iGEM 2020 — Team Grenoble_Alpes

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

# Team:Grenoble Alpes/Notebook

[![PyoBusters](https://static.igem.org/mediawiki/2020/7/7a/T--Grenoble_Alpes--PyoBustersText.png)](https://2020.igem.org/Team%3AGrenoble_Alpes)

* About us

  [Team](https://2020.igem.org/Team%3AGrenoble_Alpes/Team)
  [Attributions](https://2020.igem.org/Team%3AGrenoble_Alpes/Attributions)
  [Collaborations](https://2020.igem.org/Team%3AGrenoble_Alpes/Collaborations)
  [Sponsors](https://2020.igem.org/Team%3AGrenoble_Alpes/Sponsors)
* Project

  [Description](https://2020.igem.org/Team%3AGrenoble_Alpes/Description)
  [Partnership](https://2020.igem.org/Team%3AGrenoble_Alpes/Partnership)
  [Engineering](https://2020.igem.org/Team%3AGrenoble_Alpes/Engineering)
  [Contribution](https://2020.igem.org/Team%3AGrenoble_Alpes/Contribution)
  [Notebook](https://2020.igem.org/Team%3AGrenoble_Alpes/Notebook)
  [Implementation](https://2020.igem.org/Team%3AGrenoble_Alpes/Implementation)
* Wet Lab

  [Protocols](https://2020.igem.org/Team%3AGrenoble_Alpes/Protocols)
  [Parts](https://2020.igem.org/Team%3AGrenoble_Alpes/Parts)
  [Design](https://2020.igem.org/Team%3AGrenoble_Alpes/Design)
  [Experiments](https://2020.igem.org/Team%3AGrenoble_Alpes/Experiments)
  [Proof
  of Concept](https://2020.igem.org/Team%3AGrenoble_Alpes/Proof_Of_Concept)
  [Results](https://2020.igem.org/Team%3AGrenoble_Alpes/Results)
* Dry Lab

  [Hardware](https://2020.igem.org/Team%3AGrenoble_Alpes/Hardware)
  [Software](https://2020.igem.org/Team%3AGrenoble_Alpes/Software)
  [Pythie](https://2020.igem.org/Team%3AGrenoble_Alpes/Pythie/SignIn)
  [Design](https://2020.igem.org/Team%3AGrenoble_Alpes/Design#incubatorDiv)
  [Experiments](https://2020.igem.org/Team%3AGrenoble_Alpes/Experiments#incubatorDiv)
  [Proof of
  Concept](https://2020.igem.org/Team%3AGrenoble_Alpes/Proof_Of_Concept#incubatorDiv)
  [Results](https://2020.igem.org/Team%3AGrenoble_Alpes/Results#incubatorDiv)
* Human Practices

  [Human
  Practices](https://2020.igem.org/Team%3AGrenoble_Alpes/Human_Practices)
  [Education](https://2020.igem.org/Team%3AGrenoble_Alpes/Education)
  [Safety](https://2020.igem.org/Team%3AGrenoble_Alpes/Safety)
  [Clinic](https://2020.igem.org/Team%3AGrenoble_Alpes/Clinic)
  [Inclusion](https://2020.igem.org/Team%3AGrenoble_Alpes/Inclusion)
* Awards

  [Medals](https://2020.igem.org/Team%3AGrenoble_Alpes/Medals)
  [Awards](https://2020.igem.org/Team%3AGrenoble_Alpes/Awards)

![](https://static.igem.org/mediawiki/2020/7/79/T--Grenoble_Alpes--glasses_Icon.png)

Protanopia

Deuteranopia

Tritanopia
Protanomaly

Deuteranomaly

Tritanomaly
Achromatopsia

Achromatomaly

![](https://static.igem.org/mediawiki/2020/7/79/T--Grenoble_Alpes--glasses_Icon.png)

Protanopia

Deuteranopia

Tritanopia
Protanomaly

Deuteranomaly

Tritanomaly
Achromatopsia

Achromatomaly

PyoBuster - Notebook

Summary

[Notebook](#notebook)
[Wet Lab Agenda](#wetLabAgenda)
[Dry Lab Agenda](#dryLabAgenda)
[Social Agenda](#socialAgenda)

[Notebook](#notebook)
[Wet Lab Agenda](#wetLabAgenda)
[Dry Lab Agenda](#dryLabAgenda)
[Social Agenda](#socialAgenda)

# Notebook

This year we have been confronted with different issues related to the SARS-CoV-2 pandemic. We were unable to work for 2 months but fortunately we ended up having access to the laboratory this summer. In view of these changes, we tried to adapt to the situation.Our work was divided into 3 calendars: wet lab work, dry lab work and a general calendar that includes the events that allowed us to fundraise, make events and human practices events.

# Wet Lab Agenda

| Week | | Survival System (Mathieu and Elouen) | | | | | | | | | | | | Therapeutic System (Noémie) | | | | | | | | | | | | Delivery System (Sara) | | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Week 01 | Dec. 30 - Jan. 5 | Bibliography | | | | | | | | | | | | Bibliography | | | | | | | | | | | | Bibliography | | | | | | | | | | | |
| Week 02 | Jan. 6 - Jan. 12 |
| Week 03 | Jan. 13 - Jan. 19 |
| Week 04 | Jan. 20 - Jan. 26 |
| Week 05 | Jan. 27 - Feb. 2 |
| Week 06 | Feb. 3 - Feb. 9 |
| Week 07 | Feb. 10 - Feb. 16 |
| Week 08 | Feb. 17 - Feb. 23 |
| Week 09 | Feb. 24 - Mar. 1 |
| Week 10 | Mar. 2 - Mar. 8 |
| Week 11 | Mar. 9 - Mar. 15 |
| Week 12 | Mar. 16 - Mar. 22 |
| Week 13 | Mar. 23 - Mar. 29 |
| Week 14 | Mar. 30 - Apr. 5 |
| Week 15 | Apr. 6 - Apr. 12 | Bibliography | | | | | | Protocol Set-up | | | | | | Bibliography | | | | | | Protocol Set-up | | | | | | Bibliography | | | | | | Protocol Set-up | | | | | |
| Week 16 | Apr. 13 - Apr. 19 |
| Week 17 | Apr. 20 - Apr. 26 |
| Week 18 | Apr. 27 - May 3 |
| Week 19 | May 4 - May 10 |
| Week 20 | May 11 - May 17 |
| Week 21 | May 18 - May 24 |
| Week 22 | May 25 - May 31 |
| Week 23 | June 1 - June 7 |
| Week 24 | June 8 - June 14 |
| Week 25 | June 15 - June 21 |
| Week 26 | June 22 - June 28 | Preparation Competent | | | | | | Transformation | | | | | |  | | | | | | | | | | | | Resuspension iGEM | | | | Clonage | | | | PCR | | | |
| Week 27 | June 29 - July 5 | Midiprep | | | | Resuspension IDT PART | | | | Resuspension IGEM part | | | | Support | | | | | | | | | | | | Clonage | | | | | | Resuspension | | | | | |
| Week 28 | July 6 - July 12 | Miniprep | | | | Resuspension IDT | | | | Clonage | | | | Clonage | | | | | | Resuspension IDT | | | | | |
| Week 29 | July 13 - July 19 | Clonage | | | | | | Fluorescence | | | | | | Clonage | | | | Digestion | | | | PCR | | | |
| Week 30 | July 20 - July 26 | Clonage | | | | Fluorescence | | | | Genomic | | | | Clonage | | | | Resuspension IDT | | | | Preparation | | | |
| Week 31 | July 27 - Aug. 2 | Clonage | | | | | | | | | | | | Resuspension | | | | | | PCR | | | | | | Clonage | | | | | | Miniprep | | | | | |
| Week 32 | Aug. 3 - Aug. 9 | Clonage | | | | | | | | | | | | Resuspension & Transformation | | | Digestion | | | PCR | | | Genome | | | Clonage | | | | Fluorescence | | | | Digestion | | | |
| Week 33 | Aug. 10 - Aug. 16 | Clonage | | | | | | Troubleshooting | | | | | | Digestion | | | Clonage | | | Transformation | | | PCR | | | Preparation | | | Clonage | | | Troubleshooting | | | Fluorescence | | |
| Week 34 | Aug. 17 - Aug. 23 | Clonage | | | | | | Fluorescence | | | | | | Clonage | | | | | | PCR | | | | | | Clonage | | | | | | Fluorescence | | | | | |
| Week 35 | Aug. 24 - Aug. 30 | Clonage | | | | | | Fluorescence | | | | | | Clonage | | | | | | PCR | | | | | | Clonage | | | | | | Fluorescence | | | | | |
| Week 36 | Aug. 31 - Sep. 6 | Clonage | | | | | | | | | | | | Transformation | | | | | | Clonage | | | | | | Fluorescence | | | | | | Clonage | | | | | |
| Week 37 | Sep. 7 - Sep. 13 | Fluorescence | | | | | | | | | | | | Pyocin S5 | | | | | | Protocol | | | | | | Optical | | | | Clonage | | | | Fluorescence | | | |
| Week 38 | Sep. 14 - Sep. 20 | Troubleshooting | | | | | | | | | | | | Pyocin S5 | | | Dispersin B | | | BIOASTERS | | | Protocol | | |  | | | | | | | | | | | |
| Week 39 | Sep. 21 - Sep. 27 |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 40 | Sep. 28 - Oct. 4 |  | | | | | | | | | | | | Set-up | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 41 | Oct. 5 - Oct. 11 |  | | | | | | | | | | | | Biofilm | | | | | | | | | | | | Optical | | | | | | | | | | | |
| Week 42 | Oct. 12 - Oct. 18 |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 43 | Oct. 19 - Oct. 25 |  | | | | | | | | | | | | BIOASTERS | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 44 | Oct. 26 - Nov. 1 |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 45 | Nov. 2 - Nov. 8 |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 46 | Nov. 9 - Nov. 15 |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 47 | Nov. 16 - Nov. 22 |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 48 | Nov. 23 - Nov. 29 |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 49 | Nov. 30 - Dec. 6 |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 50 | Dec. 7 - Dec. 13 |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 51 | Dec. 14 - Dec. 20 |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 52 | Dec. 21 - Dec. 27 |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 53 | Dec. 28 - Jan. 3, 2021 |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |

# Dry Lab Agenda

| Week | | Environment Module | | | | | | | | | | | | Agitation Module | | | | | | | | | | | | Fluorescence Module | | | | | | | | | | | | Software | | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Week 01 | Dec. 30 - Jan. 5 |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 02 | Jan. 6 - Jan. 12 |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 03 | Jan. 13 - Jan. 19 |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 04 | Jan. 20 - Jan. 26 |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 05 | Jan. 27 - Feb. 2 |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 06 | Feb. 3 - Feb. 9 |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 07 | Feb. 10 - Feb. 16 |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 08 | Feb. 17 - Feb. 23 |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 09 | Feb. 24 - Mar. 1 |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 10 | Mar. 2 - Mar. 8 |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 11 | Mar. 9 - Mar. 15 |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 12 | Mar. 16 - Mar. 22 |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 13 | Mar. 23 - Mar. 29 | Bibliography | | | | | | | | | | | |  | | | | | | | | | | | | Bibliography | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 14 | Mar. 30 - Apr. 5 |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 15 | Apr. 6 - Apr. 12 |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 16 | Apr. 13 - Apr. 19 | Bibliography | | | | | | Design | | | | | |  | | | | | | | | | | | | Bibliography | | | | | | Design | | | | | |  | | | | | | | | | | | |
| Week 17 | Apr. 20 - Apr. 26 |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 18 | Apr. 27 - May 3 |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 19 | May 4 - May 10 |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 20 | May 11 - May 17 |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 21 | May 18 - May 24 |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 22 | May 25 - May 31 |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 23 | June 1 - June 7 |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 24 | June 8 - June 14 |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 25 | June 15 - June 21 | Construction | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 26 | June 22 - June 28 | Construction | | | | | | Experiments | | | | | |  | | | | | | | | | | | | Experiments | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 27 | June 29 - July 5 | Construction | | | | | | Experiments | | | | | |  | | | | | | | | | | | | Experiments | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 28 | July 6 - July 12 | Construction | | | | | | Experiments | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 29 | July 13 - July 19 | Construction | | | | Implementation | | | | Sponsor | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 30 | July 20 - July 26 | Implementation | | | | | | Experiments | | | | | |  | | | | | | | | | | | | Implementation | | | | | | Experiments | | | | | |  | | | | | | | | | | | |
| Week 31 | July 27 - Aug. 2 | Implementation | | | | Construction | | | | Experiments | | | |  | | | | | | | | | | | | Experiments | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 32 | Aug. 3 - Aug. 9 | Experiments | | | | | | | | | | | | Bibliography | | | | | | | | | | | | Image process | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 33 | Aug. 10 - Aug. 16 | Construction | | | | | | Motors | | | | | |  | | | | | | | | | | | | Experiments | | | | | | Design | | | | | |
| Week 34 | Aug. 17 - Aug. 23 |  | | | | | | | | | | | | Construction | | | | | | Motors | | | | | |  | | | | | | | | | | | | Implementation | | | | | | Design | | | | | |
| Week 35 | Aug. 24 - Aug. 30 | Implementation | | | | | | Design | | | | | | Implementation | | | | | | | | | | | | Experiments | | | | | | | | | | | | Implementation | | | | | | Experiments | | | | | |
| Week 36 | Aug. 31 - Sep. 6 |  | | | | | | | | | | | | Implementation | | | | | | | | | | | | Implementation | | | | | | | | | | | |
| Week 37 | Sep. 7 - Sep. 13 |  | | | | | | | | | | | | Construction | | | | | | | | | | | | Implementation | | | | | | Design | | | | | | Experiments | | | | | | Design | | | | | |
| Week 38 | Sep. 14 - Sep. 20 | Design | | | | | | Implementation | | | | | | Design | | | | | | | | | | | | Implementation | | | | | | Design | | | | | | Implementation | | | | | | | | | | | |
| Week 39 | Sep. 21 - Sep. 27 | Implementation | | | | | | | | | | | | Construction | | | | | | | | | | | | Implementation | | | | | | | | | | | | Implementation | | | | | | | | | | | |
| Week 40 | Sep. 28 - Oct. 4 | Human Practices | | | | | | | | | | | | Calibration | | | | | | | | | | | |  | | | | | | | | | | | | Implementation | | | | | | | | | | | |
| Week 41 | Oct. 5 - Oct. 11 |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 42 | Oct. 12 - Oct. 18 |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | | Implementation | | | | | | | | | | | |
| Week 43 | Oct. 19 - Oct. 25 |  | | | | | | | | | | | |  | | | | | | | | | | | | Experiments | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 44 | Oct. 26 - Nov. 1 |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 45 | Nov. 2 - Nov. 8 |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 46 | Nov. 9 - Nov. 15 |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 47 | Nov. 16 - Nov. 22 |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 48 | Nov. 23 - Nov. 29 |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 49 | Nov. 30 - Dec. 6 |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 50 | Dec. 7 - Dec. 13 |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 51 | Dec. 14 - Dec. 20 |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 52 | Dec. 21 - Dec. 27 |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 53 | Dec. 28 - Jan. 3, 2021 |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |

# Social Agenda

| Week | | | Funding | | | | | | | | | | | | Communication | | | | | | | | | | | | Human Practices | | | | | | | | | | | | Events | | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Week 01 | Dec. 30 - Jan. 5 | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 02 | Jan. 6 - Jan. 12 | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 03 | Jan. 13 - Jan. 19 | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 04 | Jan. 20 - Jan. 26 | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 05 | Jan. 27 - Feb. 2 | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 06 | Feb. 3 - Feb. 9 | | Application | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 07 | Feb. 10 - Feb. 16 | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 08 | Feb. 17 - Feb. 23 | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 09 | Feb. 24 - Mar. 1 | |  | | | | | | | | | | | | Identity | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 10 | Mar. 2 - Mar. 8 | | Application | | | | | | | | | | | | Team presentation | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 11 | Mar. 9 - Mar. 15 | |  | | | | | | | | | | | | Hôpital des nounours | | | | | | | | | | | | Science Fair | | | | | | | | | | | |
| Week 12 | Mar. 16 - Mar. 22 | | Hôpital des nounours | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 13 | Mar. 23 - Mar. 29 | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 14 | Mar. 30 - Apr. 5 | |  | | | | | | | | | | | |  | | | | | | | | | | | | iGEM FR | | | | | | | | | | | |
| Week 15 | Apr. 6 - Apr. 12 | |  | | | | | | | | | | | | Quiz | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 16 | Apr. 13 - Apr. 19 | |  | | | | | | | | | | | | Quiz | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 17 | Apr. 20 - Apr. 26 | |  | | | | | | | | | | | | Website | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 18 | Apr. 27 - May 3 | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 19 | May 4 - May 10 | | Application | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 20 | May 11 - May 17 | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 21 | May 18 - May 24 | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 22 | May 25 - May 31 | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 23 | June 1 - June 7 | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 24 | June 8 - June 14 | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 25 | June 15 - June 21 | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 26 | June 22 - June 28 | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 27 | June 29 - July 5 | |  | | | | | | | | | | | | Crowdfunding | | | | | | Sponsors | | | | | | WFA | | | | | | | | | | | | iGEM FR | | | | | | | | | | | |
| Week 28 | July 6 - July 12 | |  | | | | | | | | | | | | PyoBusters | | | | | | WFA | | | | | | | | | | | | iGEM FR | | | | | | | | | | | |
| Week 29 | July 13 - July 19 | |  | | | | | | | | | | | | Cystic fibrosis | | | | Sponsors | | | | Presentation | | | | WFA | | | | | | | | | | | | iGEM FR | | | | | | | | | | | |
| Week 30 | July 20 - July 26 | |  | | | | | | | | | | | | Cystic fibrosis | | | | Sponsors | | | | Crowdfunding | | | | iGEM FR | | | | | | | | | | | |
| Week 31 | July 27 - Aug. 2 | |  | | | | | | | | | | | | Cystic fibrosis | | | | | | CFTR | | | | | | iGEM FR | | | | | | | | | | | |
| Week 32 | Aug. 3 - Aug. 9 | |  | | | | | | | | | | | | Team | | | | | | Presentation | | | | | | iGEM FR | | | | | | | | | | | |
| Week 33 | Aug. 10 - Aug. 16 | |  | | | | | | | | | | | | Pseudomonas aeruginosa | | | | | | | | | | | | iGEM FR | | | | | | | | | | | |
| Week 34 | Aug. 17 - Aug. 23 | |  | | | | | | | | | | | | Our Project | | | | | | | | | | | | WFA | | | | | | | | | | | | iGEM FR | | | | | | | | | | | |
| Week 35 | Aug. 24 - Aug. 30 | |  | | | | | | | | | | | | Biofilms | | | | | | | | | | | | WFA | | | | | | | | | | | | iGEM FR | | | | | | | | | | | |
| Week 36 | Aug. 31 - Sep. 6 | |  | | | | | | | | | | | | WFA | | | | | | Collaboration | | | | | | Cystic Fibrosis | | | | | | | | | | | | iGEM FR | | | | | | | | | | | |
| Week 37 | Sep. 7 - Sep. 13 | |  | | | | | | | | | | | | iGEM FR | | | | | | | | | | | | Cystic Fibrosis | | | | | | WFA | | | | | | iGEM FR | | | | | | | | | | | |
| Week 38 | Sep. 14 - Sep. 20 | |  | | | | | | | | | | | | iGEM FR | | | | | | | | | | | |  | | | | | | | | | | | | iGEM FR | | | | | | | | | | | |
| Week 39 | Sep. 21 - Sep. 27 | |  | | | | | | | | | | | |  | | | | | | | | | | | | Cystic Fibrosis | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 40 | Sep. 28 - Oct. 4 | |  | | | | | | | | | | | | Sponsors | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 41 | Oct. 5 - Oct. 11 | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | | Midi Minatec | | | | | | | | | | | |
| Week 42 | Oct. 12 - Oct. 18 | |  | | | | | | | | | | | | Midi Minatec | | | | | | | | | | | |  | | | | | | | | | | | | Interview | | | | | | | | | | | |
| Week 43 | Oct. 19 - Oct. 25 | |  | | | | | | | | | | | | Wiki | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 44 | Oct. 26 - Nov. 1 | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 45 | Nov. 2 - Nov. 8 | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 46 | Nov. 9 - Nov. 15 | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 47 | Nov. 16 - Nov. 22 | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 48 | Nov. 23 - Nov. 29 | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 49 | Nov. 30 - Dec. 6 | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 50 | Dec. 7 - Dec. 13 | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 51 | Dec. 14 - Dec. 20 | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 52 | Dec. 21 - Dec. 27 | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |
| Week 53 | Dec. 28 - Jan. 3, 2021 | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |  | | | | | | | | | | | |

##### New message

×

### Team Grenoble\_Alpes

![](https://static.igem.org/mediawiki/2020/e/ea/T--Grenoble_Alpes--Logo_final_Grenoble.png)
![](https://static.igem.org/mediawiki/2020/7/7e/T--Grenoble_Alpes--Logo_UGAINP.png)

![](https://static.igem.org/mediawiki/2020/7/7a/T--Grenoble_Alpes--PyoBustersText.png)

### Contact

* #### Igem grenoble Place du Commandant Nal 38700, La Tronche, France
* #### igem.grenoble.2020@gmail.com

[![](https://static.igem.org/mediawiki/2020/4/41/T--Grenoble_Alpes--Logo_facebook.png)](https://www.facebook.com/igemfrancegrenoble)
[![](https://static.igem.org/mediawiki/2020/4/4d/T--Grenoble_Alpes--Logo_instagram.png)](https://www.instagram.com/igem_grenoble/)
[![](https://static.igem.org/mediawiki/2020/3/3a/T--Grenoble_Alpes--Logo_linkedIn.png)](https://www.linkedin.com/in/igem-grenoble/)
[![](https://static.igem.org/mediawiki/2020/6/62/T--Grenoble_Alpes--Logo_twitter.png)](https://twitter.com/igem_grenoble)

This wiki was completely designed and coded by the team members of the 2020 Grenoble\_Alpes' Team, all content on this wiki is available under the Creative Commons [Attribution 4.0](https://creativecommons.org/licenses/by/4.0/) license (or any later version).

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

# Team:Grenoble Alpes/Engineering

[![PyoBusters](https://static.igem.org/mediawiki/2020/7/7a/T--Grenoble_Alpes--PyoBustersText.png)](https://2020.igem.org/Team%3AGrenoble_Alpes)

* About us

  [Team](https://2020.igem.org/Team%3AGrenoble_Alpes/Team)
  [Attributions](https://2020.igem.org/Team%3AGrenoble_Alpes/Attributions)
  [Collaborations](https://2020.igem.org/Team%3AGrenoble_Alpes/Collaborations)
  [Sponsors](https://2020.igem.org/Team%3AGrenoble_Alpes/Sponsors)
* Project

  [Description](https://2020.igem.org/Team%3AGrenoble_Alpes/Description)
  [Partnership](https://2020.igem.org/Team%3AGrenoble_Alpes/Partnership)
  [Engineering](https://2020.igem.org/Team%3AGrenoble_Alpes/Engineering)
  [Contribution](https://2020.igem.org/Team%3AGrenoble_Alpes/Contribution)
  [Notebook](https://2020.igem.org/Team%3AGrenoble_Alpes/Notebook)
  [Implementation](https://2020.igem.org/Team%3AGrenoble_Alpes/Implementation)
* Wet Lab

  [Protocols](https://2020.igem.org/Team%3AGrenoble_Alpes/Protocols)
  [Parts](https://2020.igem.org/Team%3AGrenoble_Alpes/Parts)
  [Design](https://2020.igem.org/Team%3AGrenoble_Alpes/Design)
  [Experiments](https://2020.igem.org/Team%3AGrenoble_Alpes/Experiments)
  [Proof
  of Concept](https://2020.igem.org/Team%3AGrenoble_Alpes/Proof_Of_Concept)
  [Results](https://2020.igem.org/Team%3AGrenoble_Alpes/Results)
* Dry Lab

  [Hardware](https://2020.igem.org/Team%3AGrenoble_Alpes/Hardware)
  [Software](https://2020.igem.org/Team%3AGrenoble_Alpes/Software)
  [Pythie](https://2020.igem.org/Team%3AGrenoble_Alpes/Pythie/SignIn)
  [Design](https://2020.igem.org/Team%3AGrenoble_Alpes/Design#incubatorDiv)
  [Experiments](https://2020.igem.org/Team%3AGrenoble_Alpes/Experiments#incubatorDiv)
  [Proof of
  Concept](https://2020.igem.org/Team%3AGrenoble_Alpes/Proof_Of_Concept#incubatorDiv)
  [Results](https://2020.igem.org/Team%3AGrenoble_Alpes/Results#incubatorDiv)
* Human Practices

  [Human
  Practices](https://2020.igem.org/Team%3AGrenoble_Alpes/Human_Practices)
  [Education](https://2020.igem.org/Team%3AGrenoble_Alpes/Education)
  [Safety](https://2020.igem.org/Team%3AGrenoble_Alpes/Safety)
  [Clinic](https://2020.igem.org/Team%3AGrenoble_Alpes/Clinic)
  [Inclusion](https://2020.igem.org/Team%3AGrenoble_Alpes/Inclusion)
* Awards

  [Medals](https://2020.igem.org/Team%3AGrenoble_Alpes/Medals)
  [Awards](https://2020.igem.org/Team%3AGrenoble_Alpes/Awards)

![](https://static.igem.org/mediawiki/2020/7/79/T--Grenoble_Alpes--glasses_Icon.png)

Protanopia

Deuteranopia

Tritanopia
Protanomaly

Deuteranomaly

Tritanomaly
Achromatopsia

Achromatomaly

![](https://static.igem.org/mediawiki/2020/7/79/T--Grenoble_Alpes--glasses_Icon.png)

Protanopia

Deuteranopia

Tritanopia
Protanomaly

Deuteranomaly

Tritanomaly
Achromatopsia

Achromatomaly

PyoBuster - Engineering

# Engineering

The main goal of our project is to destroy the biofilm of *P. aeruginosa*. The wet lab that creates a
modified *E. coli* which should destroy the biofilm. The dry lab that creates a benchmark for testing
our modified *E. coli*. On this page, we will present one important step: first the detection of the
biofilm by *E. coli*, and second the environment module of the benchmark.

