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− | <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 A. niger morphology knockout strains were constructed, of which five of them 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> |
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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. | | 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. |
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| <div class="title"> Model and Software</div> | | <div class="title"> Model and Software</div> |
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− | <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 Aspergillus niger, both based on our experimental data. </p> |
| + | <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> Here, we demonstrate our final simulation of 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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| <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 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></figcaption></figure> |
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| <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/2020/1/1e/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> |
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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, 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/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> |
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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> |