iGEM 2019 — Team ULaval

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:ULaval/Notebook

Team:ULaval - 2019.igem.org

×

# Notebook

[Lab Journal](https://2019.igem.org/Team%3AULaval/Notebook)
[Experiments](https://2019.igem.org/Team%3AULaval/Experiments)

# Parts

[Overview](https://2019.igem.org/Team%3AULaval/Parts)
[Basic Parts](https://2019.igem.org/Team%3AULaval/Basic_Part)

# Team

[Team members](https://2019.igem.org/Team%3AULaval/Team)
[Attributions](https://2019.igem.org/Team%3AULaval/Attributions)
 [Achievements](https://2019.igem.org/Team%3AULaval/Achievements.html)
[Collaborations](https://2019.igem.org/Team%3AULaval/Collaborations)

# Human Practices

[Human Practices](https://2019.igem.org/Team%3AULaval/Human_Practices)
[Pathogens Detections](https://2019.igem.org/Team%3AULaval/Human_Practices/Pathogens_Detections)
[Our Approach](https://2019.igem.org/Team%3AULaval/Human_Practices/Our_Approach)
[Public Engagement](https://2019.igem.org/Team%3AULaval/Public_Engagement)
[Expert Insight](https://2019.igem.org/Team%3AULaval/Human_Practices/ExpertInsight)
[Market Consultation](https://2019.igem.org/Team%3AULaval/Human_Practices/Market_Consultation)
[Safety](https://2019.igem.org/Team%3AULaval/Safety)

# Project

[Description](https://2019.igem.org/Team%3AULaval/Description)
[Design](https://2019.igem.org/Team%3AULaval/Design)
[Characterization](https://2019.igem.org/Team%3AULaval/Contribution)
[Results](https://2019.igem.org/Team%3AULaval/Results)
[Model](https://2019.igem.org/Team%3AULaval/Model)
[Software](https://2019.igem.org/Team%3AULaval/Software)
[Hardware](https://2019.igem.org/Team%3AULaval/Hardware)

[![](https://static.igem.org/mediawiki/2019/e/e6/T--Ulaval--TeamLogo.png)](https://2019.igem.org/Team%3AULaval)

[

](https://static.igem.org/mediawiki/2019/6/6e/T--Ulaval--videoNavbar.mp4)

[Home](https://2019.igem.org/Team%3AULaval)

Project

[Description](https://2019.igem.org/Team%3AULaval/Description)
[Design](https://2019.igem.org/Team%3AULaval/Design)
[Characterization](https://2019.igem.org/Team%3AULaval/Contribution)
[Results](https://2019.igem.org/Team%3AULaval/Results)
[Model](https://2019.igem.org/Team%3AULaval/Model)
[Software](https://2019.igem.org/Team%3AULaval/Software)
[Hardware](https://2019.igem.org/Team%3AULaval/Hardware)

Parts

[Overview](https://2019.igem.org/Team%3AULaval/Parts)
[Basic Parts](https://2019.igem.org/Team%3AULaval/Basic_Part)

[\_ \_](https://2019.igem.org/Team%3AULaval)

Notebook

[Lab Journal](https://2019.igem.org/Team%3AULaval/Notebook)
[Experiments](https://2019.igem.org/Team%3AULaval/Experiments)

Human Practices

[Human Practices](https://2019.igem.org/Team%3AULaval/Human_Practices)
[Pathogens Detections](https://2019.igem.org/Team%3AULaval/Human_Practices/Pathogens_Detections)
[Our Approach](https://2019.igem.org/Team%3AULaval/Human_Practices/Our_Approach)
[Public Engagement](https://2019.igem.org/Team%3AULaval/Public_Engagement)
[Expert Insight](https://2019.igem.org/Team%3AULaval/Human_Practices/ExpertInsight)
[Market Consultation](https://2019.igem.org/Team%3AULaval/Human_Practices/Market_Consultation)
[Safety](https://2019.igem.org/Team%3AULaval/Safety)

Team

[Team members](https://2019.igem.org/Team%3AULaval/Team)
[Attributions](https://2019.igem.org/Team%3AULaval/Attributions)
[Achievements](https://2019.igem.org/Team%3AULaval/Achievements.html)
[Collaborations](https://2019.igem.org/Team%3AULaval/Collaborations)

☰ open

![head](https://static.igem.org/mediawiki/2019/6/63/T--Ulaval--HeaderNoteBook.png)

# Notebook

### Show all

### Preliminary organization

### Science

### Dry lab

### Wet lab

### Collaborations

### Public Engagement/Funding

### January 23rd 2019

**Helping during Career Day at Université Laval to fund the team**

François, Nathan, Pascale, Marianne, Florian and Catherine

### January 2019 - February 2019

**Recruitment and committee formation**

François, Nathan, Élodie, Catherine, Florian, Marianne, Pascale, Lucas

Planned our comitee's objectives and size, tried to broaden the disciplins from which the new students could come from, as last year it was mostly from our biochemitry, microbiology and bioinformatic's department.

### February 14th, 2019

**Project design and planification**

Whole team

Meetings discussing our objectives and the parts of the project.

