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| <img src="https://static.igem.org/mediawiki/2020/2/26/T--UCopenhagen--Poster_Description.png"> | | <img src="https://static.igem.org/mediawiki/2020/2/26/T--UCopenhagen--Poster_Description.png"> |
− | <figcaption style="font-family: Avenir, Arial, Helvetica, sans-serif; font-size: 12px; line-height: 1.25; text-align: center; margin-top: 1%;"><b>Fig. 1. Visual representation of the usage of the CIDosis patch and the engineering and scientific approach. </b>1: The patch collecting sweat from the skin. 2: The layers in the patch. 3: The scientific receptor engineering. 4: The patch after it has changed color. 5: The image-analyzing app.</figcaption> | + | <figcaption style="font-family: Arial, Helvetica, sans-serif; font-size: 12px; line-height: 1.25; text-align: center; margin-top: 1%;"><b>Fig. 1. Visual representation of the usage of the CIDosis patch and the engineering and scientific approach. </b>1: The patch collecting sweat from the skin. 2: The layers in the patch. 3: The scientific receptor engineering. 4: The patch after it has changed color. 5: The image-analyzing app.</figcaption> |
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| <img src="https://static.igem.org/mediawiki/2020/0/03/T--UCopenhagen--Poster_ubiquitin.png"> | | <img src="https://static.igem.org/mediawiki/2020/0/03/T--UCopenhagen--Poster_ubiquitin.png"> |
− | <figcaption style="font-family: Avenir, Arial, Helvetica, sans-serif; font-size: 12px; line-height: 1.25;text-align: center; margin-top: 1%;"><b>Fig. 2. Minimal biosensor design. </b> | + | <figcaption style="font-family: Arial, Helvetica, sans-serif; font-size: 12px; line-height: 1.25;text-align: center; margin-top: 1%;"><b>Fig. 2. Minimal biosensor design. </b> |
| Complementation of the C- and N-terminal halves of Ubiquitin results in their recognition by deubiquitinating enzymes, leading to cleavage and release of the bound LexA-VP16 transcription factor.</figcaption> | | Complementation of the C- and N-terminal halves of Ubiquitin results in their recognition by deubiquitinating enzymes, leading to cleavage and release of the bound LexA-VP16 transcription factor.</figcaption> |
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| <img src="https://static.igem.org/mediawiki/2020/3/3c/T--UCopenhagen--Poster_int.png"> | | <img src="https://static.igem.org/mediawiki/2020/3/3c/T--UCopenhagen--Poster_int.png"> |
− | <figcaption style="font-family: Avenir, Arial, Helvetica, sans-serif; font-size: 12px; line-height: 1.25; text-align: center;margin-top: 1%;"><b>Fig. 3. Intermediate biosensor design. </b> | + | <figcaption style="font-family: Arial, Helvetica, sans-serif; font-size: 12px; line-height: 1.25; text-align: center;margin-top: 1%;"><b>Fig. 3. Intermediate biosensor design. </b> |
| Split TEV protease complementation leading to cleavage of the linker tethering the transcription factor LexA-VP16 to the membrane.</figcaption> | | Split TEV protease complementation leading to cleavage of the linker tethering the transcription factor LexA-VP16 to the membrane.</figcaption> |
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| <img src="https://static.igem.org/mediawiki/2020/8/88/T--UCopenhagen--Poster_G_alpha_Design.png"> | | <img src="https://static.igem.org/mediawiki/2020/8/88/T--UCopenhagen--Poster_G_alpha_Design.png"> |
− | <figcaption style="font-family: Avenir, Arial, Helvetica, sans-serif; font-size: 12px; line-height: 1.25;text-align: center;margin-top: 1%;"><b>Fig. 4. Advanced biosensor design.</b> | + | <figcaption style="font-family: Arial, Helvetica, sans-serif; font-size: 12px; line-height: 1.25;text-align: center;margin-top: 1%;"><b>Fig. 4. Advanced biosensor design.</b> |
| Split TEV protease complementation leading to cleavage of a TEV protease cut site in GPA1, thereby initiating gene expression through the yeast pheromone pathway.</figcaption> | | Split TEV protease complementation leading to cleavage of a TEV protease cut site in GPA1, thereby initiating gene expression through the yeast pheromone pathway.</figcaption> |
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| <img src="https://static.igem.org/mediawiki/2020/4/48/T--UCopenhagen--Poster_dyn.png"> | | <img src="https://static.igem.org/mediawiki/2020/4/48/T--UCopenhagen--Poster_dyn.png"> |
