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| + | <img src="https://static.igem.org/mediawiki/2020/4/4c/T--DTU-Denmark--Poster_banner.svg" width="100%"> |
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| <div style="padding: 20px 0px; width:95%; margin:auto; font-size:14px;"> | | <div style="padding: 20px 0px; width:95%; margin:auto; font-size:14px;"> |
− | <h1> DTU-Denmark Poster</h1> | + | <!--<h1> DTU-Denmark Poster</h1> |
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− | <!--<h3> Poster Template </h3>
| + | <h3> Poster Template </h3> |
| <p> This poster template will let you create an interactive poster! The poster is divided in two parts: the visual overview on the left and the documentation on the right. The visual overview is broken down into sections that the user can click on. When a user clicks on a poster section in the visual overview, the documentation on the right will display the text and graphics associated with this section of the poster. You can find the documentation on how to use this template, as well as an example here: <a href="https://2020.igem.org/Competition/Deliverables/Poster">https://2020.igem.org/Competition/Deliverables/Poster</a></p>--> | | <p> This poster template will let you create an interactive poster! The poster is divided in two parts: the visual overview on the left and the documentation on the right. The visual overview is broken down into sections that the user can click on. When a user clicks on a poster section in the visual overview, the documentation on the right will display the text and graphics associated with this section of the poster. You can find the documentation on how to use this template, as well as an example here: <a href="https://2020.igem.org/Competition/Deliverables/Poster">https://2020.igem.org/Competition/Deliverables/Poster</a></p>--> |
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| <b>Presented by DTU BioBuilders 2020</b> | | <b>Presented by DTU BioBuilders 2020</b> |
− | <p style="font-size:13px">Daniel Bavnhøj<sup>a</sup>, Peter Bing<sup>a</sup>, Clara Drachmann<sup>b</sup>, Martí M. Gómez<sup>b</sup>, Cecilia D. V. Graae<sup>a</sup>, Bira A. Khan<sup>a</sup>, Niels M. Knudsen<sup>a</sup>, Lucas Levassor<sup>a</sup>, Margrethe Mærsk-Møller<sup>b</sup>, Victoria V. Nissen<sup>a</sup>, Christine Pedersen<sup>a</sup>, Cecilie A. N. Thystrup<sup>b</sup>, Timian Rindal<sup>a</sup> </p> | + | <p style="font-size:13px">Daniel Bavnhøj<sup>a</sup>, Peter Bing<sup>a</sup>, Clara Drachmann<sup>b</sup>, Martí M. Gómez<sup>b</sup>, Cecilia D. V. Graae<sup>a</sup>, Bira A. Khan<sup>a</sup>, Niels MK<sup>a</sup>, Lucas Levassor<sup>a</sup>, Margrethe Mærsk-Møller<sup>b</sup>, Victoria V. Nissen<sup>a</sup>, Christine Pedersen<sup>a</sup>, Cecilie A. N. Thystrup<sup>b</sup>, Timian Rindal<sup>a</sup> </p> |
| <p style="font-size:10px"> <sup>a</sup>DTU Bioengineering, <sup>b</sup>DTU Health Tech, Technical University of Denmark </p> | | <p style="font-size:10px"> <sup>a</sup>DTU Bioengineering, <sup>b</sup>DTU Health Tech, Technical University of Denmark </p> |
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| <!--Write the text explaining this section --> | | <!--Write the text explaining this section --> |
| <div class="text"> | | <div class="text"> |
− | <p align="justify">Non-renewable natural resources are being depleted at an alarming rate, and the by-products of industrial production are contributing to climate change. This stand in the way of reaching the UN sustainability goals, particularly goal 12: Ensure sustainable consumption and production patterns and goal 8: Decent work and economic growth. To combat these issues, production practices must be changed. | + | <p align="justify">Non-renewable natural resources are being depleted at an alarming rate, and the by-products of industrial production are contributing to climate change. This stands in the way of reaching the UN sustainability goals, particularly goal 12: Ensure sustainable consumption and production patterns and goal 8: Decent work and economic growth. To combat these issues, production practices must be changed. |
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− | Shifting production of industrially important compounds to be developed through enzymes and therefore less reliant on petrochemicals, would alleviate some of the stress which is put on the current supply chains. One way of making this shift is by using filamentous fungi as cell factories. For many compounds which could be produced by cell factories, however, the cost and nuisance of working with filamentous fungi is prohibitive to their widespread use. | + | Shifting production of industrially important compounds to be developed through enzymes, and therefore be less reliant on petrochemicals, would alleviate some of the stress which is put on the current supply chains. One way of making this shift is by using filamentous fungi as cell factories. For many compounds which could be produced by cell factories, however, the cost and nuisance of working with filamentous fungi is prohibitive to their widespread use. |