### February 27th, 2019

**Science committee job distribution**

François, Angel, Carla, Pascale, Guillaume, Ahmed

We decided who would focus their efforts on which scientific aspects of the project.

### March 19th, 2019

**First experience with NUPACK to model RNA secondary structure**

Angel, Elodie

Learning how to use NUPACK by modeling structures in their server and with their command-line tool.

### April 1st, 2019

**First Toehold designed by hand**

François, Elodie

First try at designing ToeHold switches by hand. This proved very complex, and was partly what motivated us to work on the tool.

### April 15th, 2019

**Project presentation in our department**

François and Nathan (presenters), Carla, Angel, Pascale, Guillaume, Elodie, Catherine

Meeting with the department's researchers to receive feedback on our design and our understanding of the problem.

### April 18th, 2019

**Best Scientific Project winner at the Gala de la vie étudiante**

Whole team

![](https://static.igem.org/mediawiki/2019/7/70/T--Ulaval--labapril.jpg)

### May 8th, 2019

**First full version of the toehold design script (designing a toehold for a trigger)**

Angel

Writing Python code to run NUPACK automatically and analyze the results.

### May 10th, 2019

**Submission of our presentation plan to the TEDxQuebec coordinator**

Martine and Nathan

We had to submit a plan and summary about our presentation. We also had to tell them how our presentation would be interesting to their public. We also had to submit a small video summarizing our talk.

### May 19th, 2019

**First 3D model of a toehold riboswitch**

Angel

Designed the model of the riboswitch with RNAComposer and evaluated its quality with MOLProbity.

### May 22th, 2019

**Pint of Science Québec organization + presentation**

Elodie, Florian and Martine

Presenting our 2018 project and vulgarizing synthetic biology to the general public in a pub.

![](https://static.igem.org/mediawiki/2019/1/10/T--Ulaval--labmay.jpg)

### June 1st, 2019

**Extension of the script to look at all candidate triggers in a gene of interest**

Angel

Tried with different triggers in a gene to study which ones have the most desirable energetic parameters.

### June 8th, 2019

**First coaching for the TEDxQuébec presentation**

Nathan and Catherine

Practicing the TEDxQuébec presentation with the other speakers and three communication coaches.

### June 23rd, 2019

**Toehold gBlocks received from IDT, cloning starts**

François

Cloning toehold inserts

### July 2nd, 2019

**First design of microfluidics circuit**

Francois, Ahmed and Nastaran

A two-level microchip with three reaction chambers, but lacked waste disposal on one step as it was fixed later.

### July 5nd, 2019

**Interview with a journalist from the Quebec Science magazine**

Martine and Karine

The magazine's goal is to inform people about the current advances in science. The journalist (Joel Leblanc) mainly asked questions about the projects the team has carried out until now; AdrenaYeast and ADN. He also asked questions about synthetic biology and its ethical implications. The article should be published between December and February.

### July 6th, 2019

**Collaborative comic submission to iGEM Wroclaw**

Martine, Jean-Michel and Angel

Taking photos in specific places for Wroclaw to build their comic

### July 14th, 2019

**Started molecular dynamics simulations (MDS) with the toehold 3D structure**

Angel, assisted by Patrick Lagüe

Ran MDS with NAMD in our server to study the dynamics of the 3D structure.

### July 16th, 2019

**Skype call to discuss collaborations with iGEM Concordia**

Nathan and Angel

Planning modelisation and computation sharing

### July 20th, 2019

**Participation in the meetup organized by iGEM Western Ontario**

Angel and François

Presenting the advancements so far with other Canadian teams.

### July 27th, 2019

**Competent cells**

Carla

We made the competent cells using the CaCl2 method.

### July 30th, 2019

**Alternative innovative funding opportunity launched**

Florian

We put remaining designs from diverse activities and new ones on the subject of synthetic biology or microbiology on shirts to both promote science and find a new way for the student community to get involved with our activities

### August 1st, 2019

**Change in cloning design**

Francois

We had a problem with one of our plasmids, so we changed the backbone on one of our PI's instruction.

### August 5th, 2019

**Participation in the meetup organized by iGEM MIT**

Angel and Nathan

We showed our general structure with human practices and public engagement, and the judge's comments helped us shape more thouroughly our approach

### August 8th, 2019

**Popular Science On microbiolgy presentation at Club des petits débrouillards Summer Camp**

Julien, Florian and Guillaume

Presentation on microbiology where kids can participate through quiz and are invited to "contaminate" petri dishes to observe what can't be seen on their hands.

![](https://static.igem.org/mediawiki/2019/7/7d/T--Ulaval--labaugust.jpg)

### August 23th, 2019

**Popular Science on microbiology presentation at Club des petits Débrouillards Summer Camp**

Julien and Florian

Presentation on microbiology where kids can participate through quiz and are invited to "contaminate" petri dishes to observe what can't be seen on their hands

### September 1st, 2019

**Cloning: Digestion and plasmid purification**

Carla

We did the digestion and purification of the plasmid with the new backbone proposed for cloning

### September 8th, 2019

**Received 3D modelling results from iGEM Concordia**

Angel

Exploiting different software allowed us to compare RNAComposer to Rosetta and verifying the RNA folding, with the essential region conserving its behaviour, and observing disparities in the non-specific part's folding

### September 9th, 2019

**Extension of the script to do alignments to reference genomes to test specificity**

Angel

Wrote the necessary code to run the alignments and made it versatile so that users could easily specify the genomes of interest.