− | <figcaption style="font-family: Avenir, Arial, Helvetica, sans-serif; font-size: 12px; line-height: 1.25;text-align: center;margin-top: 1%;"><b>Fig. 5. Comparison of the dynamic ranges of our designs. </b> Plotting reporter signal strength against interleukin concentration in uM reveals a significant difference in sensitivity between the three designs. | + | <figcaption style="font-family: Arial, Helvetica, sans-serif; font-size: 12px; line-height: 1.25;text-align: center;margin-top: 1%;"><b>Fig. 5. Comparison of the dynamic ranges of our designs. </b> Plotting reporter signal strength against interleukin concentration in uM reveals a significant difference in sensitivity between the three designs. |
| </figcaption> | | </figcaption> |
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| <img src="https://static.igem.org/mediawiki/2020/a/a4/T--UCopenhagen--Poster_rep.png"> | | <img src="https://static.igem.org/mediawiki/2020/a/a4/T--UCopenhagen--Poster_rep.png"> |
− | <figcaption style="font-family: Avenir, Arial, Helvetica, sans-serif; font-size: 12px; line-height: 1.25;text-align: center;margin-top: 1%;"><b>Fig. 6. The effect of reporter toxicity on the reporter concentration. </b>The amount of noise applied to all variables increases with reporter concentration, which leads to highly variable reporter concentrations (illustrative) | + | <figcaption style="font-family: Arial, Helvetica, sans-serif; font-size: 12px; line-height: 1.25;text-align: center;margin-top: 1%;"><b>Fig. 6. The effect of reporter toxicity on the reporter concentration. </b>The amount of noise applied to all variables increases with reporter concentration, which leads to highly variable reporter concentrations (illustrative) |
| </figcaption> | | </figcaption> |
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| <img src="https://static.igem.org/mediawiki/2020/8/87/T--UCopenhagen--Poster_m124.png"> | | <img src="https://static.igem.org/mediawiki/2020/8/87/T--UCopenhagen--Poster_m124.png"> |
− | <figcaption style="font-family: Avenir, Arial, Helvetica, sans-serif; font-size: 12px; line-height: 1.25;text-align: center;margin-top: 1%;"><b>Fig. 7. Change in Gibbs free energy of fragments produced after cleavage of GPA1 mutant m124. </b>The positions of each fragment in the original protein is denoted on the x-axis. Negative values suggest increased affinity to the beta subunit of the yeast G protein. | + | <figcaption style="font-family: Arial, Helvetica, sans-serif; font-size: 12px; line-height: 1.25;text-align: center;margin-top: 1%;"><b>Fig. 7. Change in Gibbs free energy of fragments produced after cleavage of GPA1 mutant m124. </b>The positions of each fragment in the original protein is denoted on the x-axis. Negative values suggest increased affinity to the beta subunit of the yeast G protein. |
| </figcaption> | | </figcaption> |
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| Our solution is a sweat-collecting patch that the patient can wear on the go. It consists of three layers. | | Our solution is a sweat-collecting patch that the patient can wear on the go. It consists of three layers. |
| <ol> | | <ol> |
− | <li><b>Porous nanofilm</b> - A porous nanofilm will allow interleukins to diffuse into the patch and prevent the yeast cells from escaping the patch. This film provides safety for the users and bio-containment.</li> | + | <li>Porous nanofilm - A porous nanofilm will allow interleukins to diffuse into the patch and prevent the yeast cells from escaping the patch. This film provides safety for the users and bio-containment.</li> |
− | <li><b>Genetically modified yeast-based biosensor</b> - Our yeast biosensor in dry-yeast form, ready to be activated upon contact with sweat.</li> | + | <li>Genetically modified yeast-based biosensor - Our yeast biosensor in dry-yeast form, ready to be activated upon contact with sweat.</li> |
− | <li><b>Adhesive patch</b> - Common transparent plastic or woven fabric (such as nylon) used by bandage manufacturers.</li> | + | <li>Adhesive patch - Common transparent plastic or woven fabric (such as nylon) used by bandage manufacturers.</li> |
| </ol> | | </ol> |
