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| To guide the development of a yeast strain with the ability to sense inflammation biomarkers we included modeling of different biosensor designs. In the dry lab, we focused on comparing the three designs in terms of their contribution to biosensor sensitivity and behavior. This resulted in identifying the most applicable design for our biosensor. Furthermore, special effort was spent on modeling and evaluating different versions of the modified GPA1 protein (advanced design), by employing computational methods for protein engineering. | | To guide the development of a yeast strain with the ability to sense inflammation biomarkers we included modeling of different biosensor designs. In the dry lab, we focused on comparing the three designs in terms of their contribution to biosensor sensitivity and behavior. This resulted in identifying the most applicable design for our biosensor. Furthermore, special effort was spent on modeling and evaluating different versions of the modified GPA1 protein (advanced design), by employing computational methods for protein engineering. |
| <br><br> | | <br><br> |
− | <b>Sensitivity Comparison</b><br> | + | <h11>Sensitivity Comparison</h11><br> |
| We compared the three designs of engineered signaling pathways in S. cerevisiae in terms of sensitivity through modeling with ordinary differential equations (ODEs). The models revealed the additional benefits of employing the yeast pheromone cascade in signal amplification (~7 orders of magnitude), thus rendering one of the designs as a clear candidate for the application in the biosensor (fig. 5). | | We compared the three designs of engineered signaling pathways in S. cerevisiae in terms of sensitivity through modeling with ordinary differential equations (ODEs). The models revealed the additional benefits of employing the yeast pheromone cascade in signal amplification (~7 orders of magnitude), thus rendering one of the designs as a clear candidate for the application in the biosensor (fig. 5). |
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− | <b>Impact of Adverse Effects</b> | + | <h11>Impact of Adverse Effects</h11> |
| <br> | | <br> |
| We modeled the effects of hypothetical cellular scenarios (e.g. reporter toxicity) on the pathways within the framework of stochastic differential equations (SDEs). There, we explored various expected and unexpected behaviors in the models, which suggested that specific failures of pathway components may lead to characteristic statistics of reporter concentrations (fig. 6). This tool has the potential to improve our troubleshooting in the future. | | We modeled the effects of hypothetical cellular scenarios (e.g. reporter toxicity) on the pathways within the framework of stochastic differential equations (SDEs). There, we explored various expected and unexpected behaviors in the models, which suggested that specific failures of pathway components may lead to characteristic statistics of reporter concentrations (fig. 6). This tool has the potential to improve our troubleshooting in the future. |
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− | <b>Protein Modeling</b><br> | + | <h11>Protein Modeling</h11><br> |
| As the most applicable design required utilization of the yeast pheromone cascade, we had to engineer a novel GPA1 protein that would allow for signal transduction from our designed receptor system. Guided by several iterations of simulations with Rosetta Software Suite, we identified multiple regions suitable for inserting cleavage sites. | | As the most applicable design required utilization of the yeast pheromone cascade, we had to engineer a novel GPA1 protein that would allow for signal transduction from our designed receptor system. Guided by several iterations of simulations with Rosetta Software Suite, we identified multiple regions suitable for inserting cleavage sites. |
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− | <b>USER Cloning</b><br> | + | <h11>USER Cloning</h11><br> |
| All plasmids used in this work were constructed using USER cloning - a ligase independent cloning technique for seamless fusion of several PCR products and a vector. The method utilizes a Uracil Specific Excision Reagent (USER) enzyme mix to cut out uracils from PCR products and create 8-10 bp 3’ overhangs on PCR products [5]. The uracils are introduced with the primers in the PCR reaction with a proofreading polymerase that has been engineered to allow uracils called X7 [6]. The products can subsequently anneal to vectors containing a “USER cassette”, meaning that the vectors contain cut-sites that can yield overhangs upon digestion. These overhangs are complementary to the overhangs on the PCR products of interest. The USER reaction products are ligated by endogenous ligation mechanisms upon E. coli transformation. | | All plasmids used in this work were constructed using USER cloning - a ligase independent cloning technique for seamless fusion of several PCR products and a vector. The method utilizes a Uracil Specific Excision Reagent (USER) enzyme mix to cut out uracils from PCR products and create 8-10 bp 3’ overhangs on PCR products [5]. The uracils are introduced with the primers in the PCR reaction with a proofreading polymerase that has been engineered to allow uracils called X7 [6]. The products can subsequently anneal to vectors containing a “USER cassette”, meaning that the vectors contain cut-sites that can yield overhangs upon digestion. These overhangs are complementary to the overhangs on the PCR products of interest. The USER reaction products are ligated by endogenous ligation mechanisms upon E. coli transformation. |
