iGEM 2020 — Team Queens_Canada

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Queens Canada/Notebook

[![](https://static.igem.org/mediawiki/2020/6/67/T--Queens_Canada--logos.png)](https://2020.igem.org/Team%3AQueens_Canada)

* [HOME](https://2020.igem.org/Team%3AQueens_Canada)
* PROJECT
  + [Description](https://2020.igem.org/Team%3AQueens_Canada/Description)
  + [Protein Construct](https://2020.igem.org/Team%3AQueens_Canada/Protein-Construct)
  + [FRET](https://2020.igem.org/Team%3AQueens_Canada/FRET)
  + [Coiled-Coils](https://2020.igem.org/Team%3AQueens_Canada/Coiled-Coils)
  + [Binding Proteins](https://2020.igem.org/Team%3AQueens_Canada/Binding-Proteins)
  + [Implementation](https://2020.igem.org/Team%3AQueens_Canada/Implementation)
  + [Results](https://2020.igem.org/Team%3AQueens_Canada/Results)
  + [Contribution](https://2020.igem.org/Team%3AQueens_Canada/Contribution)
* HUMAN
  + [Human Practices](https://2020.igem.org/Team%3AQueens_Canada/Human_Practices)
  + [Education](https://2020.igem.org/Team%3AQueens_Canada/Education)
  + [Collaborations](https://2020.igem.org/Team%3AQueens_Canada/Collaborations)
  + [Partnership](https://2020.igem.org/Team%3AQueens_Canada/Partnership)
  + [Safety](https://2020.igem.org/Team%3AQueens_Canada/Safety)
* MODEL
  + [Overview](https://2020.igem.org/Team%3AQueens_Canada/Model)
  + [Protein Stability](https://2020.igem.org/Team%3AQueens_Canada/Static-Dynamic)
  + [Coiled-Coil Stability](https://2020.igem.org/Team%3AQueens_Canada/Coiled-Coil-Stability)
  + [Cysteine Modification](https://2020.igem.org/Team%3AQueens_Canada/Directed-Cysteine)
  + [Software](https://2020.igem.org/Team%3AQueens_Canada/Software)
  + [Mutaguide](https://2020.igem.org/Team%3AQueens_Canada/Mutaguide)
* PROTOTYPE
  + [Overview](https://2020.igem.org/Team%3AQueens_Canada/Prototype-Overview)
  + [Fluorometer](https://2020.igem.org/Team%3AQueens_Canada/Fluorometer)
  + [Fluid Dynamics](https://2020.igem.org/Team%3AQueens_Canada/Fluid-Dynamics)
  + [Mobile App](https://2020.igem.org/Team%3AQueens_Canada/App)
* PARTS
  + [Overview](https://2020.igem.org/Team%3AQueens_Canada/Parts-Overview)
  + [Expression Plasmid](https://2020.igem.org/Team%3AQueens_Canada/Expression-Plasmid)
  + [Purification Summary](https://2020.igem.org/Team%3AQueens_Canada/Purification-Summary)
* NOTEBOOK
  + [Protocols](https://2020.igem.org/Team%3AQueens_Canada/Protocols)
  + [Posters and Presentations](https://2020.igem.org/Team%3AQueens_Canada/Posters-Presentations)
* TEAM
  + [Team Members](https://2020.igem.org/Team%3AQueens_Canada/Team)
  + [Our Sponsors](https://2020.igem.org/Team%3AQueens_Canada/Sponsors)
  + [Attributions](https://2020.igem.org/Team%3AQueens_Canada/Attributions)

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2014 ATOMS-Turkiye](https://2014.igem.org/Team%3AATOMS-Turkiye/Notebook)
* [2014 Tec Monterrey](https://2014.igem.org/Team%3ATec-Monterrey/ITESM14_project.html#tab_notebook)
* [2014 Kyoto](https://2014.igem.org/Team%3AKyoto/Notebook/Magnetosome_Formation#title)
* [2014 Cornell](https://2014.igem.org/Team%3ACornell/notebook)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Queens Canada/Engineering