## How to detect *P. aeruginosa*’s biofilm in a specific manner?

During the development of our project we stumbled on a problem: how to detect *P. aeruginosa*’s
biofilm in a specific manner? Indeed, biofilms of different bacteria have a lot of similarities and
therefore it is difficult to create a specific therapeutic device for *P. aeruginosa*. We needed to
think of other molecules or physical parameters that could indicate the presence of the pathogen at
the biofilm life stage that wasn’t the biofilm itself. This is how we got to the quorum sensing of
the bacteria. Since some molecules of Pseudomonas quorum sensing are specific to the pathogen and
are secreted in particular environmental conditions, it was the perfect solution to not only
specifically target *P. aeruginosa* but also target a specific stage of the biofilm formation and
avoid spreading elsewhere. As the quorum sensing molecule controls the expression of *P. aeruginosa*
genes, we recovered the quorum sensing gene regulation mechanisms of the pathogen and put our
construction under their control.

As our system involved multiple cascades that aim to activate the expression of a lysis gene, it was
important that each actor of the cascades is expressed in a sufficient amount. We needed a system to
modulate the expression of every gene involved in the lysis of our bacteria, in a way that it would
be activated at a certain threshold. One solution could have been to design promoters with different
expression levels, but as we had inducible promoters it wasn't ideal. This is why we thought of
designing RBSs with different 5' UTRs for all the actors. It allowed us to use our inducible
promoters and control the expression through translations and not through transcription. Using these
RBSs, we found a combination that triggered the lysis of our bacteria at the desired threshold. As a
consequence, if our device is used in different conditions with different sensing conditions, we
wouldn't have to redesign our constructions, we would just change the RBSs.

When the constructions were determined and our bacteria was ready to be tested on the biofilm of *P.
aeruginosa*, we stumbled upon the problem of following our bacterial population through the lysis
cycles and quantifying the biofilm destruction. This is why we added a constitutively expressed
fluorescence gene in our PyoBusters and a camera as well as appropriate fluorescence filters into
our testing bench. The biofilm would be coloured with crystal violet and our PyoBusters would
express GFP, so we could visually follow their real time evolution in the testing cell without
having to remove the cultures from the infection conditions for measures.

## How could we create a stable atmosphere inside an incubator?

In our project we created a benchmark for bacteria growth experiments and it also illustrates the
different steps of the cycle “Research - Imagine - Build - Test - Learn - Improve - Research”. The
dry lab project is made of 4 parts: the environment module, the agitation module, the fluorescent
module and the software that operates the 3 modules. The environment module perfectly illustrates
the cycle.

We made research to understand what had been done before us and we created the very first outlines
of our box. To create a stable atmosphere with the desired temperature and humidity, we thought
about building a hardware with a primary and a secondary room. Temperature and humidity would be
created in the secondary room and thanks to fans it would be sent to the primary room where the
experiment would take place. Though we never build this idea because of the discovery of a new
device: the ultrasonic water atomizer. It simplified a lot the creation of the humidity parameter
that we built everything in one box. You can see on the figure our first prototype which is only the
environment module.

![Photo of our prototype without the cover](https://static.igem.org/mediawiki/2020/c/c1/T--Grenoble_Alpes--firstPrototype.jpeg)

Photo of our prototype without the cover.

As a first prototype, we chose this box because it was easier than building
our own box with its impermeable cover (not on the photo). This prototype is made of sensors
(numbered in white from 0 to 5, number 5 is under the plate), heating resistors (the 6
orange parallelepipeds), fans (2 faces one each other), the 96 wells plate for cell cultures
and hidden under the right fan there is the humidity module. Outside the box, you can see
the microcontroller (one arduino) and the white plate is for electronic wire connections.

To put it simply: an external 20V electronic supply is powering the 6 heaters. Thanks
to an electric
relay, the microcontroller can manage the heater supply. The 6 sensors allow the microcontroller to
monitor what is happening inside the box. Thanks to this prototype, we were allowed to nicely
monitor and control our box. The usb connection is for the pc connection. Our first box goes with
its own software implemented on the Arduino. Based on a PID corrector, this code should ensure a
smooth monitoring and controlling of our box.

Now, the “test step” can begin and we will discover if we can really control the
atmosphere inside
our box and how it evolves. You can see the results on the next graphes. The experience was to reach
37°C (310°K) and 100% of relative humidity.

![Typical results of one experiment](https://static.igem.org/mediawiki/2020/2/22/T--Grenoble_Alpes--drylab_graphs.png)

Typical results of one experiment.

This figure tells us everything that we need to understand our prototype. The
different graphs show humidity and temperature datas for the 6 sensors, the duty cycle
chosen by the Arduino code and the order of the brumisateur. All abscisses are the time in
seconds.

Sensors are placed at different locations inside the box and you can see that all
sensors show the same result: around 20 minutes, the box has a stable atmosphere at the desired
temperature. Humidity is still in process but it is around 96% of relative humidity. The duty cycle
of our code is also stable, it means that the permanent system is reached: it is heated enough to
compensate for the loss.
The brumisateur is a boolean value, it is either 1 for on and 0 for off. It says that it hasn’t
reached the desired humidity value but we are higher than 95% which is really nice.

The “test step” is going on with many other experiments to understand how our system
reacts for
longer duration, when there are disruptions such as opening the cover, or trying other PID corrector
values.

The “improve step” is our actual hardware: the Automated Measurement Incubator (AMI). Too see the
hardware page click [here](https://2020.igem.org/Team%3AGrenoble_Alpes/Hardware).

### Team Grenoble\_Alpes

![](https://static.igem.org/mediawiki/2020/e/ea/T--Grenoble_Alpes--Logo_final_Grenoble.png)
![](https://static.igem.org/mediawiki/2020/7/7e/T--Grenoble_Alpes--Logo_UGAINP.png)

![](https://static.igem.org/mediawiki/2020/7/7a/T--Grenoble_Alpes--PyoBustersText.png)

### Contact

* #### Igem grenoble Place du Commandant Nal 38700, La Tronche, France
* #### igem.grenoble.2020@gmail.com

[![](https://static.igem.org/mediawiki/2020/4/41/T--Grenoble_Alpes--Logo_facebook.png)](https://www.facebook.com/igemfrancegrenoble)
[![](https://static.igem.org/mediawiki/2020/4/4d/T--Grenoble_Alpes--Logo_instagram.png)](https://www.instagram.com/igem_grenoble/)
[![](https://static.igem.org/mediawiki/2020/3/3a/T--Grenoble_Alpes--Logo_linkedIn.png)](https://www.linkedin.com/in/igem-grenoble/)
[![](https://static.igem.org/mediawiki/2020/6/62/T--Grenoble_Alpes--Logo_twitter.png)](https://twitter.com/igem_grenoble)

This wiki was completely designed and coded by the team members of the 2020 Grenoble\_Alpes' Team, all content on this wiki is available under the Creative Commons [Attribution 4.0](https://creativecommons.org/licenses/by/4.0/) license (or any later version).

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

# Team:Grenoble Alpes/Experiments

[![PyoBusters](https://static.igem.org/mediawiki/2020/7/7a/T--Grenoble_Alpes--PyoBustersText.png)](https://2020.igem.org/Team%3AGrenoble_Alpes)

* About us

  [Team](https://2020.igem.org/Team%3AGrenoble_Alpes/Team)
  [Attributions](https://2020.igem.org/Team%3AGrenoble_Alpes/Attributions)
  [Collaborations](https://2020.igem.org/Team%3AGrenoble_Alpes/Collaborations)
  [Sponsors](https://2020.igem.org/Team%3AGrenoble_Alpes/Sponsors)
* Project

  [Description](https://2020.igem.org/Team%3AGrenoble_Alpes/Description)
  [Partnership](https://2020.igem.org/Team%3AGrenoble_Alpes/Partnership)
  [Engineering](https://2020.igem.org/Team%3AGrenoble_Alpes/Engineering)
  [Contribution](https://2020.igem.org/Team%3AGrenoble_Alpes/Contribution)
  [Notebook](https://2020.igem.org/Team%3AGrenoble_Alpes/Notebook)
  [Implementation](https://2020.igem.org/Team%3AGrenoble_Alpes/Implementation)
* Wet Lab

  [Protocols](https://2020.igem.org/Team%3AGrenoble_Alpes/Protocols)
  [Parts](https://2020.igem.org/Team%3AGrenoble_Alpes/Parts)
  [Design](https://2020.igem.org/Team%3AGrenoble_Alpes/Design)
  [Experiments](https://2020.igem.org/Team%3AGrenoble_Alpes/Experiments)
  [Proof
  of Concept](https://2020.igem.org/Team%3AGrenoble_Alpes/Proof_Of_Concept)
  [Results](https://2020.igem.org/Team%3AGrenoble_Alpes/Results)
* Dry Lab

  [Hardware](https://2020.igem.org/Team%3AGrenoble_Alpes/Hardware)
  [Software](https://2020.igem.org/Team%3AGrenoble_Alpes/Software)
  [Pythie](https://2020.igem.org/Team%3AGrenoble_Alpes/Pythie/SignIn)
  [Design](https://2020.igem.org/Team%3AGrenoble_Alpes/Design#incubatorDiv)
  [Experiments](https://2020.igem.org/Team%3AGrenoble_Alpes/Experiments#incubatorDiv)
  [Proof of
  Concept](https://2020.igem.org/Team%3AGrenoble_Alpes/Proof_Of_Concept#incubatorDiv)
  [Results](https://2020.igem.org/Team%3AGrenoble_Alpes/Results#incubatorDiv)
* Human Practices

  [Human
  Practices](https://2020.igem.org/Team%3AGrenoble_Alpes/Human_Practices)
  [Education](https://2020.igem.org/Team%3AGrenoble_Alpes/Education)
  [Safety](https://2020.igem.org/Team%3AGrenoble_Alpes/Safety)
  [Clinic](https://2020.igem.org/Team%3AGrenoble_Alpes/Clinic)
  [Inclusion](https://2020.igem.org/Team%3AGrenoble_Alpes/Inclusion)
* Awards

  [Medals](https://2020.igem.org/Team%3AGrenoble_Alpes/Medals)
  [Awards](https://2020.igem.org/Team%3AGrenoble_Alpes/Awards)

![](https://static.igem.org/mediawiki/2020/7/79/T--Grenoble_Alpes--glasses_Icon.png)

Protanopia

Deuteranopia

Tritanopia
Protanomaly

Deuteranomaly

Tritanomaly
Achromatopsia

Achromatomaly

![](https://static.igem.org/mediawiki/2020/7/79/T--Grenoble_Alpes--glasses_Icon.png)

Protanopia

Deuteranopia

Tritanopia
Protanomaly

Deuteranomaly

Tritanomaly
Achromatopsia

Achromatomaly

PyoBuster - Experiments

Constitutive +
​
Therapeutic
molecules

Plsr + E7 lysis

Constitutive + RhlR

Prhlr + LacI

Genomic DNA
Ptac + E7 lysis

Constitutive +
​
Therapeutic
molecules

Plsr + E7 lysis

Constitutive + RhlR

Prhlr + LacI

Genomic DNA
Ptac + E7 lysis

# Experiments

PyoBusters is a genetically engineered *E. coli* strain, fulfilling a
therapeutic
action against *P. aeruginosa* and its biofilm. Our PyoBusters system works thanks to a
plasmid
comprising 4 different sets of genes, as well as a gene inserted in the genome.