### September 10th, 2019

**Expo Synthbio - Morrin Centre**

Julien, Florian and Guillaume

Exploiting different software allowed us to compare RNAComposer to Rosetta and verifying the RNA folding, with the essential region conserving its behaviour, and observing disparities in the non-specific part's folding

![](https://static.igem.org/mediawiki/2019/5/57/T--Ulaval--labseptembre.jpg)

### September 11th, 2019

**Day of the committees of the AESGUL**

Angel, François, Florian and Carla

We showed our project to the members of the university community and we did a survey with people to complete our part of human practices.

![](https://static.igem.org/mediawiki/2019/6/65/T--Ulaval--labseptembre11.jpg)

### September 16th, 2019

**First round of parts characterization**

Francois and Pascale

Expression of freen fluorescent protein previously characterized in vivo, here measured in cell-free media, but the results were confusing.

### September 17th, 2019

**Presentation at Cabaret ad Lab (Université Laval)**

Florian and Nathan

Ultra-vulgarized presentation for students at the Département de biochimie, microbiologie et bio-informatique.

### September 19th, 2019

**Second coaching for the TEDxQuébec presentation**

Nathan and Catherine

Practicing the TEDxQuébec presentation with the other speakers and three communication coaches.

### September 24th, 2019

**Prepared the Github repository**

Angel

Uploaded our scripts to Github to make them available to the synthetic biology community.

### September 27th-28th, 2019

**Participation in the meetup organized by iGEM Concordia**

Angel, François, Florian and Nathan

Presented our project and poster at a Mini Jamboree hosted by Concordia that also included Queen's.

![](https://static.igem.org/mediawiki/2019/0/04/T--Ulaval--labseptembre2728.jpg)

### October 3th, 2019

**Bartending at a university party to fund the team**

Catherine, Jean-Michel, Florian, François, Guillaume, Angel, Marianne, Nathan and Christina, a friend of us

![](https://static.igem.org/mediawiki/2019/9/98/T--Ulaval--laboct0319.jpg)

### October 5th, 2019

**Third coaching for the TEDxQuébec presentation**

Nathan and Catherine

Practicing the TEDxQuébec presentation with the other speakers and three communication coaches.

### October 8th, 2019

**Second design of microfluidics circuit**

Nastaran and Francois

We modified our microfluidics circuit after feedback from experts. The design was streamlined and the protocol adapted.

### October 8th, 2019

**Successfully finished raising money to participate at the competition.**

Catherine, Martine, Jean-Michel and Lucas

### October 9th, 2019

**Successful clones for ToeHolds**

Francois

Final parts that were selected for characterization were tested in cell-free media and their efficiency was assessed.

### October 10th, 2019

**Final round of parts characerization**

Pascale and Francois

Fluorescence calibration curve and testing the GFP parts in cell free media, found what was missing the last try.

### October 11th, 2019

**Toehold test in presence of trigger sequence, confirmation of system efficiency**

Francois

We tested our most efficient ToeHold switch in cell-free expression system using isolated bacterial mRNA to test our design in conditions closer that what they will be in our tool.

### October 11th, 2019

**Finished modeling collaboration with Concordia**

Angel

Wrote MATLAB code that Concordia could use to model the difusion of amilCP in their gel.

### October 11th, 2019

**Hydrogen bond analysis from molecular dynamics simulations.**

Angel and Florian

Analyzed the hydrogen bonds in the simulations to look at the most stable ones and the least stable ones. Designed a figure to convey this information clearly.

### October 14th, 2019

**Building microlfuidics device chip**

Nastaran

The newly designed microfluidics design was prepared and printed according to the protocol.

### October 15th, 2019

**Wrote code to obtain consensus secondary structures.**

Angel

This new code allows us to see the consensus structures from the designed toeholds to prove that they are similar to the intended one.

### October 16th, 2019

**Designed new toeholds for measles virus, chickenpox virus, and norovirus**

Angel and Pascale

We used our software to produce new toeholds that would allow the detection of viruses of interest and submitted them to the iGEM registry.

### October 16th, 2019

**Presentation at the Interlabs at Université Laval**

Nathan, François, Angel and Florian

Presenting the advancements so far to faculty members and students at Université Laval.