[![](https://static.igem.org/mediawiki/2020/6/67/T--Queens_Canada--logos.png)](https://2020.igem.org/Team%3AQueens_Canada)

* [HOME](https://2020.igem.org/Team%3AQueens_Canada)
* PROJECT
  + [Description](https://2020.igem.org/Team%3AQueens_Canada/Description)
  + [Protein Construct](https://2020.igem.org/Team%3AQueens_Canada/Protein-Construct)
  + [FRET](https://2020.igem.org/Team%3AQueens_Canada/FRET)
  + [Coiled-Coils](https://2020.igem.org/Team%3AQueens_Canada/Coiled-Coils)
  + [Binding Proteins](https://2020.igem.org/Team%3AQueens_Canada/Binding-Proteins)
  + [Implementation](https://2020.igem.org/Team%3AQueens_Canada/Implementation)
  + [Results](https://2020.igem.org/Team%3AQueens_Canada/Results)
  + [Contribution](https://2020.igem.org/Team%3AQueens_Canada/Contribution)
* HUMAN
  + [Human Practices](https://2020.igem.org/Team%3AQueens_Canada/Human_Practices)
  + [Education](https://2020.igem.org/Team%3AQueens_Canada/Education)
  + [Collaborations](https://2020.igem.org/Team%3AQueens_Canada/Collaborations)
  + [Partnership](https://2020.igem.org/Team%3AQueens_Canada/Partnership)
  + [Safety](https://2020.igem.org/Team%3AQueens_Canada/Safety)
* MODEL
  + [Overview](https://2020.igem.org/Team%3AQueens_Canada/Model)
  + [Protein Stability](https://2020.igem.org/Team%3AQueens_Canada/Static-Dynamic)
  + [Coiled-Coil Stability](https://2020.igem.org/Team%3AQueens_Canada/Coiled-Coil-Stability)
  + [Cysteine Modification](https://2020.igem.org/Team%3AQueens_Canada/Directed-Cysteine)
  + [Software](https://2020.igem.org/Team%3AQueens_Canada/Software)
  + [Mutaguide](https://2020.igem.org/Team%3AQueens_Canada/Mutaguide)
* PROTOTYPE
  + [Overview](https://2020.igem.org/Team%3AQueens_Canada/Prototype-Overview)
  + [Fluorometer](https://2020.igem.org/Team%3AQueens_Canada/Fluorometer)
  + [Fluid Dynamics](https://2020.igem.org/Team%3AQueens_Canada/Fluid-Dynamics)
  + [Mobile App](https://2020.igem.org/Team%3AQueens_Canada/App)
* PARTS
  + [Overview](https://2020.igem.org/Team%3AQueens_Canada/Parts-Overview)
  + [Expression Plasmid](https://2020.igem.org/Team%3AQueens_Canada/Expression-Plasmid)
  + [Purification Summary](https://2020.igem.org/Team%3AQueens_Canada/Purification-Summary)
* NOTEBOOK
  + [Protocols](https://2020.igem.org/Team%3AQueens_Canada/Protocols)
  + [Posters and Presentations](https://2020.igem.org/Team%3AQueens_Canada/Posters-Presentations)
* TEAM
  + [Team Members](https://2020.igem.org/Team%3AQueens_Canada/Team)
  + [Our Sponsors](https://2020.igem.org/Team%3AQueens_Canada/Sponsors)
  + [Attributions](https://2020.igem.org/Team%3AQueens_Canada/Attributions)

# Prototype Overview

![](https://static.igem.org/mediawiki/2020/9/9f/T--Queens_Canada--Prototype_Drawing.svg)