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| We hoped to contribute to the development of more sustainable production practices by improving the filamentous fungus <i>Aspergillus niger</i> as a cell factory. </p> | | We hoped to contribute to the development of more sustainable production practices by improving the filamentous fungus <i>Aspergillus niger</i> as a cell factory. </p> |
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| <figure class="has-text-centered"><img src="https://static.igem.org/mediawiki/2020/a/ad/T--DTU-Denmark--implementation.svg" width="auto"></figure> | | <figure class="has-text-centered"><img src="https://static.igem.org/mediawiki/2020/a/ad/T--DTU-Denmark--implementation.svg" width="auto"></figure> |
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− | <p align="justify">We approached this task from several angles, as seen below. We both worked to alter the morphology of <i>A. Niger</i> by targeting relevant gene alterations, while simultaneously working to increase its ability as a protein producer. Results gathered from these alterations were further used to create modelling tools that could be used for predicting growth of mycelia. </p> | + | <p align="justify">We approached this task from several angles, as seen below. We both worked to alter the morphology of <i>A. Niger</i> by targeting relevant gene alterations and simultaneously worked to increase its ability as a protein producer. Results gathered from these alterations were further used to create modelling tools that could be used for predicting growth of mycelia. </p> |
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| <img src="https://static.igem.org/mediawiki/2020/2/21/T--DTU-Denmark--Description.svg" width="auto"> | | <img src="https://static.igem.org/mediawiki/2020/2/21/T--DTU-Denmark--Description.svg" width="auto"> |
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| <!--Write the text explaining this section --> | | <!--Write the text explaining this section --> |
| <div class="text"> | | <div class="text"> |
− | <p align="justify">During this project, nine different A. niger morphology knockout strains were constructed, of which five of them were novel. | + | <p align="justify">During this project, nine different <i>A. niger</i> morphology knockout strains were constructed and characterized, of which five were novel. </p> |
| + | <center><figure> <img style="width:100%" src="https://static.igem.org/mediawiki/2020/d/dc/T--DTU-Denmark--Poster_WETlabflow.png" class="center"></figure></center> |
| + | <p align="justify">All strains were constructed using a CRISPR-Cas9 system (Nødvig et al., 2015). The CRISPR knockout vectors were constructed using USER-cloning and were designed to knockout seven chosen genes related to morphology. Six of the mutants were successful and three additional mutants were constructed by combining the knockouts of genes from three of the most promising strains, thereby creating three double knockout strains. |
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− | All strains were constructed using USER cloning and fusion in combination with the CRISPR-Cas9 system (Nødvig et al., 2015). CRISPR knockout vectors were designed to knock out seven chosen genes related to morphology. Six of the mutants were successful and three additional mutants were constructed by combining the genes from three of the most promising strains, thereby creating three double knockout strains.
| + | To assess the effects of the mutations, the strains were tested through a variety of experiments. These included growth on different solid media, microscopy, growth in a BioLector and in 1L bioreactors. Samples from the bioreactor were analyzed through glucoamylase and BCA assays along with HPLC analysis. </p> |
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− | To access the effects of the mutations, the strains were tested through a variety of experiments. These included growth on different solid media, microscopy, growth in a BioLector and in 1L stirred tank bioreactors. Samples from the bioreactor were analyzed through glucoamylase and BCA assays along with HPLC analysis. </p> | + | |
| </div> | | </div> |
| </div> | | </div> |
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| <div class="title"> Model and Software</div> | | <div class="title"> Model and Software</div> |
| <!--Write the text explaining this section --> | | <!--Write the text explaining this section --> |
− | <div class="text"> <p align="justify">Explain the model </p></div> | + | <div class="text"> <p align="justify">To aid our parts characterization, we developed two models for studying the morphological features of <i>Aspergillus niger</i>, both based on our experimental data. The first model, <b>Morphologizer</b>, automatically analyzes microscopic images of mycelia. The second model, <b>Mycemulator</b>, is a stochastic model that simulates mycelial growth based on morphological parameters, including some from the Morphologizer. </p> |