### October 31st to November 5th, 2019

**GIANT JAMBOREE!**

Nathan, François, Florian, Julien, Guillaume, Angel, Carla, Martine, Catherine, Pascale, Lucas and Marianne

### November 7th, 2019

**Presenting iGEM to High School students and teachers at Musée de la civilisation**

Martine and François

![head](https://static.igem.org/mediawiki/2019/e/e3/T--Ulaval--labnov0719.jpg)

### January 11th, 2020

**TEDxQuébec Presentation**

Nathan and Catherine

Presenting a vulgarized presentation about synthetic biology and its benefits and the importance of communicating science to the general public. The presentation will be filmed and available on the Web.

igem@bcm.ulaval.ca


[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:ULaval/Experiments

Team:ULaval - 2019.igem.org

×

# Notebook

[Lab Journal](https://2019.igem.org/Team%3AULaval/Notebook)
[Experiments](https://2019.igem.org/Team%3AULaval/Experiments)

# Parts

[Overview](https://2019.igem.org/Team%3AULaval/Parts)
[Basic Parts](https://2019.igem.org/Team%3AULaval/Basic_Part)

# Team

[Team members](https://2019.igem.org/Team%3AULaval/Team)
[Attributions](https://2019.igem.org/Team%3AULaval/Attributions)
 [Achievements](https://2019.igem.org/Team%3AULaval/Achievements.html)
[Collaborations](https://2019.igem.org/Team%3AULaval/Collaborations)

# Human Practices

[Human Practices](https://2019.igem.org/Team%3AULaval/Human_Practices)
[Pathogens Detections](https://2019.igem.org/Team%3AULaval/Human_Practices/Pathogens_Detections)
[Our Approach](https://2019.igem.org/Team%3AULaval/Human_Practices/Our_Approach)
[Public Engagement](https://2019.igem.org/Team%3AULaval/Public_Engagement)
[Expert Insight](https://2019.igem.org/Team%3AULaval/Human_Practices/ExpertInsight)
[Market Consultation](https://2019.igem.org/Team%3AULaval/Human_Practices/Market_Consultation)
[Safety](https://2019.igem.org/Team%3AULaval/Safety)

# Project

[Description](https://2019.igem.org/Team%3AULaval/Description)
[Design](https://2019.igem.org/Team%3AULaval/Design)
[Characterization](https://2019.igem.org/Team%3AULaval/Contribution)
[Results](https://2019.igem.org/Team%3AULaval/Results)
[Model](https://2019.igem.org/Team%3AULaval/Model)
[Software](https://2019.igem.org/Team%3AULaval/Software)
[Hardware](https://2019.igem.org/Team%3AULaval/Hardware)

[![](https://static.igem.org/mediawiki/2019/e/e6/T--Ulaval--TeamLogo.png)](https://2019.igem.org/Team%3AULaval)

[

](https://static.igem.org/mediawiki/2019/6/6e/T--Ulaval--videoNavbar.mp4)

[Home](https://2019.igem.org/Team%3AULaval)

Project

[Description](https://2019.igem.org/Team%3AULaval/Description)
[Design](https://2019.igem.org/Team%3AULaval/Design)
[Characterization](https://2019.igem.org/Team%3AULaval/Contribution)
[Results](https://2019.igem.org/Team%3AULaval/Results)
[Model](https://2019.igem.org/Team%3AULaval/Model)
[Software](https://2019.igem.org/Team%3AULaval/Software)
[Hardware](https://2019.igem.org/Team%3AULaval/Hardware)

Parts

[Overview](https://2019.igem.org/Team%3AULaval/Parts)
[Basic Parts](https://2019.igem.org/Team%3AULaval/Basic_Part)

[\_ \_](https://2019.igem.org/Team%3AULaval)

Notebook

[Lab Journal](https://2019.igem.org/Team%3AULaval/Notebook)
[Experiments](https://2019.igem.org/Team%3AULaval/Experiments)

Human Practices

[Human Practices](https://2019.igem.org/Team%3AULaval/Human_Practices)
[Pathogens Detections](https://2019.igem.org/Team%3AULaval/Human_Practices/Pathogens_Detections)
[Our Approach](https://2019.igem.org/Team%3AULaval/Human_Practices/Our_Approach)
[Public Engagement](https://2019.igem.org/Team%3AULaval/Public_Engagement)
[Expert Insight](https://2019.igem.org/Team%3AULaval/Human_Practices/ExpertInsight)
[Market Consultation](https://2019.igem.org/Team%3AULaval/Human_Practices/Market_Consultation)
[Safety](https://2019.igem.org/Team%3AULaval/Safety)

Team

[Team members](https://2019.igem.org/Team%3AULaval/Team)
[Attributions](https://2019.igem.org/Team%3AULaval/Attributions)
[Achievements](https://2019.igem.org/Team%3AULaval/Achievements.html)
[Collaborations](https://2019.igem.org/Team%3AULaval/Collaborations)

☰ open

![achievements head](https://static.igem.org/mediawiki/2019/b/ba/T--Ulaval--ExperimentsHeader.png)

# EXPERIMENTS

Microfluidics design ⮟

The PDF plug-in does not seem to work.[Click here to see the PDF.](https://static.igem.org/mediawiki/2019/1/1e/T--Ulaval--microfluidics.pdf)

Competent cells preparation (CaCl2 Method) ⮟

The PDF plug-in does not seem to work.[Click here to see the PDF.](https://static.igem.org/mediawiki/2019/a/aa/T--Ulaval--CompetentCell.pdf)

Gibson assembly of parts ⮟

The PDF plug-in does not seem to work.[Click here to see the PDF.](https://static.igem.org/mediawiki/2019/8/8f/T--Ulaval--Gibson.pdf)