### Overview

CKD is a very severe and prevalent disease, affecting 1 in 8 Canadian adults. Given the absence of tools available for frequent monitoring of CKD metabolites, and the correlation between this and rates of cardiovascular disease in CKD patients, team Queen’s Canada aims to bridge the gap between patients and clinical practice in providing patients a tool for frequent monitoring of their CKD status. We designed an accessible and portable transdermal biosensor measure levels of parathyroid hormone (PTH), phosphate, potassium, glucose, and fibroblast growth factor 23 (FGF23) at physiologically relevant concentrations, and frequently, to provide patients with the best possible health outcomes. The objective of Velcrion was to have the ability to excite the fluorescent protein in an antibody solution bound to the metabolite of interest, collected through a patient’s interstitial fluid (ISF), to measure the amount of metabolite based on a concentration vs fluorescence relationship. The results would be communicated to an accompanying app, which could collect additional critical and trackable health factors. The patient’s data could then be communicated to their healthcare provider for a holistic and patient-centered approach to care.

To learn more about how our prototype works, check out our fluorometer process!

[Fluorometer](https://2020.igem.org/Team%3AQueens_Canada/Fluorometer)

[![](https://static.igem.org/mediawiki/2020/6/6d/T--Queens_Canada--two-tone-logo.png)](https://2020.igem.org/Team%3AQueens_Canada "Go to Home")
[![](https://static.igem.org/mediawiki/2020/8/89/T--Queens_Canada--to-top-circle.svg)](#to-nav "Jump to Top")

![](https://static.igem.org/mediawiki/2020/e/ed/T--Queens_Canada--AMS.png)
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![](https://static.igem.org/mediawiki/2020/1/16/T--Queens_Canada--CFAC.png)
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[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Queens Canada/Experiments

[![](https://static.igem.org/mediawiki/2020/6/67/T--Queens_Canada--logos.png)](https://2020.igem.org/Team%3AQueens_Canada)

* [HOME](https://2020.igem.org/Team%3AQueens_Canada)
* PROJECT
  + [Description](https://2020.igem.org/Team%3AQueens_Canada/Description)
  + [Protein Construct](https://2020.igem.org/Team%3AQueens_Canada/Protein-Construct)
  + [FRET](https://2020.igem.org/Team%3AQueens_Canada/FRET)
  + [Coiled-Coils](https://2020.igem.org/Team%3AQueens_Canada/Coiled-Coils)
  + [Binding Proteins](https://2020.igem.org/Team%3AQueens_Canada/Binding-Proteins)
  + [Implementation](https://2020.igem.org/Team%3AQueens_Canada/Implementation)
  + [Results](https://2020.igem.org/Team%3AQueens_Canada/Results)
  + [Contribution](https://2020.igem.org/Team%3AQueens_Canada/Contribution)
* HUMAN
  + [Human Practices](https://2020.igem.org/Team%3AQueens_Canada/Human_Practices)
  + [Education](https://2020.igem.org/Team%3AQueens_Canada/Education)
  + [Collaborations](https://2020.igem.org/Team%3AQueens_Canada/Collaborations)
  + [Partnership](https://2020.igem.org/Team%3AQueens_Canada/Partnership)
  + [Safety](https://2020.igem.org/Team%3AQueens_Canada/Safety)
* MODEL
  + [Overview](https://2020.igem.org/Team%3AQueens_Canada/Model)
  + [Protein Stability](https://2020.igem.org/Team%3AQueens_Canada/Static-Dynamic)
  + [Coiled-Coil Stability](https://2020.igem.org/Team%3AQueens_Canada/Coiled-Coil-Stability)
  + [Cysteine Modification](https://2020.igem.org/Team%3AQueens_Canada/Directed-Cysteine)
  + [Software](https://2020.igem.org/Team%3AQueens_Canada/Software)
  + [Mutaguide](https://2020.igem.org/Team%3AQueens_Canada/Mutaguide)
* PROTOTYPE
  + [Overview](https://2020.igem.org/Team%3AQueens_Canada/Prototype-Overview)
  + [Fluorometer](https://2020.igem.org/Team%3AQueens_Canada/Fluorometer)
  + [Fluid Dynamics](https://2020.igem.org/Team%3AQueens_Canada/Fluid-Dynamics)
  + [Mobile App](https://2020.igem.org/Team%3AQueens_Canada/App)
* PARTS
  + [Overview](https://2020.igem.org/Team%3AQueens_Canada/Parts-Overview)
  + [Expression Plasmid](https://2020.igem.org/Team%3AQueens_Canada/Expression-Plasmid)
  + [Purification Summary](https://2020.igem.org/Team%3AQueens_Canada/Purification-Summary)
* NOTEBOOK
  + [Protocols](https://2020.igem.org/Team%3AQueens_Canada/Protocols)
  + [Posters and Presentations](https://2020.igem.org/Team%3AQueens_Canada/Posters-Presentations)
* TEAM
  + [Team Members](https://2020.igem.org/Team%3AQueens_Canada/Team)
  + [Our Sponsors](https://2020.igem.org/Team%3AQueens_Canada/Sponsors)
  + [Attributions](https://2020.igem.org/Team%3AQueens_Canada/Attributions)