| <br><br> | | <br><br> |
− | <b>Yeast Genomic Integration</b><br> | + | <h11>Yeast Genomic Integration</h11><br> |
| Yeast strains were created through stable integration by homologous recombination using a 5-assembler system (unpublished). The yeast is transformed with five vectors that are integrated into site 3 in chromosome X of Saccharomyces cerevisiae in a directional manner. Before transformation the vectors are linearized so that flanking 3’ and 5’ regions in each vector can be recombined with the other vectors and the integration site in the yeast genome. The 3’ region of each vector is identical to the 5’ region of the next vector in the system or an upstream region of the integration site. The 5’ region of each vector is identical to the 3’ region of the previous vector in the system or a downstream region of the integration site. See figure 9. | | Yeast strains were created through stable integration by homologous recombination using a 5-assembler system (unpublished). The yeast is transformed with five vectors that are integrated into site 3 in chromosome X of Saccharomyces cerevisiae in a directional manner. Before transformation the vectors are linearized so that flanking 3’ and 5’ regions in each vector can be recombined with the other vectors and the integration site in the yeast genome. The 3’ region of each vector is identical to the 5’ region of the next vector in the system or an upstream region of the integration site. The 5’ region of each vector is identical to the 3’ region of the previous vector in the system or a downstream region of the integration site. See figure 9. |
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− | <b>Luciferase Assays</b><br> | + | <h11>Luciferase Assays</h11><br> |
| To evaluate the functionality of our biosensors we used luciferase assays. The biosensor strains were incubated with a dilution series of the interleukin of interest. To measure the amount of signalling from our biosensors, nanoLuc luciferase [7] was used as a reporter. After incubation with the interleukin, the amount of luminesce was measured using a plate reader as a measure of the amount of reporter produced by the biosensors. | | To evaluate the functionality of our biosensors we used luciferase assays. The biosensor strains were incubated with a dilution series of the interleukin of interest. To measure the amount of signalling from our biosensors, nanoLuc luciferase [7] was used as a reporter. After incubation with the interleukin, the amount of luminesce was measured using a plate reader as a measure of the amount of reporter produced by the biosensors. |
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− | <b>Testing Receptor Localization</b><br> | + | <h11>Testing Receptor Localization</h11><br> |
| Through our extensive subcellular localization assays for all the three designs, we found that localization issues of the interleukin receptor proteins and accessory proteins were prominent for several of the proteins. <br><br> | | Through our extensive subcellular localization assays for all the three designs, we found that localization issues of the interleukin receptor proteins and accessory proteins were prominent for several of the proteins. <br><br> |
| <img src="https://static.igem.org/mediawiki/2020/e/ec/T--UCopenhagen--Poster_cells.png"> | | <img src="https://static.igem.org/mediawiki/2020/e/ec/T--UCopenhagen--Poster_cells.png"> |
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− | <b>Cleavage of membrane-bound transcription factor</b><br> | + | <h11>Cleavage of membrane-bound transcription factor</h11><br> |
| Induction of the TEV protease was shown to effectively cleave our membrane-bound transcription factor containing a TEV recognition site in a flexible linker, thereby inducing the expression of our reporter gene of interest.<br><br> | | Induction of the TEV protease was shown to effectively cleave our membrane-bound transcription factor containing a TEV recognition site in a flexible linker, thereby inducing the expression of our reporter gene of interest.<br><br> |
| <img src="https://static.igem.org/mediawiki/2020/c/c3/T--UCopenhagen--Poster_TMD_TF.png"> | | <img src="https://static.igem.org/mediawiki/2020/c/c3/T--UCopenhagen--Poster_TMD_TF.png"> |
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− | <b>GPA1 Switch Functionality</b><br> | + | <h11>GPA1 Switch Functionality</h11><br> |