Those sets of genes can be separated into 3 systems, operating in parallel with different
objectives.

* Survival system: Specifically target the *P. aeruginosa’s* biofilm to grow on its
  surface.
* Therapeutic system: Produce intracellular therapeutic molecules to destroy
  *P. aeruginosa* and
  its biofilm.
* Delivery system: Work as a delivery system allowing the *in-situ* release of
  therapeutic
  molecules.

Summary

[Bacteria](#bacteriaDiv)
[Wet Lab](#wetLabIntroduction)

[Introduction](#wetLabIntroduction)
[Survival System](#survivalSystem)
[Therapeutic System](#therapeuticSystem)
[Delivery System](#deliverySystem)
[DNA sequences](#tableDNA)
[Incubator](#incubatorDiv)

[Introduction](#dryLabIntroduction)

[Bacteria](#bacteriaDiv)
[Wet Lab](#wetLabIntroduction)

[Introduction](#wetLabIntroduction)
[Survival System](#survivalSystem)
[Therapeutic System](#therapeuticSystem)
[Delivery System](#deliverySystem)
[DNA sequences](#tableDNA)
[Incubator](#incubatorDiv)

[Introduction](#dryLabIntroduction)

# WetLab Introduction

Before starting the experiments, we used Benchling and UTR Designer for respectively building our
plasmid constructions and designing the DNA sequences. Indeed, UTR Designer is a web site able to
provide different 5’UTR sequences for a specific gene and estimate with a score their translational
strength.
To simplify the insertions of these 5’UTR sequences, we had them synthesized following the
respective
promoters by IDT. In order to differentiate the different strengths of 5’UTR sequences we added an
adjective (high or low) or a percentage of relative expression followed by the noun of the gene for
which it was designed.

To standardize the cloning strategy we chose to work with pUCBB. This plasmid contains restriction
sites
shown in the illustration 1 that were used to insert specific parts. For example, all the promoters
were
cloned between Xbal and Ndel and all
the genes were cloned between Ndel and Nsil. For the terminators, we used two different approaches; we cloned
them either between Nsil and Xhol if the
next slot was used or Nsil and Spel
if not.
Sequences were previously amplified by PCR with primers that permit to add the respective
restriction
enzyme
sites and then have been digested.
All the constructions were checked by digestion and PCR to validate them.
All the constructions were made in *E. coli* DH5 α.

![restrictions sites](https://static.igem.org/mediawiki/2020/5/5b/T--Grenoble_Alpes--Expe_INTRO.png)

Figure 1 represents the different restriction sites
contained in the plasmid pUCBB and the cloning strategy used to insert promoters, genes and
terminators.

# Survival system

### 1. Selection of the strongest promoter to produce the RhlR protein

So that our system is as sensitive as possible to BHL, we needed to have the highest production of
rhlR in the engineered *E. coli* strain. Thus, we tested three constitutive promoters with
different
strengths found in the iGEM Bank: J23100, J23102 and J23106. To identify the strongest one, we
integrated the promoters into pUCBB-*eGFP* and transformed *E. coli* DH5 α
strains.

![J23100](https://static.igem.org/mediawiki/2020/5/56/T--Grenoble_Alpes--Expe_Survival_J23100.png)

Figure 2 representing the part [BBa\_K3463006](http://parts.igem.org/Part%3ABBa_K3463006)

![J23102](https://static.igem.org/mediawiki/2020/0/0e/T--Grenoble_Alpes--Expe_Survival_J23102.png)

Figure 3 representing the part [BBa\_K3463004](http://parts.igem.org/Part%3ABBa_K3463004)

![J23102](https://static.igem.org/mediawiki/2020/4/41/T--Grenoble_Alpes--Expe_Survival_J23106.png)

Figure 4 representing the part [BBa\_K3463005](http://parts.igem.org/Part%3ABBa_K3463005)

From this step, we monitored both the fluorescence and the absorbance of the bacterial cultures
(transformed *E. coli* DH5 α). Indeed, we quantified the level of expression of eGFP
over time for
the different constructions in order to select the strongest promoter. Then, we replaced the
reporter gene by the *rhlR* gene to build the pUCBB-J13100-*RhlR* [BBa\_K3463007](http://parts.igem.org/Part%3ABBa_K3463007). To finish, we added the
terminator B0015 behind *rhlR* [BBa\_K3463008](http://parts.igem.org/Part%3ABBa_K3463008).

![J2310àxB0015](https://static.igem.org/mediawiki/2020/8/88/T--Grenoble_Alpes--Expe_Survival_RHLR.png)

Figure 5 representing the part [BBa\_K3463008](http://parts.igem.org/Part%3ABBa_K3463008)

### 2. Inducible promoter *rhlR* (P*RhlR*)

The coding sequence for the inducible promoter *RhlR* comes from the IGEM Bank [BBa\_K1949060](http://parts.igem.org/Part%3ABBa_K3463060). This
promoter is activated by the presence of BHL via the formation of a BHL/RhlR complex. Indeed, it
is
known that the binding of the BHL/RhlR complex on P*rhlR* can trigger specific
downstream genes. Thus,
before using this sequence in the final system, we wanted to ensure that P*rhlR* couldn't
trigger the
transcription of the downstream gene without its specific transcriptional activator RhlR.
Therefore,
we cloned this promoter into the plasmid pUCBB with the reporter gene eGFP in *E. coli*
Nissle strain
[BBa\_K3463011](http://parts.igem.org/Part%3ABBa_K3463011).

![PrhlRxterminator](https://static.igem.org/mediawiki/2020/1/17/T--Grenoble_Alpes--Expe_Survival_PrhlR.png)

Figure 6 representing the part [BBa\_K3463011](http://parts.igem.org/Part%3ABBa_K3463011)

We chose this particular *E. coli* strain because it hasn’t any endogenous production of RhlR.
Thus,
with or without BHL in the medium, P*rhlR* shouldn’t be activated and no eGFP should be
produced. So,
we monitored the fluorescence of the bacterial cultures to quantify the expression of eGFP over
time
in absence or in presence of 100µM of BHL in the medium. The construct will be validated only if
no
fluorescence is detected in both conditions.

### 3. BHL sensing

Thanks to the Standard Assembly technique, the two previous parts described above were cloned in
the
same vector to obtain the plasmid pUCBB-J23100-*RhlR*-B0015-P*rhlR*-*eGFP*
[BBa\_K3463019](http://parts.igem.org/Part%3ABBa_K3463019).

![J23100xB0015xPrhlRxterminator](https://static.igem.org/mediawiki/2020/2/26/T--Grenoble_Alpes--Expe_Survival_Composite.png)

Figure 7 representing the part [BBa\_K3463019](http://parts.igem.org/Part%3ABBa_K3463019)

Indeed, by digesting J23100-*RhlR*-B0015 [BBa\_K3463008](http://parts.igem.org/Part%3ABBa_K3463008)
with EcoRI/SpeI, P*rhlR*-*eGFP* [BBa\_K3463011](http://parts.igem.org/Part%3ABBa_K3463011)
with
XbaI/PstI and the backbone pUCBB with EcoRI/PstI, we built a new part able to both produce RhlR
and
sense BHL.
Therefore, 6 bacterial cultures of transformed *E. coli* Nissle were set up with an
increasing amount
of synthetic BHL : 100µM / 10µM / 1µM / 100nM / 10nM / without BHL. In addition, a culture of
wild
type *E. coli* Nissle was used as negative control.
The purpose of this experiment was to evaluate the correlation between the BHL concentration in
the
medium and the intensity of the fluorescence emitted. From this step, the same construction with
eGFP replaced by the LacI gene can be built.

### 4. P*tac* construction and verification

In order to integrate the “validation” lysis system into the genome, we built two different
plasmids. Both contain the *eGFP* gene followed by the terminator B0017 between NsiI and XhoI
(leaving
an emplacement for the future genomic insertion procedure) from the iGEM Bank and either the
promoter P*tac*High or P*tac*Low.

![PtacxutrHigh](https://static.igem.org/mediawiki/2020/5/5c/T--Grenoble_Alpes--Expe_Survival_Ptac_High.png)

Figure 8 representing the part [BBa\_K3463018](http://parts.igem.org/Part%3ABBa_K3463018)

![PtacxutrLow](https://static.igem.org/mediawiki/2020/b/b5/T--Grenoble_Alpes--Expe_Survival_Ptac_Low.png)

Figure 9 representing the part [BBa\_K3463017](http://parts.igem.org/Part%3ABBa_K3463017)

To ensure that the 5’UTR selection and terminator B0017 were relevant, we monitored the eGFP
expression of the four constructions in *E. coli* DH5 α. We measured both the
fluorescence and the
absorbance of the bacterial culture for 4 to 5 hours of the construction
P*tac*High-*eGFP*,
P*tac*Low-*eGFP*, P*tac*High-*eGFP*-B0017,
P*tac*Low-*eGFP*-B0017,
and a
non-transformed strain as a negative
control.
This experiment was initially planned for constructions integrated into the genome. However due
to a
lack of time and a technical issue, the experience was made with plasmid constructions.

### 5. Genomic integration of the constructions

The genomic integration was made following the Datsenko et al. protocol
(<https://doi.org/10.1073/pnas.120163297> )
adjusted in order to fit our needs as described in the
illustration.

![expe_survival_genomic](https://static.igem.org/mediawiki/2020/5/5e/T--Grenoble_Alpes--Expe_Survival_genomic.png)
![expe_survival_genomic](https://static.igem.org/mediawiki/2020/d/da/T--Grenoble_Alpes--Expe_Survival_genomic2.png)

Figure 10 representing the plasmids PKD3,
PKD46,
and
PKD20 were used respectively to:
(i) enable the selection of the recombinant clone
(ii) make the recombination
(iii) eliminate the resistance gene

Initially, the integration protocol was carried out without using the resistance gene to limit
the
use of antibiotics and avoid the creation of strain carrying a resistance gene into its genome.
This approach was proved ineffective.
Consequently, the protocol was carried out following the initial protocol as described in the
following figure. Schéma The PKD3 chloramphenicol gene resistance flanked by the 2 FRTsites, was
amplified by PCR and added to the Plasmid pUCBB-P*tac*High-*eGFP*-B0017
or
P*tac*Low-*eGFP*-B0017 between
the restriction sites XhoI and SpeI.
The overall constructs that needed to be integrated into the genome namely
pUCBB-P*tac*High/Low-*eGFP*-B0017 were then unsuccessfully
amplified by PCR. Due to the
lack of time, we conceived a new experiment to overcome this issue.

### 6. Proof of concept construction

To get a proof of concept for our entire genetic circuit, we decided to build a plasmid
containing
all the parts of the survival system:

* J23100-*RhlR*-B0015 [BBa\_K3463008](http://parts.igem.org/Part%3ABBa_K3463008)
* P*rhlR*-*LacI* [BBa\_K3463031](http://parts.igem.org/Part%3ABBa_K3463031)
* P*tac*-*eGFP*-B0017 [BBa\_K3463018](http://parts.igem.org/Part%3ABBa_K3463018)

![expe_survival_POC](https://static.igem.org/mediawiki/2020/6/68/T--Grenoble_Alpes--Expe_Survival_proof_of_concept.png)

Figure 11 representing the part [BBa\_K3463020](http://parts.igem.org/Part%3ABBa_K3463020)

Then, we transformed a *E. coli* BL21 strain with [BBa\_K3463020](http://parts.igem.org/Part%3ABBa_K3463020)
and monitored the expression of the
eGFP by measuring the fluorescence with an increasing amount of synthetic BHL : 100µM / 10µM /
1µM /
100nM / without BHL. In addition, a culture of wild type *E. coli* BL21 was used as
negative
control.

[GO TO RESULTS](https://2020.igem.org/Team%3AGrenoble_Alpes/Results#survivalSystem)

[GO TO DESIGN](https://2020.igem.org/Team%3AGrenoble_Alpes/Design#survivalSystem)

# Therapeutic system

### 1. Parts extraction and constructions

To deliver the therapeutics molecules to the biofilm of *P. aeruginosa*, we transformed the
engineered *E. coli* BL21 with the necessary genes.

We got the *Dispersin B* (*DspB*) from the part [BBa\_K1659200](http://parts.igem.org/Part%3ABBa_K1659200), designed by Wei Chung Kong
iGEM15\_Oxford15
(2015-08-28).

The *Alginate lyase* (*AlgL*, [BBa\_K3463002](http://parts.igem.org/Part%3ABBa_K3463002))
and the *Pyocin S5* (*PyoS5*, [BBa\_K3463001](http://parts.igem.org/Part%3ABBa_K3463001))
have been extracted
from the PAO1’s genome. To do so, a kit of DNA extraction was used to lyse PAO1.