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Queens Canada/Results

[![](https://static.igem.org/mediawiki/2020/6/67/T--Queens_Canada--logos.png)](https://2020.igem.org/Team%3AQueens_Canada)

* [HOME](https://2020.igem.org/Team%3AQueens_Canada)
* PROJECT
  + [Description](https://2020.igem.org/Team%3AQueens_Canada/Description)
  + [Protein Construct](https://2020.igem.org/Team%3AQueens_Canada/Protein-Construct)
  + [FRET](https://2020.igem.org/Team%3AQueens_Canada/FRET)
  + [Coiled-Coils](https://2020.igem.org/Team%3AQueens_Canada/Coiled-Coils)
  + [Binding Proteins](https://2020.igem.org/Team%3AQueens_Canada/Binding-Proteins)
  + [Implementation](https://2020.igem.org/Team%3AQueens_Canada/Implementation)
  + [Results](https://2020.igem.org/Team%3AQueens_Canada/Results)
  + [Contribution](https://2020.igem.org/Team%3AQueens_Canada/Contribution)
* HUMAN
  + [Human Practices](https://2020.igem.org/Team%3AQueens_Canada/Human_Practices)
  + [Education](https://2020.igem.org/Team%3AQueens_Canada/Education)
  + [Collaborations](https://2020.igem.org/Team%3AQueens_Canada/Collaborations)
  + [Partnership](https://2020.igem.org/Team%3AQueens_Canada/Partnership)
  + [Safety](https://2020.igem.org/Team%3AQueens_Canada/Safety)
* MODEL
  + [Overview](https://2020.igem.org/Team%3AQueens_Canada/Model)
  + [Protein Stability](https://2020.igem.org/Team%3AQueens_Canada/Static-Dynamic)
  + [Coiled-Coil Stability](https://2020.igem.org/Team%3AQueens_Canada/Coiled-Coil-Stability)
  + [Cysteine Modification](https://2020.igem.org/Team%3AQueens_Canada/Directed-Cysteine)
  + [Software](https://2020.igem.org/Team%3AQueens_Canada/Software)
  + [Mutaguide](https://2020.igem.org/Team%3AQueens_Canada/Mutaguide)
* PROTOTYPE
  + [Overview](https://2020.igem.org/Team%3AQueens_Canada/Prototype-Overview)
  + [Fluorometer](https://2020.igem.org/Team%3AQueens_Canada/Fluorometer)
  + [Fluid Dynamics](https://2020.igem.org/Team%3AQueens_Canada/Fluid-Dynamics)
  + [Mobile App](https://2020.igem.org/Team%3AQueens_Canada/App)
* PARTS
  + [Overview](https://2020.igem.org/Team%3AQueens_Canada/Parts-Overview)
  + [Expression Plasmid](https://2020.igem.org/Team%3AQueens_Canada/Expression-Plasmid)
  + [Purification Summary](https://2020.igem.org/Team%3AQueens_Canada/Purification-Summary)
* NOTEBOOK
  + [Protocols](https://2020.igem.org/Team%3AQueens_Canada/Protocols)
  + [Posters and Presentations](https://2020.igem.org/Team%3AQueens_Canada/Posters-Presentations)
* TEAM
  + [Team Members](https://2020.igem.org/Team%3AQueens_Canada/Team)
  + [Our Sponsors](https://2020.igem.org/Team%3AQueens_Canada/Sponsors)
  + [Attributions](https://2020.igem.org/Team%3AQueens_Canada/Attributions)