| The different GPA1 mutants were tested in the context of an adenosine biosensor employing the human adenosine GPCR receptor A2A(R199A) (unpublished) in S. cerevisiae, previously developed in the Kampranis lab. By comparing our engineered GPA1 proteins to the wildtype, we observed that the amount of luminescence produced by the biosensor was higher when cells were incubated in galactose + raffinose media (and the TEV protease was induced) for all versions of GPA1, including the wildtype. This infers that the rise in luminescence with the change of media might be caused by another mechanism than cleavage of GPA1. Thus, it was not possible to determine whether cleavage of our modified GPA1 variants by the TEV protease was the reason for reporter gene expression in our induction assays. | | The different GPA1 mutants were tested in the context of an adenosine biosensor employing the human adenosine GPCR receptor A2A(R199A) (unpublished) in S. cerevisiae, previously developed in the Kampranis lab. By comparing our engineered GPA1 proteins to the wildtype, we observed that the amount of luminescence produced by the biosensor was higher when cells were incubated in galactose + raffinose media (and the TEV protease was induced) for all versions of GPA1, including the wildtype. This infers that the rise in luminescence with the change of media might be caused by another mechanism than cleavage of GPA1. Thus, it was not possible to determine whether cleavage of our modified GPA1 variants by the TEV protease was the reason for reporter gene expression in our induction assays. |
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− | <b>Complete Minimal Biosensor Interleukin Assay</b><br> | + | <h11>Complete Minimal Biosensor Interleukin Assay</h11><br> |
| No correlation between interleukin concentration and luminescence was observed for the minimal IL-6 and IL-10 biosensor strains at any incubation time. | | No correlation between interleukin concentration and luminescence was observed for the minimal IL-6 and IL-10 biosensor strains at any incubation time. |
| <br><br><img src="https://static.igem.org/mediawiki/2020/1/17/T--UCopenhagen--Poster_IL6.png"> | | <br><br><img src="https://static.igem.org/mediawiki/2020/1/17/T--UCopenhagen--Poster_IL6.png"> |
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− | <b>Future</b><br> | + | <h11>Future</h11><br> |
| Several experiments were identified for optimization of the biosensors. Our future work must focus on improving the subcellular localization of the interleukin receptors to allow efficient detection of interleukins in sweat for color production through expression of our reporter gene of interest. | | Several experiments were identified for optimization of the biosensors. Our future work must focus on improving the subcellular localization of the interleukin receptors to allow efficient detection of interleukins in sweat for color production through expression of our reporter gene of interest. |
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| Human practices were instrumental in both developing the science behind the CIDosis patch, and placing/establishing it in the larger context of healthcare. Both in the initial phases of the project and all throughout the project period, we have engaged with stakeholders and experts that could guide our decisions. | | Human practices were instrumental in both developing the science behind the CIDosis patch, and placing/establishing it in the larger context of healthcare. Both in the initial phases of the project and all throughout the project period, we have engaged with stakeholders and experts that could guide our decisions. |
| <br><br> | | <br><br> |
− | <b>Conceptualizing CIDosis</b><br> | + | <h11>Conceptualizing CIDosis</h11><br> |
| From the conception of the project, our choice to dedicate the patch to measure general inflammation rather than for diagnostic purposes was guided by expert advice from doctors and researchers within the field. Through conversing with specialists in this initial phase of the biosensor design, we decided to make the following wet lab modifications: | | From the conception of the project, our choice to dedicate the patch to measure general inflammation rather than for diagnostic purposes was guided by expert advice from doctors and researchers within the field. Through conversing with specialists in this initial phase of the biosensor design, we decided to make the following wet lab modifications: |
| <ul style="font-family: Avenir, Arial, Helvetica, sans-serif;"> | | <ul style="font-family: Avenir, Arial, Helvetica, sans-serif;"> |
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| </ul> | | </ul> |
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− | <b>Designing the CIDosis Patch</b><br> | + | <h11>Designing the CIDosis Patch</h11><br> |
| In total, we engaged with 10 experts ranging from researchers at the University of Copenhagen to doctors in the United States, and received input from 86 CID patients across four interviews and two surveys. While the first survey was aimed at CID patients in general, our second survey specifically targeted Crohn’s and Colitis patients.<br> | | In total, we engaged with 10 experts ranging from researchers at the University of Copenhagen to doctors in the United States, and received input from 86 CID patients across four interviews and two surveys. While the first survey was aimed at CID patients in general, our second survey specifically targeted Crohn’s and Colitis patients.<br> |