We used the plasmid pUCBB containing the J23100 promoter (pUCBB-J23100-*eGFP*) because we had
characterized it by ourselves. The *Dispersin B*, *Pyocin S5* and *Alginate
lyase* were cloned inside
instead of the rapporter gene in *E. coli* DH5α. Unfortunately, it went wrong for both
*Pyocin
S5*,
and *Alginate lyase*. Thus, we decided to clone them in the pUCBB-ntH6-*eGFP*
containing a
stronger
promoter than J23100.

![expe_therap_PyoS5](https://static.igem.org/mediawiki/2020/3/3c/T--Grenoble_Alpes--Expe_Therap_PyoS5.png)

Figure 12 representing the part [BBa\_K3463001](http://parts.igem.org/Part%3ABBa_K3463001)

![expe_therap_dispersinB](https://static.igem.org/mediawiki/2020/5/51/T--Grenoble_Alpes--Expe_Therap_DispB.png)

Figure 13 representing the part [BBa\_K1659200](http://parts.igem.org/Part%3ABBa_K1659200)

As soon as we obtained the plasmid pUCBB-ntH6-*PyoS5* and pUCBB-J23100-*DspB*, we
transformed each of
them in *E. coli* BL21 to optimize protein expression.

### 2. Creation of *Pseudomonas aeruginosa*’s biofilm

To test our therapeutics molecules on the PAO1's biofilm, we decided to create a biofilm on a
96-wells plate. We started a preculture that grew overnight. The next day, we inoculated a 96-well
plate and incubated for 20 hours at 30°C. Nevertheless, we didn’t manage to create this biofilm in
our laboratory. From this step, we decided to contact our two partners (Bioaster and I&L Biosystems)
to produce the PAO1’s biofilm and test our therapeutic molecules in their laboratories.

### 3. Protein expression, extraction and storage

In our system, the engineered *E. coli* should lysate itself to deliver the therapeutics
molecules. In
order to test these two therapeutics molecules (Pyocin S5 and Dispersin B) on *P. aeruginosa*
and its
biofilm, we needed to produce and extract them from *E. coli* BL21. For each growing culture,
we
started the lysis protocol when the OD reached 0,9. After sonication, all the samples of bacterial
lysis cultures were stored at -80°C in a glycerol buffer.

### 4. Tests on PAO1

As we didn’t manage to create the PAO1’s biofilm in our laboratory, we sent our therapeutic
molecules for testing to Bioaster.
According to the article [A steam-based method
to investigate biofilm](https://doi.org/10.1038/s41598-018-31437-y)[1], they use a steam washing
concept. This technique allows the removal of planktonic bacteria without altering the biofilm in
contrast to pipette-based washing. They grow biofilms in a 96-well plate, with a cooling system,
which is held upside down above a boiling system. The steam gently washes biofilms and removes
planktonic bacteria.

Concerning the quantification of the biofilm, they use the resuspension of the biofilm in
phosphate-buffered saline instead of dye staining which can be toxic. They realised successive
dilutions until 10-10 and spoted 10µL of each dilutions in COS geloses to assess the CFU/mL per
replicate.

The tested conditions were:

* Lysis Buffer (used to obtain the *E. coli* lysates)
* Lysate Dispersin B
* Lysate Pyocin S5
* Lysate non-transformed *E. coli* BL21

These conditions were both inoculated at 25% (163µL of component + 487µL of Dulbecco's phosphate-buffered saline) or 50% (325µL of component + 325µL of DPBS) in triplicate and compared to a non-treated condition.

Furthermore, we wanted to evaluate if the Pyocin S5 was able to kill planktonic *Pseudomonas
aeruginosa*. For this experiment, we used four different growing cultures:

* BL21 wt 20mL (control -)
* BL21 PyoS5 20mL
* BL21 PyoS5 10mL
* BL21 PyoS5 5mL

After reaching 0.9 OD, the sonication protocol was executed to prepare four protein samples of
500µL.
In addition, two negative control samples of 500µL were also prepared:

* Buffer
* TSP (culture medium)

From this step, six growing cultures (3,5mL) of *P. aeruginosa* were set up at 0.1 OD and
all of
these previous samples were added inside.
Indeed, 450µL of each protein and control samples were used to evaluate if the Pyocin S5 was
able to kill *P. aeruginosa* by monitoring the OD overtime at 37°C (2H).

![therapeutic_falcon](https://static.igem.org/mediawiki/2020/1/10/T--Grenoble_Alpes--Expe_Therap_tubes_bleu.png)

Figure 14: Protocols experiments used to determine the killing effect of the Pyocin S5 on planktonic *pseudomonas aeruginosa*.

In addition, another experiment on an agar plate was performed to complement the previous one.
Using the disk diffusion test, we wanted to show if the Pyocin S5 was able to block the growth
of *P. aeruginosa* on agar plates. Firstly, we spread 100µL of *P. aeruginosa* on
an agar plate and
we deposited 50µL of each protein and control samples in six different discs. Then, these six
discs were put on the plate and this one was incubated at 37°C overnight.

![therap_petri](https://static.igem.org/mediawiki/2020/f/f8/T--Grenoble_Alpes--Expe_Therap_petri.png)

Figure 15: Schema representing the experiment on
an agar plate to measure the killing effect of five different samples on *pseudomonas
aeruginosa*.

[GO TO RESULTS](https://2020.igem.org/Team%3AGrenoble_Alpes/Results#therapeuticSystem)

[GO TO DESIGN](https://2020.igem.org/Team%3AGrenoble_Alpes/Design#therapeuticSystem)

# Delivery system

In order to permit PyoBusters to control its population and release therapeutic
molecules it is necessary to modulate the transcription of the lysis proteins (E7), but
according to the design of our system, it was not possible to change the promoter. The unique
option was to modulate the transcription of the colicin protein through the selection of 5’UTR
sequences using UTR designer.

### 1. 5’UTRs comparaison

It was important to validate the power value of the transcription given by the software. To do so,
initially, we used *lsrA* promoter and two different 5’UTR fused to the *mCherry* reporter
gene.
First, we built pUCBB-*mCherry* plasmid combining the igem part [BBa\_J06504](http://parts.igem.org/Part%3ABBa_J06504) containing *mCherry* and the
pUCBB backbone from pUCBB-*eGFP*.
The next step was the integration into the plasmid pUCBB-*mCherry* of the promoter
P*lsrA* with
the
respective 5’UTR. The first 5’UTR has the highest transcriptional rate (5’UTR *mCherry*High
=
600
thousand) and the second one has a lower transcriptional rate, 50% less powerful than the high one.
We used the igem part [Bba\_K649100](http://parts.igem.org/Part%3ABba_K649100) as promoter
*lsrA*.
P*lsrA* was cloned in the pUCBB-*mCherry* plasmid and we obtained both
pUCBB-P*lsr*High-*mCherry*
and
pUCBB-P*lsr*Low-*mCherry* .

![utrHighXterminator](https://static.igem.org/mediawiki/2020/d/de/T--Grenoble_Alpes--Expe_Delivery_UTR_HIGH.png)

Figure 16 representing the part [BBa\_K3463023](http://parts.igem.org/Part%3ABBa_K3463023).

![utrLowXterminator](https://static.igem.org/mediawiki/2020/0/0b/T--Grenoble_Alpes--Expe_Delivery_UTR_LOW.png)

Figure 17 representing the part [BBa\_K3463024](http://parts.igem.org/Part%3ABBa_K3463024).

After obtaining the two plasmids that express mCherry at different rates, we needed a mCherry
constitutive expression to compare it to the P*lsrA* expression and demonstrate that it is
related to
the density of the bacterial population. For this purpose, we designed pUCBB-J23100-*mCherry*.

*E. coli* DH5 alpha strain doesn't produce AI-2 that is why the previous construction was used to
transform *E. coli* BL21, a strain which produces AI-2 therefore allowing the expression of
the
reporter gene.

BL21 with the plasmid of interest, were grown overnight and were used to inoculate a culture at OD =
0.5 to follow the mCherry expression hourly.

### 2. *E. coli* lysis

The challenge of the delivery system we designed is to induce bacterial lysis according to the
growth which is monitored thanks to the optical density. In order to test different 5’UTR as we did
for modulating the expression of mCherry, we selected three 5’UTR and we named them 100%, 50%, and
25% as the percentage of the power value of the E7 transcription. Notice that 100% is equivalent to
2 million values.
First, we cloned each P*lsrA* fused to the 5’UTR sequence in pUCBB-*eGFP*, then we
inserted the
*E7* gene [Bba\_K117000](http://parts.igem.org/Part%3ABBa_K117000). We obtained
pUCBB-P*lsr*100%-*E7* [BBa\_K3463025](http://parts.igem.org/Part%3ABBa_K3463025),
pUCBB-P*lsr*50%-*E7* [BBa\_K3463026](http://parts.igem.org/Part%3ABBa_K3463026)
and
pUCBB-P*lsr*25%-*E7* [BBa\_K3463027](http://parts.igem.org/Part%3ABBa_K3463027).

![UTR100%_X_terminator](https://static.igem.org/mediawiki/2020/1/1e/T--Grenoble_Alpes--Expe_Delivery_E7.png)

Figure 18 representing the part [BBa\_K3463025](http://parts.igem.org/Part%3ABBa_K3463025).

Once we obtained the 3 constructs, we followed hourly the OD to evaluate the lysis in the same
conditions as we did previously.
It is admitted that this is not the best method to evaluate the lysis but due to the lack of time,
we were not able to perform another more quantitative method. If we could have experimented
differently we would have used propidium iodide and Cyber Green labeling or we would have simply
counted the colonies on plates after serial dilutions.

[GO TO RESULTS](https://2020.igem.org/Team%3AGrenoble_Alpes/Results#deliverySystem)

[GO TO DESIGN](https://2020.igem.org/Team%3AGrenoble_Alpes/Design#deliverySystem)

# Summary of the sequences and the primers to amplify iGEM parts

| 5’UTR name | | | | | | | | | | | | Sequences | | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| P*lsr*High*-mCherry* | | | | | | | | | | | | 5'ATTGTGATCTATTCGTCGGAAATATGTGCAATGTCCACCTAAGGTTATGAACAAATTA   AAAGCAGAAATACATTTGTTCAAAACTCACCTGCAAAACCGTCGAAAACGTGAGGGG   GTTGTCAT 3' | | | | | | | | | | | |
| P*lsr*Low*-mCherry* | | | | | | | | | | | | 5'ATTGTGATCTATTCGTCGGAAATATGTGCAATGTCCACCTAAGGTTATGAACAAATTA   AAAGCAGAAATACATTTGTTCAAAACTCACCTGCAAAACCGTCGCGAACGTGAGGG   GGTTGTCAT 3' | | | | | | | | | | | |
| P*lsr*100%*-E7* | | | | | | | | | | | | 5'ATTGTGATCTATTCGTCGGAAATATGTGCAATGTCCACCTAAGGTTATGAACAAATTA   AAAGCAGAAATACATTTGTTCAAAACTCACCTGCAAAACAAAAAAACACAAAGGAGC   ATCACAT 3' | | | | | | | | | | | |
| P*lsr*50%*-E7* | | | | | | | | | | | | 5'ATTGTGATCTATTCGTCGGAAATATGTGCAATGTCCACCTAAGGTTATGAACAAATTA   AAAGCAGAAATACATTTGTTCAAAACTCACCTGCAAAACTAAACCCATCAAAGGAGCA   TCACAT 3' | | | | | | | | | | | |
| P*lsr*25%*-E7* | | | | | | | | | | | | 5'ATTGTGATCTATTCGTCGGAAATATGTGCAATGTCCACCTAAGGTTATGAACAAATTA   AAAGCAGAAATACATTTGTTCAAAACTCACCTGCAAAACTAAGCTCATCAAAGGAGCA   TCACAT 3' | | | | | | | | | | | |
| J23100 | | | | | | | | | | | | 5’GTCTCTAGAATGCAATGAGGCCATTTAAGGCCATTACGCGATTAGCCTTGACGGCT   AGCTCAGTCCTAGGTACAGTGCTAGCGAAATGGGGTTGGGAGGTAGAACATATGTAG 3’ | | | | | | | | | | | |

| gene | FRW | REV |
| --- | --- | --- |
| **Delivery system** | | |
| *mCherry* | 5'CCACATATGGTGAGCAAGGGCGA 3' | 5'AATATGCATTTATTACTTGTAC   AGCTCGTCCATGC 3' |
| *E7* | 5'ATCCATATGAAAAAAATAACAGGGAT 3' | 5'ATGCATTTACTGCGTTTCCACTC 3' |
| P*lsr* with *mCherry* UTR | 5'CTAGAATTCTAGGATCCATCTAGAATT GTGATCTATTCGTCGG 3' | 5'CTACATATGACAACCCCCTCAC 3' |
| **Therapeutic system** | | |
| *Dispersin B* | 5'GTACATATGTTATTAATGATGA TGGTGGTG 3' | 5'CGAATGCATACAACCCCCTCAC 3' |
| *Alginate lyase* | 5'GATCATATGCGAATCAGCATCTTT 3' | 5'AGAATGCATCTACCAGCAGATGCCCT 3' |
| *Pyocin S5* | 5'TTGCATATGTCCAATGACAACGAAG 3' | 5'CGAATGCATTTAAATGGATATT ACAAGATTGTTTG 3' |

![](https://static.igem.org/mediawiki/2020/c/c6/T--Grenoble_Alpes--incubatorSchema.png)

![](https://static.igem.org/mediawiki/2020/c/c6/T--Grenoble_Alpes--incubatorSchema.png)

# Experiments

PyoBusters’ goal is to destroy the biofilm of *P. aeruginosa* which acts in
different environments. As engineers we wanted to provide the experimenters a way to
grow their biofilms and insert the modified *E. coli* in an automated experience. Hence
we
built an incubator called the Automated Measurement Incubator (AMI) which is built and
implemented in 4 parts:

* The environment module that controls and monitor the atmosphere inside the box.
* The agitation module that operates the plate movements.
* The fluorescent module that makes the analysis during the whole experiment.
* The software that controls the 3 previous modules and allows a Human Computer Interface.