# Results

WHAT DID WE FIND?

To determine the most conserved substitutions for free cysteines in the binding proteins, amino acid sequences were inserted into Phyre2, an online server for protein homology which runs an intensive BLAST search, giving a sequence homology output in FASTA format. Sequences for phosphate binding protein, potassium binding protein, glucose/galactose binding protein, alpha-klotho, and the parathyroid hormone receptor (N-termini) were inserted.

Compared to other binding proteins, alpha-klotho had the most free cysteines, requiring the most substitutions. Interestingly, glucose, potassium, and the parathyroid hormone receptor N-termini had no free cysteines, so only one substitution was made – inserting a cysteine to later bind to a coil. Both glucose and potassium binding proteins had their C-termini trimmed by 23 and 8 amino acids, respectively, a recommendation found in literature. To read more about how we mutated, trimmed, and identified residues for cysteine type immobilization refer to our protocol section.

After substitutions took place, mNeonGreen and mCherry were added to the N- and C- terminis of our binding proteins respectively, to allow for fluorescent detection of biomarkers. With phosphate binding protein, a 2 amino acid glycine-threonine linker was inserted between the fluorophores and binding protein, a recommendation from literature. It is worth noting that the parathyroid hormone receptor and alpha-klotho only have a N- termini fluorophore attached. This is because they are involved in competition type assays, with either PTH or FGF23 tagged with the partner fluorophore. More information on this may be found in our contributions or protocol pages.

![](https://static.igem.org/mediawiki/2020/e/e6/T--Queens_Canada--results-1.png)

![](https://static.igem.org/mediawiki/2020/0/04/T--Queens_Canada--results-2.png)

![](https://static.igem.org/mediawiki/2020/e/e4/T--Queens_Canada--results-3.png)

![](https://static.igem.org/mediawiki/2020/2/2c/T--Queens_Canada--results-4.png)

![](https://static.igem.org/mediawiki/2020/8/8b/T--Queens_Canada--results-5.png)

**Fluorescent binding protein constructs with mutations.** Cysteine based substitutions were made after intensive homology searches for five biomarker binding proteins. From top to bottom; phosphate, potassium, glucose, PTH, and FGF23 binding proteins. Fluorophore(s) were added to the N- and C- termini’s. DNA basic and composite part sequences may be found on the ‘parts page’ on this wiki or the Registry of Standard Biological Parts.

The one remaining free cysteine on the surface of the binding protein was placed in a position opposite to the active site and away from the fluorophore tails, as to not interfere with either ligand binding or fluorescent activity. Cysteines were not added to areas of intrinsic disorder, as to avoid a binding that may be uncontrolled in spatial space, and further, avoid hindering protein conformational adjustments. Binding proteins with this cysteine were modelled using PyMOL, a molecular viewing software, to ensure that the substitution and cysteine would face away from the protein, towards solvent, and was free of steric clash. Interestingly, all of our cysteines were mutated onto an alpha helix residue facing towards solvent, regardless of the binding protein.