| The results confirmed the wish for more frequent and accessible testing for inflammation (fig. 15) and provided us many ideas for central design features of the patch, including: | | The results confirmed the wish for more frequent and accessible testing for inflammation (fig. 15) and provided us many ideas for central design features of the patch, including: |
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| <figcaption style="font-family: Arial, Helvetica, sans-serif; font-size: 12px; line-height: 1.25;text-align: center;margin-top: 1%;"><b>Fig. 15. Snippet of survey results. </b>Survey results showing the frequency of CID testing and desired frequency of wearing the CIDosis patch.</figcaption> | | <figcaption style="font-family: Arial, Helvetica, sans-serif; font-size: 12px; line-height: 1.25;text-align: center;margin-top: 1%;"><b>Fig. 15. Snippet of survey results. </b>Survey results showing the frequency of CID testing and desired frequency of wearing the CIDosis patch.</figcaption> |
| <br><br> | | <br><br> |
− | <b>Ethical Considerations</b><br> | + | <h11>Ethical Considerations</h11><br> |
| Due to the nature of our project, ethical concerns surrounding working with patients with CIDs and developing a “device” such as ours quickly surfaced. Our considerations spanned from our patch potentially causing mental distress to the patients, to ensuring the safety surrounding GMO usage. Patient interviews, literature findings, and discussions with our fellow Nordic iGEM teams were invaluable in resolving these issues. | | Due to the nature of our project, ethical concerns surrounding working with patients with CIDs and developing a “device” such as ours quickly surfaced. Our considerations spanned from our patch potentially causing mental distress to the patients, to ensuring the safety surrounding GMO usage. Patient interviews, literature findings, and discussions with our fellow Nordic iGEM teams were invaluable in resolving these issues. |
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| Apart from the work directly related to developing the CIDosis patch, a lot of our iGEM journey was dedicated to interacting with the iGEM community, as well as different local communities to spread the word of science, help each other learn and grow, and develop new and exciting skills ourselves. Under here you’ll find a handful of the things you can read more about on our wiki! | | Apart from the work directly related to developing the CIDosis patch, a lot of our iGEM journey was dedicated to interacting with the iGEM community, as well as different local communities to spread the word of science, help each other learn and grow, and develop new and exciting skills ourselves. Under here you’ll find a handful of the things you can read more about on our wiki! |
| <br><br> | | <br><br> |
− | <b>Our Ethics Guide</b><br> | + | <h11>Our Ethics Guide</h11><br> |
| Since ethical considerations surrounding working with chronically ill people was such a big part of our project this year, we decided to develop an ethics guide as our contribution to the iGEM community. The guide was developed in collaboration with SynthEthics, and serves as a six step guide for future iGEM teams to analyze and reduce moral ambiguities in their iGEM projects and ideas. We used CIDosis and the iGEM Lund 2020 team’s project, Protecto, as example cases for easy understanding and interpretation. Do give it a read! | | Since ethical considerations surrounding working with chronically ill people was such a big part of our project this year, we decided to develop an ethics guide as our contribution to the iGEM community. The guide was developed in collaboration with SynthEthics, and serves as a six step guide for future iGEM teams to analyze and reduce moral ambiguities in their iGEM projects and ideas. We used CIDosis and the iGEM Lund 2020 team’s project, Protecto, as example cases for easy understanding and interpretation. Do give it a read! |
| <br><br> | | <br><br> |
− | <b>Children’s Book</b><br> | + | <h11>Children’s Book</h11><br> |
| To inculcate passion for science and synthetic biology among children from an early age, we made the first installment of what we envision to be a series of children’s books. The first book, “My Sister Can Talk with Bacteria”, explains the concept of bacterial transformation, while future books will explain other synbio techniques, such as PCRs or protein fusions, or cellular processes such as cell division. With this, we hope to sensitize and educate young minds on genetic engineering, while emphasizing the importance of women in science. Other iGEM teams also helped us in translating the book to Arabic, Dutch and Japanese! | | To inculcate passion for science and synthetic biology among children from an early age, we made the first installment of what we envision to be a series of children’s books. The first book, “My Sister Can Talk with Bacteria”, explains the concept of bacterial transformation, while future books will explain other synbio techniques, such as PCRs or protein fusions, or cellular processes such as cell division. With this, we hope to sensitize and educate young minds on genetic engineering, while emphasizing the importance of women in science. Other iGEM teams also helped us in translating the book to Arabic, Dutch and Japanese! |
| <br><br> | | <br><br> |