# DryLab Introduction

Concerning the Dry Lab, experiments are done all along the building phase. Unlike the
manipulation of bacteria, modifying parameters or code is fast, and testing the consequence of
those modifications is also very fast.
This is why it is unlikely to index every little run tests that are done, as many of them only
concerns very little modifications.

[GO TO RESULTS](https://2020.igem.org/Team%3AGrenoble_Alpes/Results#DryLab)

[GO TO DESIGN](https://2020.igem.org/Team%3AGrenoble_Alpes/Design#DryLab)

### Team Grenoble\_Alpes

![](https://static.igem.org/mediawiki/2020/e/ea/T--Grenoble_Alpes--Logo_final_Grenoble.png)
![](https://static.igem.org/mediawiki/2020/7/7e/T--Grenoble_Alpes--Logo_UGAINP.png)

![](https://static.igem.org/mediawiki/2020/7/7a/T--Grenoble_Alpes--PyoBustersText.png)

### Contact

* #### Igem grenoble Place du Commandant Nal 38700, La Tronche, France
* #### igem.grenoble.2020@gmail.com

[![](https://static.igem.org/mediawiki/2020/4/41/T--Grenoble_Alpes--Logo_facebook.png)](https://www.facebook.com/igemfrancegrenoble)
[![](https://static.igem.org/mediawiki/2020/4/4d/T--Grenoble_Alpes--Logo_instagram.png)](https://www.instagram.com/igem_grenoble/)
[![](https://static.igem.org/mediawiki/2020/3/3a/T--Grenoble_Alpes--Logo_linkedIn.png)](https://www.linkedin.com/in/igem-grenoble/)
[![](https://static.igem.org/mediawiki/2020/6/62/T--Grenoble_Alpes--Logo_twitter.png)](https://twitter.com/igem_grenoble)

This wiki was completely designed and coded by the team members of the 2020 Grenoble\_Alpes' Team, all content on this wiki is available under the Creative Commons [Attribution 4.0](https://creativecommons.org/licenses/by/4.0/) license (or any later version).

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

# Team:Grenoble Alpes/Results

[![PyoBusters](https://static.igem.org/mediawiki/2020/7/7a/T--Grenoble_Alpes--PyoBustersText.png)](https://2020.igem.org/Team%3AGrenoble_Alpes)

* About us

  [Team](https://2020.igem.org/Team%3AGrenoble_Alpes/Team)
  [Attributions](https://2020.igem.org/Team%3AGrenoble_Alpes/Attributions)
  [Collaborations](https://2020.igem.org/Team%3AGrenoble_Alpes/Collaborations)
  [Sponsors](https://2020.igem.org/Team%3AGrenoble_Alpes/Sponsors)
* Project

  [Description](https://2020.igem.org/Team%3AGrenoble_Alpes/Description)
  [Partnership](https://2020.igem.org/Team%3AGrenoble_Alpes/Partnership)
  [Engineering](https://2020.igem.org/Team%3AGrenoble_Alpes/Engineering)
  [Contribution](https://2020.igem.org/Team%3AGrenoble_Alpes/Contribution)
  [Notebook](https://2020.igem.org/Team%3AGrenoble_Alpes/Notebook)
  [Implementation](https://2020.igem.org/Team%3AGrenoble_Alpes/Implementation)
* Wet Lab

  [Protocols](https://2020.igem.org/Team%3AGrenoble_Alpes/Protocols)
  [Parts](https://2020.igem.org/Team%3AGrenoble_Alpes/Parts)
  [Design](https://2020.igem.org/Team%3AGrenoble_Alpes/Design)
  [Experiments](https://2020.igem.org/Team%3AGrenoble_Alpes/Experiments)
  [Proof
  of Concept](https://2020.igem.org/Team%3AGrenoble_Alpes/Proof_Of_Concept)
  [Results](https://2020.igem.org/Team%3AGrenoble_Alpes/Results)
* Dry Lab

  [Hardware](https://2020.igem.org/Team%3AGrenoble_Alpes/Hardware)
  [Software](https://2020.igem.org/Team%3AGrenoble_Alpes/Software)
  [Pythie](https://2020.igem.org/Team%3AGrenoble_Alpes/Pythie/SignIn)
  [Design](https://2020.igem.org/Team%3AGrenoble_Alpes/Design#incubatorDiv)
  [Experiments](https://2020.igem.org/Team%3AGrenoble_Alpes/Experiments#incubatorDiv)
  [Proof of
  Concept](https://2020.igem.org/Team%3AGrenoble_Alpes/Proof_Of_Concept#incubatorDiv)
  [Results](https://2020.igem.org/Team%3AGrenoble_Alpes/Results#incubatorDiv)
* Human Practices

  [Human
  Practices](https://2020.igem.org/Team%3AGrenoble_Alpes/Human_Practices)
  [Education](https://2020.igem.org/Team%3AGrenoble_Alpes/Education)
  [Safety](https://2020.igem.org/Team%3AGrenoble_Alpes/Safety)
  [Clinic](https://2020.igem.org/Team%3AGrenoble_Alpes/Clinic)
  [Inclusion](https://2020.igem.org/Team%3AGrenoble_Alpes/Inclusion)
* Awards

  [Medals](https://2020.igem.org/Team%3AGrenoble_Alpes/Medals)
  [Awards](https://2020.igem.org/Team%3AGrenoble_Alpes/Awards)

![](https://static.igem.org/mediawiki/2020/7/79/T--Grenoble_Alpes--glasses_Icon.png)

Protanopia

Deuteranopia

Tritanopia
Protanomaly

Deuteranomaly

Tritanomaly
Achromatopsia

Achromatomaly

![](https://static.igem.org/mediawiki/2020/7/79/T--Grenoble_Alpes--glasses_Icon.png)

Protanopia

Deuteranopia

Tritanopia
Protanomaly

Deuteranomaly

Tritanomaly
Achromatopsia

Achromatomaly

PyoBuster - Results

Constitutive +
​
Therapeutic
molecules

Plsr + E7 lysis

Constitutive + RhlR

Prhlr + LacI

Genomic DNA
Ptac + E7 lysis

Constitutive +
​
Therapeutic
molecules

Plsr + E7 lysis

Constitutive + RhlR

Prhlr + LacI

Genomic DNA
Ptac + E7 lysis

# Wet Lab Results

PyoBusters is a genetically engineered *E. coli* strain, fulfilling a
therapeutic
action against *P. aeruginosa* and its biofilm. Our PyoBusters system works thanks to a
plasmid
comprising 4 different sets of genes, as well as a gene inserted in the genome.

Those sets of genes can be separated into 3 systems, operating in parallel with different
objectives.

* Survival system: Specifically target the *P. aeruginosa’s* biofilm to grow on its
  surface.
* Therapeutic system: Produce intracellular therapeutic molecules to destroy
  *P. aeruginosa* and
  its biofilm.
* Delivery system: Work as a delivery system allowing the *in-situ* release of
  therapeutic
  molecules.

Summary

[Bacteria](#bacteriaDiv)
[Wet Lab](#wetLabIntroduction)

[Introduction](#wetLabIntroduction)
[Survival System](#survivalSystem)
[Therapeutic System](#therapeuticSystem)
[Delivery System](#deliverySystem)
[Incubator](#incubatorDiv)
[Dry Lab](#dryLabIntroduction)

[Introduction](#dryLabIntroduction)
[Environment
Control](#automatedEnvironmentControl)
[References](#references)

[Bacteria](#bacteriaDiv)
[Wet Lab](#wetLabIntroduction)

[Introduction](#wetLabIntroduction)
[Survival System](#survivalSystem)
[Therapeutic System](#therapeuticSystem)
[Delivery System](#deliverySystem)
[Incubator](#incubatorDiv)
[Dry Lab](#dryLabIntroduction)

[Introduction](#dryLabIntroduction)
[Environment
Control](#automatedEnvironmentControl)
[References](#references)

# WetLab Introduction

The main objective of our experiments was to design a PyoBusters able to detect *Pseudomonas
aeruginosa*’s biofilm in order to trigger gene expression. We intended, besides, to produce two
therapeutic molecules capable of destroying the biofilm and killing *P. aeruginosa*. We also
wanted to
show that it is possible to use *E. coli*'s sensing quorum to induce the PyoBusters lysis
thus
enabling the release of the therapeutic molecules.

# Survival system

### 1. Constitutive promoter for the production of the RhlR

In order to compare the strength of three promoters, we have monitored four DH5alpha
cultures:

* DH5α-pUCBB-J23100-*eGFP* [BBa\_K3463006](http://parts.igem.org/Part%3ABBa_K3463006)
* DH5α-pUCBB-J23102-*eGFP* [BBa\_K3463004](http://parts.igem.org/Part%3ABBa_K3463004)
* DH5α-pUCBB-J23106-*eGFP* [BBa\_K3463005](http://parts.igem.org/Part%3ABBa_K3463005)
* DH5α (Control-)

These 4 cultures were inoculated in triplicata (OD600nm=0.1) in a semi-transparent 96-wells plate
and incubated at 37°C in Varioskan LUX six hours long. This experiment was repeated three times and
data were collected and analysed together to build the following graphs.

Every ten minutes, both OD600nm (Figure 1) and *eGFP* fluorescence (Figure 2) were
measured in each well and triplicata's mean were plotted.

![evolution of DH5a OD overtime](https://static.igem.org/mediawiki/2020/a/a6/T--Grenoble_Alpes--Res_Survival_OD.png)

Figure 1: Optical density of DH5α
over time according to the J23 promoter

Growth curves are presented Figure 1. We can see that all these curves have the same pattern of
evolution over time. Therefore, when compared to the growth curve of the control culture, we can
conclude that the strength of a promoter has no effect on the growth of the bacterial culture
(p-value=0.896).

![fluo J23](https://static.igem.org/mediawiki/2020/7/78/T--Grenoble_Alpes--Res_Survival_Fluo_J23.png)

Figure 2: kinetics of eGFP expression
under the control of various promoters

Averages: J23100>J23102>J23106>DH5wt

The figure 2 shows the fluorescence intensity over time. For each curve, the first measurement point
(T0) was set at 0 and subtracted to the following ones.
Firstly, no fluorescence was detected in the DH5 alpha wt control culture. In contrast, in all the
cultures expressing *eGFP* under the control of a specific constitutive promoter, we detected
an
significant increasing amount of fluorescence over time (p-value<2.10-16). The total amount of *eGFP*
produced increased according to the promoter tested with 15.2, 9.7 and 2.5 RFU for J23100, J23102
and J23106 respectively. Significantly, the promoter J23100 seems to be the best one to produce *eGFP*
in large quantities. Thus, it will be used to produce RhlR [BBa\_K3463007](http://parts.igem.org/Part%3ABBa_K3463007).

### 2. Inducible promoter *rhlR* (P*rhlR*)

The Figure 3 illustrates the fluorescence intensity over time for 2 growing cultures of *E.
coli*
Nissle transformed with the plasmid pUCBB Pr*hlR-eGFP* [BBa\_K3463011](http://parts.igem.org/Part%3ABBa_K3463007). Cultures were performed in
absence or in presence of 100µM of synthetic BHL.
In addition, a culture of wild type *E. coli* Nissle (not transformed) was used as a negative
control.
The fluorescence intensity was monitored for 15 hours. Each time point was performed hourly in
triplicata. For each curve, the first measurement (T0) was set at 0 and subtracted to following
ones.
This experiment was repeated 3 times.