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| + | <center><figure> <img style="width:80%" src="https://static.igem.org/mediawiki/2020/5/53/T--DTU-Denmark--Poster_imageana.png" class="center"><figcaption><p align="justify"><i> Showcase of our first model, Morphologizer. The model analyzes microscope images of mycelia, converts them into graph objects, and extracts morphological features of interest from the graph, such as the number of branches, branching angles, and hyphal curvature. </i></p></figcaption></figure></center> |
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| + | <center><figure> <img style="width:80%" src="https://static.igem.org/mediawiki/2020/0/06/T--DTU-Denmark--Poster_ATCC.gif" class="center"><figcaption><p align="justify"><i>Showcase of our second model, Mycemulator. A stochastic simulation of background strain ATCC 1015 based on parameters obtained from both experimental measurements and microscopic image analysis. The substrate gradient is shown in green, i.e. the greener the higher substrate concentration and the mycelium is shown in purple with newer hyphal elements in lighter colors. </i></p></figcaption></figure></center> |
| + | </div> |
| </div> | | </div> |
| </div> | | </div> |
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| <p align="justify">During the project we managed to make nine new morphology strains and nine mycelium growth simulations based on real life data. The results from the three most promising strains are shown here with the reference strain ATCC 1015:</p> | | <p align="justify">During the project we managed to make nine new morphology strains and nine mycelium growth simulations based on real life data. The results from the three most promising strains are shown here with the reference strain ATCC 1015:</p> |
− | <br><br>
| + | <figure><img src="https://static.igem.org/mediawiki/2020/8/8c/T--DTU-Denmark--Poster_plates.png"> <figcaption><p align="justify"><i>The three most promising morphology strains and the reference strain ATCC 1015 plated on CYA media. They are made by knocking out gul-1 separately and in combination with either chsC or spaA. All three strains are novel work.</i></p></figcaption></figure> |
− | <figure><img src="https://static.igem.org/mediawiki/2020/8/8c/T--DTU-Denmark--Poster_plates.png"> <figcaption><p align="justify"><i>The three most promising morphology strains and the reference strain ATCC 1015 plated on CYA media. They are made by knocking-out gul-1 and in combination with either of chsC and spaA. All three strains are novel work.</i></p></figcaption></figure> | + | <figure> <img src="https://static.igem.org/mediawiki/2020/d/db/T--DTU-Denmark--Poster_micro.png"><figcaption><p align="justify"><i>Microscope pictures of the three strains with the reference strain ATCC 1015. The pictures (stained version with Calcofluor White) were used by Morphologizer to estimate the parameters used in the Mycemulator simulations below.</i></p></figcaption></figure> |
− | <figure> <img src="https://static.igem.org/mediawiki/2020/d/db/T--DTU-Denmark--Poster_micro.png"><figcaption><p align="justify"><i>Microscopic pictures of the three strains with the reference strain ATCC 1015. The pictures (stained version with Calcofluor White) were used by the Morphologizer to estimate the parameters used in the Mycemulator simulations below.</i></p></figcaption></figure> | + | |
| <img style="width:24%" src="https://static.igem.org/mediawiki/2020/0/06/T--DTU-Denmark--Poster_ATCC.gif"> | | <img style="width:24%" src="https://static.igem.org/mediawiki/2020/0/06/T--DTU-Denmark--Poster_ATCC.gif"> |
| <img style="width:24%" src="https://static.igem.org/mediawiki/2020/5/59/T--DTU-Denmark--Poster_gul1.gif"> | | <img style="width:24%" src="https://static.igem.org/mediawiki/2020/5/59/T--DTU-Denmark--Poster_gul1.gif"> |
| <img style="width:24%" src="https://static.igem.org/mediawiki/2020/7/70/T--DTU-Denmark--Poster_chsC_gul1.gif"> | | <img style="width:24%" src="https://static.igem.org/mediawiki/2020/7/70/T--DTU-Denmark--Poster_chsC_gul1.gif"> |
| <img style="width:24%" src="https://static.igem.org/mediawiki/2020/5/52/T--DTU-Denmark--Poster_spaA_gul1.gif"> | | <img style="width:24%" src="https://static.igem.org/mediawiki/2020/5/52/T--DTU-Denmark--Poster_spaA_gul1.gif"> |
− | <figure><figcaption><p> <i>12h Mycemulator simulations of the three strain and reference strain ATCC 1015 in the order (ATCC 1015, Δgul-1, ΔchsC_Δgul-1 and ΔspaA_Δgul-1). Parameters for the simulations are estimated by the Morphlogizer and growth rate is found from the BioLector growth data. </i></p></i></p></figcaption></figure> | + | <figure><figcaption><p align="justify"> <i>12h Mycemulator simulations of the three strains and reference strain ATCC 1015 in the order (ATCC 1015, Δgul-1, ΔchsC_Δgul-1 and ΔspaA_Δgul-1). Parameters for the simulations are estimated by Morphlogizer and growth rate is found from the BioLector growth data.</i></p></figcaption></figure> |