− | <b>Partnership with iGEM Team Aalto-Helsinki</b><br> | + | <h11>Partnership with iGEM Team Aalto-Helsinki</h11><br> |
| We partnered with iGEM Team Aalto-Helsinki for the most part of our iGEM journey. We met in the spring for a coffee hour, and the professional relationship strengthened through the summer and fall as we partnered on troubleshooting each other’s dry lab models and organized an ethics workshop for various Nordic teams. | | We partnered with iGEM Team Aalto-Helsinki for the most part of our iGEM journey. We met in the spring for a coffee hour, and the professional relationship strengthened through the summer and fall as we partnered on troubleshooting each other’s dry lab models and organized an ethics workshop for various Nordic teams. |
| <br><br> | | <br><br> |
− | <b>Publications</b><br> | + | <h11>Publications</h11><br> |
| As part of iGEM Team MSP-Maastricht’s initiative to produce a peer-reviewed journal, we wrote an article explaining the science behind our project and were voted to be published in the journal. We are thankful to the MSP team for allowing us this opportunity and helping us hone our academic writing skills through peer review. | | As part of iGEM Team MSP-Maastricht’s initiative to produce a peer-reviewed journal, we wrote an article explaining the science behind our project and were voted to be published in the journal. We are thankful to the MSP team for allowing us this opportunity and helping us hone our academic writing skills through peer review. |
| We were also approached by the iGEM Taiwan teams for their “I’ve Gotta PhD” initiative for which we contributed an article on the mental health effects of devices such as our patch on patients with CIDs. | | We were also approached by the iGEM Taiwan teams for their “I’ve Gotta PhD” initiative for which we contributed an article on the mental health effects of devices such as our patch on patients with CIDs. |
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− | <b>Entrepreneurship</b><br> | + | <h11>Entrepreneurship</h11><br> |
| The University of Copenhagen has at least three different innovation and incubator hubs that guide students, employees, and others on the fundamentals of planning and launching a start-up through various workshops spread throughout the year. For CIDosis, we approached the hub at the Faculty of Health and Medical Sciences, called SUND Hub, to mentor our team. We joined the annual SUND Hub Incubator Program where we were enlightened on a wide range of topics from engagement with stakeholders and customer segmentation to regulatory pathways for medical devices. | | The University of Copenhagen has at least three different innovation and incubator hubs that guide students, employees, and others on the fundamentals of planning and launching a start-up through various workshops spread throughout the year. For CIDosis, we approached the hub at the Faculty of Health and Medical Sciences, called SUND Hub, to mentor our team. We joined the annual SUND Hub Incubator Program where we were enlightened on a wide range of topics from engagement with stakeholders and customer segmentation to regulatory pathways for medical devices. |
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| <div class="title"><h10>Attributions</h10></div> | | <div class="title"><h10>Attributions</h10></div> |
| <div class="text"> | | <div class="text"> |
− | <b>Supervisors:</b> Sotirios Kampranis, Nanna Heinz, Karel Miettinen, Jon Fugl, Nattawat Leelahakorn, Cecilie Cetti Hansen, Iben Egebæk Nikolajsen & Jonas Hansen.
| + | Supervisors: Sotirios Kampranis, Nanna Heinz, Karel Miettinen, Jon Fugl, Nattawat Leelahakorn, Cecilie Cetti Hansen, Iben Egebæk Nikolajsen & Jonas Hansen. |
| <br> | | <br> |
| <br> | | <br> |
− | <b>Thank you to our sponsors!</b> | + | <h11>Thank you to our sponsors!</h11> |
| <br> | | <br> |
| <img src="https://static.igem.org/mediawiki/2020/5/54/T--UCopenhagen--Poster_sponsors.png"> | | <img src="https://static.igem.org/mediawiki/2020/5/54/T--UCopenhagen--Poster_sponsors.png"> |
| <br> | | <br> |
| <br> | | <br> |
− | <b>Scientific references</b><br><br> | + | <h11>Scientific references</h11><br><br> |
| [1] - Snider, J., Kittanakom, S., Curak, J., & Stagljar, I. (2010). Split-ubiquitin based membrane yeast two-hybrid (MYTH) system: A powerful tool for identifying protein-protein interactions. Journal of Visualized Experiments. https://doi.org/10.3791/1698<br> | | [1] - Snider, J., Kittanakom, S., Curak, J., & Stagljar, I. (2010). Split-ubiquitin based membrane yeast two-hybrid (MYTH) system: A powerful tool for identifying protein-protein interactions. Journal of Visualized Experiments. https://doi.org/10.3791/1698<br> |
| [2] - Dossani, Zain & Apel, Amanda & Szmidt‐Middleton, Heather & Hillson, Nathan & Deutch, Samuel & Keasling, Jay & Mukhopadhyay, Aindrila. (2017). A combinatorial approach to Synthetic Transcription Factor-Promoter combinations for yeast strain engineering. Yeast. 35. 10.1002/yea.3292. <br> | | [2] - Dossani, Zain & Apel, Amanda & Szmidt‐Middleton, Heather & Hillson, Nathan & Deutch, Samuel & Keasling, Jay & Mukhopadhyay, Aindrila. (2017). A combinatorial approach to Synthetic Transcription Factor-Promoter combinations for yeast strain engineering. Yeast. 35. 10.1002/yea.3292. <br> |