![anova results](https://static.igem.org/mediawiki/2020/7/72/T--Grenoble_Alpes--Res_Survival_fluo_prhlR.png)

Figure 3: *eGFP* expression
according
to the BHL and over time. Two conditions with 100µM of BHL and without BHL were performed to
evaluate the effect of BHL in *E. coli* Nissle. With and without BHL, no *eGFP*
expression should be
detected because there is no production of *RhlR* protein.

Averages: Without BHL=100µM BHL=Control -

As it can be seen in figure 3, all cultures have similar levels of *eGFP* expression over time
(p-value=0,44). Indeed, we can see that the fluorescence intensity of *E. coli* Nissle P*rhlR-eGFP*
increases slightly whatever the presence of BHL in the medium. In addition, the curve of negative
control also increases in the same manner over time. Therefore, there is no significant expression
of eGFP in *E. coli* Nissle P*rhlR-eGFP*, even with a large amount of BHL in the
medium.

To conclude, we can significantly ensure that our P*rhlR* construction [BBa\_K3463011](http://parts.igem.org/Part%3ABBa_K3463011) does not allow *E. coli*
Nissle to express downstream genes without RhlR, whatever the presence of BHL.

From this step, the proof of concept’s experiment using pUCBB J23100-RhlR-B0015-P*rhlR*-*eGFP*
can be done.

### 3. BHL sensing

To evaluate if our system works, we performed 6 bacterial cultures of transformed E. coli Nissle
[BBa\_K3463019](http://parts.igem.org/Part%3ABBa_K3463019) incubated with increasing
amounts of synthetic BHL in the medium from 10nM to 100µM.
Once all the media have been set up at T0 with 100µM / 10µM / 1µM / 100nM / 10nM and without BHL,
bacteria were inoculated at OD600nm=0.1.Then, fluorescence measurements of eGFP were performed for
15H (hourly in triplicata until 7H) in opaque 96-wells (FluoStars). For each curve in figure 4, the
first measurement point (T0) was set at 0 and subtracted to the following ones.
This experiment was repeated 3 times.

In addition, a culture of wild type *E. coli* Nissle (not transformed) was used as a negative
control.

![BHL fluo](https://static.igem.org/mediawiki/2020/8/8c/T--Grenoble_Alpes--Res_Survival_BHL_fluo.png)

Figure 4: eGFP expression according
to the BHL concentration and over time. Different concentrations of BHL were performed to
evaluate the effect of BHL in E. coli Nissle. Thanks to the BHL, the engineered *E. coli* should
be able to express eGFP. Without BHL, no eGFP expression should be detected.

Averages: 100µM/10µM/1µM > others

The Figure 4 shows the fluorescence intensity of each bacterial culture according to
the BHL
concentration and over time. While the wild type *E. coli* Nissle auto-fluorescence is used
as blank,
all the other transformed cultures show significant eGFP expression.

First, we can see that transformed *E. coli* Nissle produce low quantities of eGFP even without BHL. However, we can also observe that the more BHL, the more eGFP expression (pvalue<2.10-16). Indeed, in the culture containing 10µM or 100µM of BHL, the fluorescent intensity starts to increase sharply after 3 hours of culture and reaches 800 RFU or more. Nevertheless, even if a curve reaches 600 RFU with 1µM, weaker concentrations of BHL (100nM) don't allow transformed *E. coli* Nissle to significantly express eGFP.

Through an ANOVA test, we can conclude that, thanks to the new part [BBa\_K3463019](http://parts.igem.org/Part%3ABBa_K3463019), our engineered *E. coli* Nissle is
able to sense low concentration of BHL (1µM) in its environment and express a specific gene under
the control of P*rhlR*.

### 4. P*tac* construction and verification

To validate the UTR designer prediction and check the B0017 relevance, we measured the fluorescence
and the absorbance of the cultures of the strains containing one of the following constructs: P*tac*High-*GFP*,
P*tac*Low-*GFP*-B0017 [BBa\_K3463018](http://parts.igem.org/Part%3ABBa_K3463018),
P*tac*Low-*GFP*-B0017
[BBa\_K3463017](http://parts.igem.org/Part%3ABBa_K3463017), and a non-transformed strain as
a negative
control for to 4,5h. The experiment was carried out 3 times.

We obtained the following result shown in figure 5.

![survival ptac](https://static.igem.org/mediawiki/2020/b/b9/T--Grenoble_Alpes--Res_Survival_Ptac_construction.png)

Figure 5: Effect of UTR prediction
and B0017 terminator on eGFP fluorescence expression.
The Figure 5 shows that no fluorescence was detected in the culture of the control non-
transformed strain. As predicted by UTR designer the strong 5’UTR sequence (high) gave higher
fluorescent levels than the weak 5’UTR sequence (Low). The terminator B0017 seems to have a
limited effect on the fluorescent levels. The last seems to only impact the eGFP production of
the strong UTR sequence.

First, the influence of the UTR over the fluorescent level obtained was confirmed by an Anova
displayed in figure 6.

![anova result](https://static.igem.org/mediawiki/2020/3/38/T--Grenoble_Alpes--Res_Survival_annova.png)

Figure 6: Anova results of
constructs influence over fluorescent. Constructs have significant influence over fluorescent:
P-value <2.10-16.

And secondly, as predicted in-silico by UTR designer, the HIGH\_5’UTR has a significantly stronger
expression level than the LOW\_5’UTR. However, during our experiment, we didn’t find any significant
impact of the B0017 terminator on the fluorescence.
It should be noted that the relative fluorescence level between the difference constructions seems
to be slightly different from our prediction with a HIGH relative expression of 100% and a LOW
relative of 50%. According to our model, the relative expression of the LOW prediction was around
75% for a HIGH fixed at 100%. A part of this difference can be explained by the prediction
incertitude. But further investigation and experiments need to be done in order to fully assess the
origin of this difference.
This experiment opens new ways to future iGEM participants that could use a reliable tool to predict
protein expression and therefore give them the ability to build complex genetic networks.

### 5. Genomic integration of the constructions

The genomic integration was made following the Datsenko et al. protocol adjusted to fit our needs.
Initially, the integration protocol was carried out without using the resistance gene to limit the
use of antibiotics and avoid the creation of strain carrying a resistance gene into its genome.
This approach was proved to be ineffective as it gave too many colonies to screen. Indeed a
1/10-6
dilution was necessary to obtain screenable colonies on LB-Agar. No colonies showed visible green
fluorescence when illuminated at 475nm. The experiment was carried out three times and gave the same
data.
Consequently, the protocol was carried out following the initial protocol Datsenko. The PKD3
chloramphenicol gene resistance flanked by the 2 FRT sites, was amplified by PCR and added to the
Plasmid pUCBB P*tac*High-*GFP*-B0017 or
P*tac*Low-*GFP*-B0017 between the restriction sites XhoI and SpeI.
The overall construct that needed to be integrated into the genome namely pUCBB
P*tac*High/Low-*eGFP*-B0017 was then unsuccessfully amplified by
PCR. This failure was identified as a
primer issue, caused by a possible mutation or recombination happening during the insertion of the
PKD3 resistance gene into the pUCBB P*tac*High-*eGFP*-B0017 or
P*tac*Low-*eGFP*-B0017.

![survival genomic integration](https://static.igem.org/mediawiki/2020/2/2f/T--Grenoble_Alpes--Res_Survival_Genomic_integration.png)

Figure 7: PCR amplification of the
genomic insert. 1: PtacHigh-eGFP-B0017-PKD3 2: control PCR 3: P*tac*High-*eGFP*-B0017.

This mutation was highlighted by PCR. The genomic insert was able to be produced only before the
addition of PKD3 gene resistance as seen in figure 7.
Knowing that the amplified zone is set a few nucleotides before the EcoRI restriction site in 5’ and
after the SpeI one in 3’ with the primer beginning at the last nucleotides of the SpeI site, we
assumed that this SpeI site was involved in the failure.
This specific SpeI site was often mutated during our cloning (cf 6. Proof of concept construction),
which suggests that this restriction site is responsible for the PCR failure.
We should have designed the insert primer out of the restriction sites to avoid any mutation
impacting the PCR.
Further investigation needs to be done to fully understand the encountered problem such as
sequencing.

### 6. Proof of concept construction

To get a proof of concept for our all genetic circuit, we decided to build a Proof of concept
plasmid [BBa\_K3463020](http://parts.igem.org/Part%3ABBa_K3463020) containing all the parts
of the survival system:

* J23100-R*hlr*-B0015 [BBa\_K3463008](http://parts.igem.org/Part%3ABBa_K3463008)
* P*rhlr*-*LacI* [BBa\_K3463031](http://parts.igem.org/Part%3ABBa_K3463031)
* P*tac*-*eGFP*-B0017 [BBa\_K3463018](http://parts.igem.org/Part%3ABBa_K3463018)

We then transformed a BL21 strain with [BBa\_K3463020](http://parts.igem.org/Part%3ABBa_K3463020)
and monitored the expression of the *eGFP* by measuring the level of fluorescence under a
gradient of
concentration of synthetic BHL: 100µM / 10µM / 1µM / 100nM and without BHL.

![res survival poc](https://static.igem.org/mediawiki/2020/9/9f/T--Grenoble_Alpes--Res_Survival_proof_of_concept.png)

Figure 8: Effect of the BHL on the
Proof of concept construct. No significant differences were obtained between the tested
concentration and the negative control.

In figure 8, the BHL didn’t have any effects on the fluorescent level (p-value=0.999). Different
hypotheses can be made to explain these data.
For instance the genetic networks didn’t function as planned. However different explanations can be
given before coming to this conclusion. First the fluorescent expression level obtained is much
higher than those obtained classically with the P*tac-GFP* construct and much in line with
the level
obtained with the classic pUCBB-*GFP* suggesting that the construct doesn’t contain the *tac*
promoter.
However the plasmid was built following the standard assembly protocol and gave the expected results
for the digestion gel control (figure 9 and 10).
Despite having the desired digestion profile some screened colonies show an usual profile with
missing restriction sites. To be more precise the PstI and SpeI restrictions were missing
as shown
in figures 9 and 10. That loss of restriction sites can be due to a potential rearrangement or
alternative cut made by the restriction enzyme.
Besides, after the second standard assembly the *tac* promoter couldn’t be amplified by PCR
whereas it
could be from the first standard assembly step (not shown).
This suggested a mutation or a modification of the P*tac* during the second standard assembly
explaining the fluorescent level and the dysfonctionnement of our genetic network.
A DNA sequencing is necessary to fully understand the issue concerning our proof of concept build,
although we had no remaining time to perform it.

![res gel survival poc](https://static.igem.org/mediawiki/2020/6/69/T--Grenoble_Alpes--Res_Survival_Gel_proof_of_concept_1.png)

Figure 9: Digestion of the positive
colonies pUCBB P*tac-eGFP*-B0017-J23100-*RhlR*-B0015 in EcoRI and PstI. All
colonies were negative
with colonies 3 and 5 as classic pUCBB but 1-2 and 5 showed a missing PstI restriction sites
after the standard assembly.

![res gel survival 2 poc](https://static.igem.org/mediawiki/2020/f/ff/T--Grenoble_Alpes--Res_Survival_Gel_proof_of_concept_2.png)

Figure 10: Digestion of the positive
colonies pUCBB P*tac-eGFP*-B0017-J23100-R*hlR*-B0015-P*rhlr-LacI*
[BBa\_K3463020](http://parts.igem.org/Part%3ABBa_K3463020) in EcoRI and PstI.
The 1-2-3-6-8-9 colonies were negative i.e. classic pUCBB. The 5 was positive and contained the
3 composite parts. The 4-7 showed an unexpected profile with a missing PstI restriction
site.

[GO TO DESIGN](https://2020.igem.org/Team%3AGrenoble_Alpes/Design#survivalSystem)

[GO TO EXPERIMENTS](https://2020.igem.org/Team%3AGrenoble_Alpes/Experiments#survivalSystem)

# Therapeutic system

### 1. Amplification of the target genes and construction

To do the construction with the *Dispersin B*, we used the part [BBa\_K1659200](http://parts.igem.org/Part%3ABBa_K1659200), designed by Wei Chung Kong
iGEM15\_Oxford15 (2015-08-28).
Concerning the constructions containing the *Alginate lyase* ([BBa\_K3463002](http://parts.igem.org/Part%3ABBa_K3463002)) and the
*Pyocin S5*
([BBa\_K3463001](http://parts.igem.org/Part%3ABBa_K3463001)) genes, we extracted them from
the PAO1’s genome. To do so, we used a kit for genome
extraction (see [protocols](https://2020.igem.org/Team%3AGrenoble_Alpes/Protocols)) then
proceeded to PCR amplification with
appropriate primers. As you can
see (figure 11), the extraction of the *Alginate lyase* shows non-specific bands but we
extracted the
band at the right size (1326 bp). We extracted the Pyocin S5 at the right size (1512 bp)
too.