− | <figure> <img style="width:100%" src="https://static.igem.org/mediawiki/2020/1/1e/T--DTU-Denmark--Poster_growth.png" class="center"><figcaption><p align="justify"><i>A comparison of growth rates for the three strains and reference strain ATCC 1015 when grown in 1L bioreactors. ΔchsC_Δgul-1 showed an minor increase in growth rate whereas the two others showed minor decrease in growth rates compared to the reference strain. None of them showed a significant difference from the growth rate of the reference strain.</i></p></figcaption></figure> | + | |
− | <figure> <img style="width:100%" src="https://static.igem.org/mediawiki/2020/d/d7/T--DTU-Denmark--Poster_protein.png" class="center"><figcaption><p align="justify"><i>Protein secretion and glucoamylase activity for Δgul-1, ΔchsC_Δgul-1 and ΔspaA_Δgul-1 compared to the reference strain ATCC 1015. Samples analyzed from the last time-point of the runs. All three strains showed an increase in specific activity, whereas Δgul-1 had the highest increased (with the exception of the one ΔspaA_Δgul-1 replicate, but results here are not conclusive.) <br><small>(Green: Glucoamylase activity in UA/mL. Blue: Specific activity in UA/mg calculated from the activity and the protein concentration. Purple: Protein concentration in mg/mL.)</small></i></p></figcaption></figure> | + | <figure> <img style="width:100%" src="https://static.igem.org/mediawiki/2020/1/1e/T--DTU-Denmark--Poster_growth.png" class="center"><figcaption><p align="justify"><i>A comparison of growth rates for the three strains and reference strain ATCC 1015 when grown in 1L bioreactors. ΔchsC_Δgul-1 showed a minor increase in growth rate whereas the two others showed minor decreases in growth rates compared to the reference strain. None of them showed a significant difference from the growth rate of the reference strain.</i></p></figcaption></figure> |
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| + | <figure> <img style="width:100%" src="https://static.igem.org/mediawiki/2020/d/d7/T--DTU-Denmark--Poster_protein.png" class="center"><figcaption><p align="justify"><i>Protein secretion and glucoamylase activity for Δgul-1, ΔchsC_Δgul-1 and ΔspaA_Δgul-1 compared to the reference strain ATCC 1015. Samples analyzed from the last time-point of the runs. All three strains showed an increase in specific activity, with Δgul-1 having the highest increase (with the exception of the one ΔspaA_Δgul-1 replicate, but results here are not conclusive.) <br><small>(Green: Glucoamylase activity in UA/mL. Blue: Specific activity in UA/mg calculated from the activity and the protein concentration. Purple: Protein concentration in mg/mL.)</small></i></p></figcaption></figure> |
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| <p align="justify">These results confirmed our original hypothesis that morphology has an impact on protein production. This is significant as increasing protein production while maintaining a similar or increased growth rate would lead to decreased production costs compared to existing systems.</p> | | <p align="justify">These results confirmed our original hypothesis that morphology has an impact on protein production. This is significant as increasing protein production while maintaining a similar or increased growth rate would lead to decreased production costs compared to existing systems.</p> |
| </div> | | </div> |
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| <p align="justify"><b>New morphology strains</b><br> | | <p align="justify"><b>New morphology strains</b><br> |
− | We have made 9 new morphology strains and characterized their performance as cell factories based on growth and protein secretion when grown in BioLectors and Bioreactors. | + | We have made 9 new morphology strains and characterized their performance as cell factories based on growth and protein secretion when grown in BioLectors and Bioreactors. We even showed that some of our new strains, the three novel strains were <i>gul-1</i> was knocked-out, had up to three-fold increased protein production compared to the reference strain. |
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| <b>Morphological toolbox for <i>A. niger</i></b><br> | | <b>Morphological toolbox for <i>A. niger</i></b><br> |
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| <b>Analysis of fungal microscopic images</b><br> | | <b>Analysis of fungal microscopic images</b><br> |
− | We have developed Morphologizer - a tool for analyzing microscope images of mycelia. Using Morphologizer, one can extract growth parameters of any fungal strain. | + | We have developed Morphologizer - a tool for analyzing microscope images of mycelia. Using Morphologizer, one can easily and automatically estimate morphological parameters for a fungal strain. |
| <br><br> | | <br><br> |
| <b>Simulation of mycelial growth</b><br> | | <b>Simulation of mycelial growth</b><br> |
− | We have developed Mycemulator - a model that can simulate mycelial growth based on real life parameters - for example extracted by Morphologizer. </p> | + | We have developed Mycemulator - a model that can simulate mycelial growth based on real-world parameters - for example the parameters estimated by our Morphologizer model. </p> |
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| </div> | | </div> |