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| <img src="https://static.igem.org/mediawiki/2020/2/2e/T--UCopenhagen--Poster_Patch_Layers.png"> | | <img src="https://static.igem.org/mediawiki/2020/2/2e/T--UCopenhagen--Poster_Patch_Layers.png"> |
− | | + | <figcaption style="font-family: Arial, Helvetica, sans-serif; font-size: 12px; line-height: 1.25;text-align: center;margin-top: 1%;"><b>Fig. 16. Structure of the CIDosis patch.</b></figcaption> |
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| General inflammation can greatly fluctuate, and infrequent testing can give a misleading picture of a patient's inflammation status, due to the snapshot nature of these tests. For example, rheumatoid arthritis patients can experience big inflammation changes between current testing, leading to irrevocable damage. We want to avoid this by giving patients easy access to weekly stress-free monitoring. The CIDosis patch can be used in the patient’s daily life and help guide treatment by providing more data to the healthcare professionals. | | General inflammation can greatly fluctuate, and infrequent testing can give a misleading picture of a patient's inflammation status, due to the snapshot nature of these tests. For example, rheumatoid arthritis patients can experience big inflammation changes between current testing, leading to irrevocable damage. We want to avoid this by giving patients easy access to weekly stress-free monitoring. The CIDosis patch can be used in the patient’s daily life and help guide treatment by providing more data to the healthcare professionals. |
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| <img src="https://static.igem.org/mediawiki/2020/5/55/T--UCopenhagen--Poster_Snapshot_graph.png"> | | <img src="https://static.igem.org/mediawiki/2020/5/55/T--UCopenhagen--Poster_Snapshot_graph.png"> |
− | | + | <figcaption style="font-family: Arial, Helvetica, sans-serif; font-size: 12px; line-height: 1.25;text-align: center;margin-top: 1%;"><b>Fig. 17. Longer spaced testing gives an incomplete picture of the disease</b></figcaption> |
− | | + | <br><br> |
| We envision the use of an app that would enable the users to read the inflammation results in a precise manner. This app will track the inflammation results over time. In this way, inflammation levels can be saved and used for disease progression analysis. Here you see a representation of our app with a color slider that allows the user to save the patch color to their calendar and follow their inflammation. | | We envision the use of an app that would enable the users to read the inflammation results in a precise manner. This app will track the inflammation results over time. In this way, inflammation levels can be saved and used for disease progression analysis. Here you see a representation of our app with a color slider that allows the user to save the patch color to their calendar and follow their inflammation. |
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| <img src="https://static.igem.org/mediawiki/2020/6/63/T--UCopenhagen--Poster_app.png"> | | <img src="https://static.igem.org/mediawiki/2020/6/63/T--UCopenhagen--Poster_app.png"> |
− | | + | <figcaption style="font-family: Arial, Helvetica, sans-serif; font-size: 12px; line-height: 1.25;text-align: center;margin-top: 1%;"><b>Fig. 18. Mock-up of the CIDosis app.</b></figcaption> |
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| </div> | | </div> |
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| <img src="https://static.igem.org/mediawiki/2020/c/c8/T--UCopenhagen--Poster_workflow.png"> | | <img src="https://static.igem.org/mediawiki/2020/c/c8/T--UCopenhagen--Poster_workflow.png"> |
− | <figcaption style="font-family: Avenir, Arial, Helvetica, sans-serif; font-size: 12px; line-height: 1.25;text-align: center;margin-top: 1%;"><b>Fig. 8. General scientific workflow.</b></figcaption> | + | <figcaption style="font-family: Arial, Helvetica, sans-serif; font-size: 12px; line-height: 1.25;text-align: center;margin-top: 1%;"><b>Fig. 8. General scientific workflow.</b></figcaption> |
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| <img src="https://static.igem.org/mediawiki/2020/c/c0/T--UCopenhagen--Poster_5ass.png"> | | <img src="https://static.igem.org/mediawiki/2020/c/c0/T--UCopenhagen--Poster_5ass.png"> |