Transformation of chemically competent E. coli ⮟

The PDF plug-in does not seem to work.[Click here to see the PDF.](https://static.igem.org/mediawiki/2019/8/88/T--Ulaval--BacterialTransformation.pdf)

Colony PCR for cloning confirmation ⮟

The PDF plug-in does not seem to work.[Click here to see the PDF.](https://static.igem.org/mediawiki/2019/c/c3/T--Ulaval--ColonyPCR.pdf)

Plasmid extraction ⮟

The PDF plug-in does not seem to work.[Click here to see the PDF.](https://static.igem.org/mediawiki/2019/0/0b/T--Ulaval--Plasmid_Extraction.pdf)

RNA extraction ⮟

The PDF plug-in does not seem to work.[Click here to see the PDF.](https://static.igem.org/mediawiki/2019/f/f4/T--Ulaval--RNAextract.pdf)

Magnetic beads DNA extraction ⮟

The PDF plug-in does not seem to work.[Click here to see the PDF.](https://static.igem.org/mediawiki/2019/5/5d/T--Ulaval--DNAextract.pdf)

Cell-free expression system ⮟

The PDF plug-in does not seem to work.[Click here to see the PDF.](https://static.igem.org/mediawiki/2019/a/a7/T--Ulaval--Cell-freesystem.pdf)

igem@bcm.ulaval.ca

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:ULaval/Results

Team:ULaval - 2019.igem.org

×

# Notebook

[Lab Journal](https://2019.igem.org/Team%3AULaval/Notebook)
[Experiments](https://2019.igem.org/Team%3AULaval/Experiments)

# Parts

[Overview](https://2019.igem.org/Team%3AULaval/Parts)
[Basic Parts](https://2019.igem.org/Team%3AULaval/Basic_Part)

# Team

[Team members](https://2019.igem.org/Team%3AULaval/Team)
[Attributions](https://2019.igem.org/Team%3AULaval/Attributions)
 [Achievements](https://2019.igem.org/Team%3AULaval/Achievements.html)
[Collaborations](https://2019.igem.org/Team%3AULaval/Collaborations)

# Human Practices

[Human Practices](https://2019.igem.org/Team%3AULaval/Human_Practices)
[Pathogens Detections](https://2019.igem.org/Team%3AULaval/Human_Practices/Pathogens_Detections)
[Our Approach](https://2019.igem.org/Team%3AULaval/Human_Practices/Our_Approach)
[Public Engagement](https://2019.igem.org/Team%3AULaval/Public_Engagement)
[Expert Insight](https://2019.igem.org/Team%3AULaval/Human_Practices/ExpertInsight)
[Market Consultation](https://2019.igem.org/Team%3AULaval/Human_Practices/Market_Consultation)
[Safety](https://2019.igem.org/Team%3AULaval/Safety)

# Project

[Description](https://2019.igem.org/Team%3AULaval/Description)
[Design](https://2019.igem.org/Team%3AULaval/Design)
[Characterization](https://2019.igem.org/Team%3AULaval/Contribution)
[Results](https://2019.igem.org/Team%3AULaval/Results)
[Model](https://2019.igem.org/Team%3AULaval/Model)
[Software](https://2019.igem.org/Team%3AULaval/Software)
[Hardware](https://2019.igem.org/Team%3AULaval/Hardware)

[![](https://static.igem.org/mediawiki/2019/e/e6/T--Ulaval--TeamLogo.png)](https://2019.igem.org/Team%3AULaval)

[

](https://static.igem.org/mediawiki/2019/6/6e/T--Ulaval--videoNavbar.mp4)

[Home](https://2019.igem.org/Team%3AULaval)

Project

[Description](https://2019.igem.org/Team%3AULaval/Description)
[Design](https://2019.igem.org/Team%3AULaval/Design)
[Characterization](https://2019.igem.org/Team%3AULaval/Contribution)
[Results](https://2019.igem.org/Team%3AULaval/Results)
[Model](https://2019.igem.org/Team%3AULaval/Model)
[Software](https://2019.igem.org/Team%3AULaval/Software)
[Hardware](https://2019.igem.org/Team%3AULaval/Hardware)

Parts

[Overview](https://2019.igem.org/Team%3AULaval/Parts)
[Basic Parts](https://2019.igem.org/Team%3AULaval/Basic_Part)

[\_ \_](https://2019.igem.org/Team%3AULaval)

Notebook

[Lab Journal](https://2019.igem.org/Team%3AULaval/Notebook)
[Experiments](https://2019.igem.org/Team%3AULaval/Experiments)

Human Practices

[Human Practices](https://2019.igem.org/Team%3AULaval/Human_Practices)
[Pathogens Detections](https://2019.igem.org/Team%3AULaval/Human_Practices/Pathogens_Detections)
[Our Approach](https://2019.igem.org/Team%3AULaval/Human_Practices/Our_Approach)
[Public Engagement](https://2019.igem.org/Team%3AULaval/Public_Engagement)
[Expert Insight](https://2019.igem.org/Team%3AULaval/Human_Practices/ExpertInsight)
[Market Consultation](https://2019.igem.org/Team%3AULaval/Human_Practices/Market_Consultation)
[Safety](https://2019.igem.org/Team%3AULaval/Safety)