![](https://static.igem.org/mediawiki/2020/8/85/T--Queens_Canada--results-6.png)

**Cysteine immobilization modifications of binding proteins.** Protein structures were obtained from the RCSB Protein Data Bank. All protein residues are shown in a blue cartoon preset with cysteine residues in red cartoon, respectively, using PyMOL. A. Phosphate binding protein with a Cys 256 modification (red). B. Potassium binding protein with a Cys 26 modification (red). C. Parathyroid hormone receptor, with a Cys 48 residue (red). D. Glucose/Galactose binding protein with a Cys 190 modification (red). E. Alpha-klotho (FGF23 receptor) with a Cys 664 modification (red).

Using Chimera, a protein modeling software, mNeonGreen and mCherry fluorophores were attached the N- and C- terminis, respectively, with the exception of the parathyroid hormone receptor and alpha-klotho. This was to ensure that the cysteine placement did not interfere with the fluorophores in 3D space – as this would either hinder fluorescence, coil binding, or both. A cartoon model preset was generated for reference while a ball and stick preset were used to observe all residues in 3D space. For all proteins, no interference was observed between any potential cysteine placement and the fluorophores.

![](https://static.igem.org/mediawiki/2020/2/24/T--Queens_Canada--results-7.png)

**Phosphate binding protein fluorescent construct.** Protein structures were obtained from the RCSB Protein Data Bank and a construct was made using Chimera software. Torsion angles between fluorescent and binding proteins are adjusted for display purposes. mNeonGreen (green), binding protein (blue), and mCherry (red) are all displayed using PyMOL. A. Phosphate binding protein with a cartoon preset coupled with two fluorophores (mNeonGreen and mCherry). B. Construct in a stick preset to provide insights regarding the construct volume in 3D space.

![](https://static.igem.org/mediawiki/2020/9/9b/T--Queens_Canada--results-8.png)

**Potassium binding protein fluorescent construct.** Protein structures were obtained from the RCSB Protein Data Bank and a construct was made using Chimera software. Torsion angles between fluorescent and binding proteins are adjusted for display purposes. mNeonGreen (green), binding protein (blue), and mCherry (red) are all displayed using PyMOL. A. Potassium binding protein with a cartoon preset coupled with two fluorophores (mNeonGreen and mCherry). B. Construct in a stick preset to provide insights regarding the construct volume in 3D space.

![](https://static.igem.org/mediawiki/2020/f/fb/T--Queens_Canada--results-9.png)

**Glucose/Galactose binding protein fluorescent construct.** Protein structures were obtained from the RCSB Protein Data Bank and a construct was made using Chimera software. Torsion angles between fluorescent and binding proteins are adjusted for display purposes. mNeonGreen (green), binding protein (blue), and mCherry (red) are all displayed using PyMOL. A. Glucose/Galactose binding protein with a cartoon preset coupled with two fluorophores (mNeonGreen and mCherry). B. Construct in a stick preset to provide insights regarding the construct volume in 3D space.

![](https://static.igem.org/mediawiki/2020/e/e0/T--Queens_Canada--results-10.png)

**Parathyroid receptor hormone fluorescent construct.** Protein structures were obtained from the RCSB Protein Data Bank and a construct was made using Chimera software. Torsion angles between fluorescent and binding proteins are adjusted for display purposes. mNeonGreen (green) and binding protein (blue), are both displayed using PyMOL. A. Parathyroid hormone receptor with a cartoon preset coupled with a fluorophore (mNeonGreen). B. Construct in a stick preset to provide insights regarding the construct volume in 3D space.

![](https://static.igem.org/mediawiki/2020/5/52/T--Queens_Canada--results-11.png)

**Alpha-Klotho (FGF23 receptor) fluorescent construct.** Protein structures were obtained from the RCSB Protein Data Bank and a construct was made using Chimera software. Torsion angles between fluorescent and binding proteins are adjusted for display purposes. mNeonGreen (green) and binding protein (blue), are both displayed using PyMOL. A. Alpha-klotho (FGF23 receptor) with a cartoon preset coupled with a fluorophore (mNeonGreen). B. Construct in a stick preset to provide insights regarding the construct volume in 3D space.