− | <figcaption style="font-family: Avenir, Arial, Helvetica, sans-serif; font-size: 12px; line-height: 1.25;text-align: center;margin-top: 1%;"><b>Fig. 9. The 5-assembler system. </b>The biosensor is assembled by co-transforming five linearized module plasmids that contain a promoter and a coding sequence, with both being flanked by homologous recombination regions.</figcaption> | + | <figcaption style="font-family: Arial, Helvetica, sans-serif; font-size: 12px; line-height: 1.25;text-align: center;margin-top: 1%;"><b>Fig. 9. The 5-assembler system. </b>The biosensor is assembled by co-transforming five linearized module plasmids that contain a promoter and a coding sequence, with both being flanked by homologous recombination regions.</figcaption> |
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| <img src="https://static.igem.org/mediawiki/2020/1/1c/T--UCopenhagen--Poster_yeast_col.png"> | | <img src="https://static.igem.org/mediawiki/2020/1/1c/T--UCopenhagen--Poster_yeast_col.png"> |
− | <figcaption style="font-family: Avenir, Arial, Helvetica, sans-serif; font-size: 12px; line-height: 1.25;text-align: center;margin-top: 1%;"><b>Fig. 10. Yeast colony PCR. </b>A band at 1500 bp meant no insertion into the locus, while a band at 1000 bp implied successful insertion of the five vectors. Arrow a and b denote positive and negative bands respectively.</figcaption> | + | <figcaption style="font-family: Arial, Helvetica, sans-serif; font-size: 12px; line-height: 1.25;text-align: center;margin-top: 1%;"><b>Fig. 10. Yeast colony PCR. </b>A band at 1500 bp meant no insertion into the locus, while a band at 1000 bp implied successful insertion of the five vectors. Arrow a and b denote positive and negative bands respectively.</figcaption> |
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| 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"> |
− | <figcaption style="font-family: Avenir, Arial, Helvetica, sans-serif; font-size: 12px; line-height: 1.25;text-align: center;margin-top: 1%;"><b>Fig. 11. Example of subcellular localization assay result. </b>sIL-10R1-Cub is shown to primarily localize to the plasma membrane, in the endoplasmic reticulum, and in small inclusion bodies near the membrane.</figcaption> | + | <figcaption style="font-family: Arial, Helvetica, sans-serif; font-size: 12px; line-height: 1.25;text-align: center;margin-top: 1%;"><b>Fig. 11. Example of subcellular localization assay result. </b>sIL-10R1-Cub is shown to primarily localize to the plasma membrane, in the endoplasmic reticulum, and in small inclusion bodies near the membrane.</figcaption> |
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| 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"> |
− | <figcaption style="font-family: Avenir, Arial, Helvetica, sans-serif; font-size: 12px; line-height: 1.25;text-align: center;margin-top: 1%;"><b>Fig. 12. Luminescence assay based on cleavage of a membrane-bound transcription factor. </b>Cells were expressed without (glucose media) and with (galactose+raffinose media) induction of the TEV protease. A 1300-fold increase in luminescence was seen upon induction of the TEV protease. The data shown is the average of three biological replicates.</figcaption> | + | <figcaption style="font-family: Arial, Helvetica, sans-serif; font-size: 12px; line-height: 1.25;text-align: center;margin-top: 1%;"><b>Fig. 12. Luminescence assay based on cleavage of a membrane-bound transcription factor. </b>Cells were expressed without (glucose media) and with (galactose+raffinose media) induction of the TEV protease. A 1300-fold increase in luminescence was seen upon induction of the TEV protease. The data shown is the average of three biological replicates.</figcaption> |
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| <img src="https://static.igem.org/mediawiki/2020/9/93/T--UCopenhagen--Poster_Ga_assays.png"> | | <img src="https://static.igem.org/mediawiki/2020/9/93/T--UCopenhagen--Poster_Ga_assays.png"> |