Team

[Team members](https://2019.igem.org/Team%3AULaval/Team)
[Attributions](https://2019.igem.org/Team%3AULaval/Attributions)
[Achievements](https://2019.igem.org/Team%3AULaval/Achievements.html)
[Collaborations](https://2019.igem.org/Team%3AULaval/Collaborations)

☰ open

![achievements head](https://static.igem.org/mediawiki/2019/f/fc/T--Ulaval--ResultsHeader.png)

# Results

## Dry lab

The design of toehold riboswitches is critical for their success as biosensors. Our project includes comprehensive modeling approaches that allowed us to gain insight into the properties of toehold riboswitches [(see Model)](https://2019.igem.org/Team%3AULaval/Model)and the development of a tool that simplifies their production [(see Software).](https://2019.igem.org/Team%3AULaval/Software)In doing so, we performed several *in silico* tests that allowed us to evaluate the efficiency of our tools and find areas of design that could be further optimized.

## Toehold design tool

Our design tool reliably produces toeholds with an appropriate secondary structure [(see Software).](https://2019.igem.org/Team%3AULaval/Software)However, we performed several rigorous tests that would allow us to evaluate the properties of riboswitch - trigger pairs and would be the best options to detect a particular gene of interest. We used the NUPACK suite (Zadeh et al., 2011) to model how our designed toeholds would bind to their intended targets. This allowed us to answer the following questions:

* How does the accessibility of the trigger sequence within the target RNA molecule affect the capacity of the toehold to bind to it?
* Is it actually more favorable for the toehold to bind to the trigger sequence than to remain unbound?
* Are our toeholds predicted to bind specifically to the trigger RNA? Could they bind elsewhere within the target RNA molecule?

To address the first question, we systematically design toehold riboswitches for trigger sequences with different accessibilities. We defined accessibility as the number of unpaired bases in the 30 bp trigger sequence in the predicted secondary structure for the target mRNA molecule and selected only those that had 10 or more unpaired bases. We then used NUPACK to model the binding between each of the designed toeholds and the target mRNA molecule. We show that the accessibility of the trigger within the target mRNA does not have an impact on the free energy of binding (Figure 1). In fact, we see a small trend towards greater accessibility resulting in a less favorable binding, but the correlation between the variables is very low, as implied by the low R2 value. This observation is contrary to our expectations, but factors involved might include the small sample size for these regions with high accessibility or sequence properties like low GC content that cause binding to them to be weaker.

The results of simulating how the toeholds bound to their corresponding triggers allowed us to address our other two questions. By comparing the free energies of the bound states to those of the unbound states, we show that the bound state is more favorable since for all our toeholds it results in a more negative free energy (Figure 2A). Next, we compared the position where the toehold binds to the target molecule to the actual position of the trigger to evaluate. We observed that a very high percentage (78%) of our riboswitches bind perfectly to the target, whereas a small percentage of them bind imperfectly to it (Figure 2B). These results further suggest that our tool can produce toehold riboswitches that bind to their targets. Since the percentage of toeholds that are predicted to bind perfectly is so high, we would suggest discarding all the others to ensure the best results.

## 3D models

Toehold riboswitches had never been modeled in 3D. We set out to do so because it would allow us to study their structural dynamics and use this new knowledge to improve their design. We started by generating 3D models from the secondary structure of the toehold riboswitch produced by Green et al. (2014) that had the highest ON/OFF ratio. This led to two alternative models, one obtained with the RNAComposer tool (Purzycka et al., 2015; Popenda et al., 2012) and the other with Rosetta (Das et al., 2010; Cheng et al., 2015) through a collaboration with Team iGEM Concordia. We validated these models using MOLProbity (Chen et al., 2010), which showed that they had an overall good quality (see [Model).](https://2019.igem.org/Team%3AULaval/Model)

Once we had 3D structures for the toehold riboswitch, we mounted a system for molecular dynamics simulations with the CHARMM-GUI server (Lee et al., 2016; Jo et al., 2008) and the NAMD simulation engine (Phillips et al., 2005). This would allow us to explore the dynamics of our structural model and to evaluate the flexibility of the ends of the molecule. We ran two simulations starting from our RNAComposer model: a computationally tractable one using an implicit solvent and a computationally intensive one using explicit solvent. Briefly, the simulation with implicit solvent uses the Born generalization to derive an approximation of the effect of solvation on each atom in the toehold’s structure, while the simulation with explicit solvent actually uses distance measurements to water molecules to calculate the magnitude of their interactions with the toehold (Anandakrishnan et al., 2015). In the simulations, we observe qualitatively that under an implicit solvent the toehold riboswitch starts unfolding after the first 40 ns (Movie 1) but not in the explicit solvent simulation (Movie 2). We analyzed the number of hydrogen bonds formed in the hairpin throughout the simulations using VMD built-in tools (Humphrey et al., 1996). In the explicit solvent simulation, the number of hydrogen bonds stays closer to the theoretical optimal expectation, which suggests a stable hairpin (Figure 3). This confirms the advantages of using an explicit solvent to obtain more accurate results from simulations. However, it also suggests that there could be potential for design improvements to prevent leaky expression caused by a spontaneous unfolding of the toehold riboswitch. We studied which hydrogen bonds were detected in most of the simulation and which were lost to find potential sites for design improvement (Figure 4). We found that the most stable hydrogen bonds were mediated by GC pairings at the base and the top of the hairpin, while the least stable ones were located closer to the AUG mismatch near the center of the hairpin. This result suggests that even more stable structures could be obtained by selecting sequences that have a higher GC content or ensuring that the last pairing at the top of the hairpin is a GC pair. Nevertheless, an excessively stable structure could bind inefficiently to the trigger sequence, so this potential trade-off between preventing leaky expression and target recognition. Further simulations will allow us to provide insight into this design aspect and further improvements to our workflow.