Using the APBS electrostatics function in PyMOL, an electrostatic potential map of all protein constructs was generated to view the potential active sites of biomarker binding. This may be viewed in our appendix on the wiki.

![](https://static.igem.org/mediawiki/2020/7/77/T--Queens_Canada--results-12.png)

**Phosphate binding protein fluorescent construct electrostatic potential map.** Protein structures were obtained from the RCSB Protein Data Bank and a construct was made using Chimera software. Torsion angles between fluorescent and binding proteins are adjusted for display purposes. mNeonGreen, binding protein, and mCherry are oriented left to right in the ‘FRONT’ figure and were generated using the APBS Electrostatics function in PyMOL. 3D volume in space can also be visualized. A high and low electrostatic potential, is indicated by blue and red, respectively. Note: A high electrostatic potential indicates an absence of electrons and is positive.

Complete constructs (fluorescent binding protein with E/K cysteine tagged coiled-coil) were built using Chimera in order to visualize the construct in 3D space. Primary concern was to ensure that the length of the five-heptad repeated coils was long enough to anchor the fluorescent binding proteins away from the gold chamber and avoid them from hitting the chamber. We believe this to be the case for our protein constructs, and based on the proposed construction protocol, the proteins should not clash with the gold chamber – granted they are solvated in buffer solution.

![](https://static.igem.org/mediawiki/2020/0/09/T--Queens_Canada--results-13.png)

**Coiled coil system bound to fluorescent binding proteins.** Protein structures were obtained from the RCSB Protein Data Bank. All protein residues are shown in a cartoon preset with the E and K coils in red and blue, respectively, using Chimera software. E and K coils each consist of five-heptad repeats with a hydrophobic core stabilized by glutamates and lysine residues surround the interface. A. Phosphate binding protein with a Cys 256 modification. B. Potassium binding protein with a Cys 26 modification. C. Parathyroid hormone receptor with a Cys 48 residue. D. Glucose/Galactose binding protein with a Cys 190 modification. E. Alpha-klotho (FGF23 receptor) with a Cys 664 modification.

Enhanced view of the disulfide binding interface between fluorescent binding proteins and the coiled-coil was collected to ensure no interference or steric clashing by either protein structure upon association. Chimera also was used to collect photos of the coiled-coil in both cartoon and solid surface presets – demonstrating a potentially tight hydrophobic association.

![](https://static.igem.org/mediawiki/2020/f/fc/T--Queens_Canada--results-14.png)

**Coiled coil system bound to fluorescent phosphate binding protein.** Protein structures were obtained from the RCSB Protein Data Bank. All protein residues are shown in a cartoon preset with the E and K coils in red and blue, respectively, using Chimera software. E and K coils each consist of five-heptad repeats with a hydrophobic core stabilized by glutamates and lysine residues surround the interface. Binding protein is a phosphate binding protein with a Cys 256 modification to bind the E coil, along with mNeonGreen and mCherry fluorophores at the N- and C- termini's, respectively. A. Entire construct structure. B. View of the Cys 256 of the phosphate binding protein attached to the E coil. C. Enhanced view of the E and K coiled coil system.

![](https://static.igem.org/mediawiki/2020/c/c0/T--Queens_Canada--results-15.png)

**E/K coiled coil system.** All protein residues are shown with the E and K coils in red and blue (B-D) , respectively, using Chimera software. E and K coils each consist of five-heptad repeats with a hydrophobic core stabilized by glutamates and lysine residues surround the interface. A. Coil’s in a cartoon preset with each coil's cysteine labelled and displayed in a ball-and-stick preset. Note the sulfur atom in yellow. B. Coiled coil system in a mesh preset to visualize volume in 3D space. C. Volume in 3D space further enhanced with a solid surface preset, further, highlighting the interwoven nature of the coiled coil. D. Standard cartoon preset with the coiled coil alpha-helices oriented vertically.

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