However, the strain is still useful in comparing mCherry production to no mCherry production. The production of mCherry from the two promoters was evaluated in a confocal microscope based on fungi grown on agar plates and although we were unable to determine the relative promoter strength quantitatively, it was evident that both promoters produced mCherry.
Sources here will also come soon