## Wet lab

### Switch cloning and testing

The first goal of this project was to demonstrate that we could produce efficient and specific ToeHold switches. To do so, we first designed switches targeting genes in *E. coli.* This design choice was so that once the switches were designed, a simple transformation and fluorescence measurement in vivo could allow us to assess switch efficiency. However, we had a lot of trouble with the initial cloning and had to change our destination vector, resulting in complications for this kind of test. Furthermore, our initial designs often targeted essential genes in *E. coli.* This was suboptimal, as the expression of the ToeHold could encourage the formation of RNA duplexes in the cell, resulting in lowered expression of essential proteins and a subsequent lowering in cell fitness. Our final design relied on the plasmid pNZ123, a small plasmid with a rolling circle replication origin and a chloramphenicol resistance.

![](https://static.igem.org/mediawiki/2019/2/2a/T--Ulaval--ResultsFig5.png)

**Figure 5.** Gel were successful clones were confirmed. Gel is annotated directly on the figure. A grey band was added in well two, as the real band was very faint.

As the switch that we decided to move forward with targeted ampicillin resistance, we had to prepare a co-transformation of the switch-containing plasmid and another plasmid with a compatible replication origin and an ampicillin resistance gene. At this point, we needed a specific strain of *E. coli*, BL21 DE3, as it possesses an insertion from the lysogenic phage T7, and can express its polymerase, which is necessary for ToeHold expression. Sadly, producing chemically competent cells for this strain proved more complex than planned, and the *in vivo* part was dropped.

We then decided to move forward with the cell-free characterization of our part BBa\_K3026001. We placed the plasmid harboring this part in myTXTL cell-free expression system from ArborBiosciences, along with a constant amount of the total mRNA sample extracted from bacterial cultures. As we wanted to replicate the detection apparatus from our tool, we decided to do four biological (BR) and three technical replicates for each biological ones, to a total of 12 replicates. For each biological replicate, we did an mRNA-less reaction to confirm the absence of signal leakage, and also did a control with only myTXTL and mRNA, to confirm that signal was not from autofluorescent proteins produced from the mRNA.

![](https://static.igem.org/mediawiki/2019/0/01/T--Ulaval--ResultsFig6.png)

**Figure 6.** Boxplot containing fluorescence units corrects according to the IGEM fluorescence measurement protocol. Fluorescence measurement were taken after 16h of incubation. Samples were prepared according to the myTXTL user manual. Negative fluorescence units are due to plate reader error and subsequence correction using the standard curve produced from fluorescein.

This figure clearly demonstrates that fluorescence was detected in only samples with both the ToeHold switch and the mRNA from ampicillin-resistant E. coli. All negative controls show no fluorescence whatsoever. This data confirms that this part works as intended and that our ToeHold design workflow produces functioning ToeHolds. However, there is a lot of variation between BR and even among technical replicates. For the design of our tool, this confirmed the design choice to incorporate several detection chambers, and treat as much of the initial sample as possible.

Sequencing of the clones used for the BR also showed that the part sequence was as designed for the entire length of the sequence, further confirming the efficiency and specificity of this part.

With these results, we knew that the sequence constraints we used for our switch design was efficient, and we moved forward to create more switches. Sadly, due to time and monetary constraints, we could not characterize other parts we designed further. **Fluorescence reference curve for part characterization is available at the bottom of the page.** For the future, we intend to test more switches that we designed to confirm our design and tool further, as well as benchmark our switches more thoroughly to assess the proper detection limit for the various switches. While there is still optimization work that needs to be done, the initial results with the tested switch show that we are on the right track and that our workflow is efficient.

### Characterization of already available parts

As we were doing most of our experiments in cell-free media, we looked for parts that had been characterized in such a media as points of reference. However, we found very few resources regarding this kind of system, especially for fluorescent protein expression. Therefore, we chose to select a part that was initially designed for *in vivo* use, and express it into our cell-free system to compare fluorescence levels and efficiency of expression. We chose parts BBa\_K567018 and BBa\_I746907 for this experiment. We produced fluorescence curves for these two proteins and made them available for the IGEM community to use as a comparison point between *in vivo* and cell-free expression of fluorescent proteins.