− | <figcaption style="font-family: Avenir, Arial, Helvetica, sans-serif; font-size: 12px; line-height: 1.25;text-align: center;margin-top: 1%;"><b>Fig. 13. Characterization of modified GPA1 proteins in adenosine biosensor. </b>The biosensors were grown in either glucose media or galactose + raffinose media which repressed or activated expression of a TEV protease. The biosensors were further grown with a dilution series of adenosine.</figcaption> | + | <figcaption style="font-family: Arial, Helvetica, sans-serif; font-size: 12px; line-height: 1.25;text-align: center;margin-top: 1%;"><b>Fig. 13. Characterization of modified GPA1 proteins in adenosine biosensor. </b>The biosensors were grown in either glucose media or galactose + raffinose media which repressed or activated expression of a TEV protease. The biosensors were further grown with a dilution series of adenosine.</figcaption> |
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| 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"> |
− | <figcaption style="font-family: Avenir, Arial, Helvetica, sans-serif; font-size: 12px; line-height: 1.25;text-align: center;margin-top: 1%;"><b>Fig. 14. Luciferase assay of the minimal interleukin-6 biosensor. </b>The amount of luminescence observed after incubation with a dilution series of interleukin-6 for 1, 3, 14 and 22 hours.</figcaption> | + | <figcaption style="font-family: Arial, Helvetica, sans-serif; font-size: 12px; line-height: 1.25;text-align: center;margin-top: 1%;"><b>Fig. 14. Luciferase assay of the minimal interleukin-6 biosensor. </b>The amount of luminescence observed after incubation with a dilution series of interleukin-6 for 1, 3, 14 and 22 hours.</figcaption> |
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| <ul style="font-family: Avenir, Arial, Helvetica, sans-serif;"> | | <ul style="font-family: Avenir, Arial, Helvetica, sans-serif;"> |
| <li>Choosing interleukins for biomarkers, as they are abundant in general inflammation.</li> | | <li>Choosing interleukins for biomarkers, as they are abundant in general inflammation.</li> |
− | <li>Making a modular design to accomodate the vast number of interleukins that experts deemed could be of clinical interest.</li> | + | <li>Making a modular design to accommodate the vast number of interleukins that experts deemed could be of clinical interest.</li> |
| </ul> | | </ul> |
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| <ul style="font-family: Avenir, Arial, Helvetica, sans-serif;"> | | <ul style="font-family: Avenir, Arial, Helvetica, sans-serif;"> |
− | <li>Patch colour - we were made aware of the counterintuitiveness of providing CID patients, who do not wish to be defined by their illness, with a very noticeable patch. Concerns about skin color differences were also brought up, leading us to make the patch see-through. This, in turn, would also make any patch color change much more visible.</li> | + | <li>Patch colour - we were made aware of the counterintuitivity of providing CID patients, who do not wish to be defined by their illness, with a very noticeable patch. Concerns about skin color differences were also brought up, leading us to make the patch see-through. This, in turn, would also make any patch color change much more visible.</li> |
| <li>Clear instructions - we made a clear user guide for the patch detailing how to use the patch correctly and record/interpret the received results, due to reported concerns regarding accidental misuse and unclear instructions.</li> | | <li>Clear instructions - we made a clear user guide for the patch detailing how to use the patch correctly and record/interpret the received results, due to reported concerns regarding accidental misuse and unclear instructions.</li> |
| <li>Wear time - our modeling results showed that the biosensor in the patch is best able to distinguish between high and low inflammation around the five hour mark, which fits perfectly within the time period users said they would be willing to wear the patch for. | | <li>Wear time - our modeling results showed that the biosensor in the patch is best able to distinguish between high and low inflammation around the five hour mark, which fits perfectly within the time period users said they would be willing to wear the patch for. |
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| <img src="https://static.igem.org/mediawiki/2020/c/cf/T--UCopenhagen--Poster_survey.png"> | | <img src="https://static.igem.org/mediawiki/2020/c/cf/T--UCopenhagen--Poster_survey.png"> |
− | <figcaption style="font-family: Avenir, 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> | | <b>Ethical Considerations</b><br> |