![](https://static.igem.org/mediawiki/2019/4/41/T--Ulaval--ResultsFig7A.png)

![](https://static.igem.org/mediawiki/2019/0/04/T--Ulaval--ResultsFig7B.png)

**Figure 7, A and B:** Fluorescence curves for the two pre-existing characterized parts.

## Hardware

The only hardware we were able to produce from scratch was the microfluidics circuits. This was a very complex part, as we had no prior experience with microfluidics. We are very proud that we were able to produce this device and test liquid flow through it. The device contains two layers, once where the sample will flow, and another where valves are present to control sample flow.

![](https://static.igem.org/mediawiki/2019/a/aa/T--Ulaval--HardwareFig3.jpg)
![](https://static.igem.org/mediawiki/2019/f/fa/T--Ulaval--HardwareFig4.jpg)

**Figure 8:** Fluidics layer (right) and Valve layer (left) of the microfluidics circuit. The valve layer is placed on top of the fluidics layer to produce the finished circuit.

Now that the circuit is ready, the next step will be to integrate all the sample treatment parts into the chip and set up the automatic sample treatment. Finally, creating the A.D.N. device itself and incorporate the cartridge system in the device. This part will require the help of more engineers, but we are hopeful that the logic of our design is sound, and that the only hurdles left are technical and monetary ones.

### References ⮟

Anandakrishnan, Ramu, Aleksander Drozdetski, Ross C. Walker, and Alexey V. Onufriev. 2015. “Speed of Conformational Change: Comparing Explicit and Implicit Solvent Molecular Dynamics Simulations.” *Biophysical Journal* 108 (5): 1153–64.

Cheng, Clarence Yu, Fang-Chieh Chou, and Rhiju Das. 2015. “Modeling Complex RNA Tertiary Folds with Rosetta.” *Methods in Enzymology* 553 (February): 35–64.

Chen, Vincent B., W. Bryan Arendall 3rd, Jeffrey J. Headd, Daniel A. Keedy, Robert M. Immormino, Gary J. Kapral, Laura W. Murray, Jane S. Richardson, and David C. Richardson. 2010. “MolProbity: All-Atom Structure Validation for Macromolecular Crystallography.” *Acta Crystallographica. Section D, Biological Crystallography* 66 (Pt 1): 12–21.

Das, Rhiju, John Karanicolas, and David Baker. 2010. “Atomic Accuracy in Predicting and Designing Noncanonical RNA Structure.” Nature Methods 7 (4): 291–94.

Green, Alexander A., Pamela A. Silver, James J. Collins, and Peng Yin. 2014. “Toehold Switches: De-Novo-Designed Regulators of Gene Expression.” *Cell* 159 (4): 925–39.

Humphrey, W., A. Dalke, and K. Schulten. 1996. “VMD: Visual Molecular Dynamics.” *Journal of Molecular Graphics* 14 (1): 33–38, 27–28.

Jo, Sunhwan, Taehoon Kim, Vidyashankara G. Iyer, and Wonpil Im. 2008. “CHARMM-GUI: A Web-Based Graphical User Interface for CHARMM.” *Journal of Computational Chemistry* 29 (11): 1859–65.

Lee, Jumin, Xi Cheng, Jason M. Swails, Min Sun Yeom, Peter K. Eastman, Justin A. Lemkul, Shuai Wei, et al. 2016. “CHARMM-GUI Input Generator for NAMD, GROMACS, AMBER, OpenMM, and CHARMM/OpenMM Simulations Using the CHARMM36 Additive Force Field.” *Journal of Chemical Theory and Computation* 12 (1): 405–13.

Phillips, James C., Rosemary Braun, Wei Wang, James Gumbart, Emad Tajkhorshid, Elizabeth Villa, Christophe Chipot, Robert D. Skeel, Laxmikant Kalé, and Klaus Schulten. 2005. “Scalable Molecular Dynamics with NAMD.” *Journal of Computational Chemistry*, Springer Seri, 26 (16): 1781–1802.

Popenda, Mariusz, Marta Szachniuk, Maciej Antczak, Katarzyna J. Purzycka, Piotr Lukasiak, Natalia Bartol, Jacek Blazewicz, and Ryszard W. Adamiak. 2012. “Automated 3D Structure Composition for Large RNAs.” *Nucleic Acids Research* 40 (14): e112.

Purzycka, K. J., M. Popenda, M. Szachniuk, M. Antczak, P. Lukasiak, J. Blazewicz, and R. W. Adamiak. 2015. “Chapter One - Automated 3D RNA Structure Prediction Using the RNAComposer Method for Riboswitches1.” In Methods in Enzymology, edited by Shi-Jie Chen and Donald H. Burke-Aguero, 553:3–34. Academic Press.

Zadeh, Joseph N., Conrad D. Steenberg, Justin S. Bois, Brian R. Wolfe, Marshall B. Pierce, Asif R. Khan, Robert M. Dirks, and Niles A. Pierce. 2011. “NUPACK: Analysis and Design of Nucleic Acid Systems.” *Journal of Computational Chemistry* 32 (1): 170–73.

igem@bcm.ulaval.ca