![res therapie gel](https://static.igem.org/mediawiki/2020/9/9a/T--Grenoble_Alpes--Res_Therap_Gel.png)

Figure 11: PCR amplification of the
*Alginate
lyase* and *Pyocin S5* from the genomic extraction material. 1: control PCR
*Alginate lyase*, 2 and 3: *Alginate lyase*, 4: control PCR Pyocin S5, 5
and 6: Pyocin S5. The amplification of these genes was conclusif.

We wanted to clone the sequence of our three therapeutic molecules in both
pUCBB-J23100-*eGFP* and
pUCBB-ntH6-*eGFP*.
As we said in the Experiment section, we used primers with particular restriction sites to
standardize the cloning strategy.
Unfortunately we didn’t succeed to clone the *Alginate lyase* gene in the
pUCBB-ntH6-*eGFP*. It may be
explained by the PCR’s product that was not pure enough. Indeed, as you can see on figure 12,
several non-specific bands are still present in the insert sample. It can be explained by the fact
that the Tm or the primers were not optimal.

![res therapie gel 2](https://static.igem.org/mediawiki/2020/6/62/T--Grenoble_Alpes--Res_Therap_Gel_2.png)

Figure 12: PCR amplification of the
*Alginate
lyase*. 1: *Alginate lyase*, 2: control PCR *Alginate lyase*. The
amplification shows several non-specific bands.

The Dispersin B and the Pyocin S5 were respectively cloned in pUCBB-J23100-*eGFP*
and pUCBB-ntH6-*eGFP* in
*E. coli* BL21 to optimize protein expression successfully.

### 2. Tests on PAO1

As we didn’t manage to create the PAO1’s biofilm in our laboratory, we sent the *Dispersin B*
[BBa\_K1659200](http://parts.igem.org/Part%3ABBa_K1659200) to evaluate its activity on *P.
aeruginosa*’s biofilm to Bioaster.
For that, their tested conditions were:

* Lysis buffer (used to obtain the *E. coli* lysates)
* Lysate Dispersin B
* Lysate Pyocin S5
* Lysate non-transformed *E. coli* BL21

These conditions were both inoculated at 25% (163µL of component + 487µL of Dulbecco's phosphate-buffered saline) or 50% (325µL of component + 325µL of DPBS) in triplicate and compared to a non-treated condition.
Then, they counted the CFU/mL per replicate and calculated the bacterial concentrations for each
condition.
As you can see (figure 13), the preliminary data are highly dispersed (cf standard deviations) for
the tested conditions. The lysis buffer condition didn’t impact bacterial concentrations and
comparable results to the non-treated conditions. Concerning the Dispersin B and the Pyocin
S5, they present a dose-effect and the bacterial concentrations are higher than the non-treated
condition. It seems that they have a stimulatory effect.

![therapeutic molecules effects](https://static.igem.org/mediawiki/2020/1/1c/T--Grenoble_Alpes--Res_Therap_Bioaster.png)

Figure 13: Effect of the therapeutic molecules on the *Pseudomonas aeruginosa*’s biofilm resuspension in phosphate-buffered saline. The CFU/mL per replicate were counted after resuspension of the *P. aeruginosa* biofilm. The bacterial concentrations were calculated for each condition.

The next step would be to reiterate these experiments by optimizing the conditions. To do so, the lysates would be purified to obtain higher concentrations of the therapeutic molecules than in the preliminary tests and optimize the sonication protocol to release the molecules. We could do a western blot with antibodies directed against both Dispersin B and Pyocin S5 to make sure that the proteins are expressed.
Moreover, the freezing/thaw process for the transport can have an impact on the protein and have to
be avoided.
Furthermore, in our experimental condition, the observed effect of the three therapeutic molecules didn’t correlate with the one observed in the literature. Therefore further experiments could allow us to fully understand and observe the mechanism of those molecules and to assess their relevance in our therapeutic system.

![therapetics tubes](https://static.igem.org/mediawiki/2020/1/10/T--Grenoble_Alpes--Expe_Therap_tubes_bleu.png)

Figure 14: Killing effect of the
Pyocin
S5 on planktonic *Pseudomonas aeruginosa*.

To evaluate the killing activity of the Pyocin S5 [BBa\_K3463001](http://parts.igem.org/Part%3ABBa_K3463001) on *P. aeruginosa*,
we performed
two different experiments:
The first one (Figure 14) aimed to show the OD decrease of *P. aeruginosa*’s growing
cultures over
time incubated with various concentrations of Pyocin S5 and 3 negative controls.

The data obtained from this experiment (Figure 15) didn’t show any differences between all the
growing cultures after 2H of incubation. Indeed, the OD of *P. aeruginosa* didn’t decrease or
decelerate over the time for cultures with Pyocin S5. Thus, we can say that the Pyocin
S5 doesn’t have any killing activity against planktonic *P. aeruginosa* in suspension at
the concentration tested.

![therapetics table](https://static.igem.org/mediawiki/2020/5/5f/T--Grenoble_Alpes--Res_Therap_tableau.png)

Figure 15: Table representing the
growth
rate of *pseudomonas aeruginosa* under six different conditions.

The second experiment (Figure 16) showed the same result on plate. We wanted to show if the Pyocin
S5 can block the growth of *P. aeruginosa* on LB agar plates.

After an overnight culture at 37°C, the plate was analysed and didn’t show any growing
inhibition around all the 5 discs soaked with Pyocin S5. Thus, we can say that the Pyocin
S5
doesn’t have any killing or inhibiting activity against *P. aeruginosa* on LB agar plate.

Nevertheless, these unexpected results can be explained by the sonication protocol (not
optimised) and the stockage at -80°C. Indeed, it is possible that these steps led to a loss of
the activity of the Pyocin S5 protein.

![therapeutics petri test](https://static.igem.org/mediawiki/2020/4/4c/T--Grenoble_Alpes--Res_Therap_petri_test.png)

Figure 16: Agar plate
representing the
killing effect of five different samples on
*Pseudomonas aeruginosa*.

[GO TO DESIGN](https://2020.igem.org/Team%3AGrenoble_Alpes/Design#therapeuticSystem)

[GO TO EXPERIMENTS](https://2020.igem.org/Team%3AGrenoble_Alpes/Experiments#therapeuticSystem)

# Delivery system

### 1. UTRs comparaison

To confirm the influence of the optical density on the *lsrA* promoter, we
compared the
fluorescent
expression of mCherry due to a constitutive J23100 and *PlsrA*.

For this experiment all the construction PUCBB-J23100-*mCherry*,
pUCBB-P*lsr*High-*mCherry* ([BBa\_K3463023](http://parts.igem.org/Part%3ABBa_K3463023)
) used were successfully obtained and transformed in *E. coli* BL21.

Each bacteria was cultivated at 0,5 OD in LB and we measured the optical density and the
mCherry
fluorescence hourly with the FluoStar BMG labtech (we made triplicate measurement ). An
Opaque-walled 96 was used and the fluorescent gain had been set to 2000 (we made triplicate
measurement).
This experience was repeated three times in the same conditions.

![rdelievry lsr promoteurA](https://static.igem.org/mediawiki/2020/5/50/T--Grenoble_Alpes--Res_Delivery_lsr_promoteur_A.png)
![delievry lsr promoteurB](https://static.igem.org/mediawiki/2020/e/e5/T--Grenoble_Alpes--Res_Delivery_lsr_promoteur_B.png)

Figure 17: mCherry fluorescence
expression
reported at bacterial optical density over time.
To represent the two curves, we used two gradations: the one on the left represents the
expression of the *lsr* promoter and the one on the right represents the J23100 promoter.
A and B
are two examples of the data obtained in the same working conditions

Figure number 17 shows the expression of the mCherry under P*lsrA*, J23100 promoter
and BL21 WT as control over time.

We can first observe that the expression of mCherry under the control of P*lsrA* occurs
between 7 and
8 hours after inoculation, while the constitutive promoter expresses mCherry earlier (after only 3
to 4 hours). In addition, the level of expression of mCherry is about 10 times lower under the
control of P*lsrA* rather than under the control of the constitutive promoter J23100 which
informs
about the strength of the promoter.
In addition, the level of expression of mCherry is about 10 times lower if it is under the
control
of P*lsr* rather than under the control of the constituent promoter J23100 which is not
surprising.

Intending to prove that the P*lsr* expression is related to Autoinducer 2 (AI-2) we performed
the same
experiment with *E. coli* DH5α. This strain is known for not producing any AI-2, and as
expected
there was no mCherry expression during the growing time (results not shown).

We also calculated the Spearman correlation between fluorescence and optical density. For the
constitutive promoter we obtained 0.6263641 and for P*lsr* 0.4615318. A higher correlation is
observed
for J23100 which is explained by a constant expression during growth unlike the expression of the
*lsr* promoter.

The delay of expression observed with *lsr* promoter in BL21 relative to the constitutive
promoter
J23100 is probably linked to the necessary accumulation of Ai-2 during growth and therefore to the
optical density.
Although this experiment was repeated several times under the same conditions we did not obtain the
same results as we can notice (figure 17). Even if the trend suggests that the expression of the
*lsr* promoter is linked to the optical density, there are probably other factors that we
have
involuntarily stimulated that could influence the expression of the protein.

We wanted to verify the expression of the mCherry protein according to the UTR sequence
generated
with the UTR designer software. Two sequences were generated, one associated with high score
(P*lsr*High) and one low score, 50% less than the last
(P*lsr*Low). For this experiment two construction,
pUCBB-P*lsr*High-*mCherry*
([BBa\_K3463023](http://parts.igem.org/Part%3ABBa_K3463023)),
pUCBB-P*lsr*Low-*mCherry* ([BBa\_K3463024](http://parts.igem.org/Part%3ABBa_K3463024)) used were successfully obtained
and transformed
in *E. coli* BL21.
The mCherry expression in *E. coli* BL21 was compared (figure 18) according to the same protocol as
described previously.

![delievry fluo](https://static.igem.org/mediawiki/2020/c/c5/T--Grenoble_Alpes--Res_Delivery_Fluorecence_solo.png)

Figure 18: mCherry fluorescence
expression under control of P*lsr*High or P*lsr*Low and
WT BL21 reported at bacterial optical
density over time.

Figure 18 shows the fluorescent expression associated with the two UTR sequences. We see that
the expression of P*lsr*High and P*lsr*Low is simultaneously
triggered around 7.5h. It can also be noted that the difference between the two becomes more and
more pronounced.

![box plot 1](https://static.igem.org/mediawiki/2020/6/6b/T--Grenoble_Alpes--Res_Delivery_boxplot_1.png)

Figure 19: Boxplot representing the
distribution of ttem under the control of P*lsr* fluorescence as a function of UTR
strength using
*E. coli* BL21.

Figure 19 shows a different distribution of mCherry fluorescence values depending on the
strength of
the UTR sequence. The median for P*lsr*High is higher than that
of P*lsr*Low.
Each time we reproduced this experiment with *E. coli* BL21 we found a significant difference
of fluorescent expression according to the UTR sequence and the 50% ratio was respected.

Once our hypothesis was validated with the *E. coli* BL21 strain, we wished to verify and see if the
difference of expression was preserved in the *E. coli* Nissle strain.
The same experiment as the previous one, under the same conditions, was reproduced with *E.
coli* Nissle transformed with pUCBB-P*lsr*High-*mCherry* ([BBa\_K3463023](http://parts.igem.org/Part%3ABBa_K3463023)) and
pUCBB-P*lsr*Low-*mCherry* ([BBa\_K3463024](http://parts.igem.org/Part%3ABBa_K3463024)).

![box plot 1](https://static.igem.org/mediawiki/2020/2/23/T--Grenoble_Alpes--Res_Delivery_boxplot_2.png)

Figure 20: Boxplot representing the
distribution of mCherry under the control of P*lsr* fluorescence as a function
of UTR
strength using *E. coli* Nissle

Figure 20 shows a similar distribution of fluorescence values; using the *E. coli* Nissle
bacterial strain we do not notice a significant difference between mCherry expression with
the UTR
sequence high or low.
*E. coli* Nissle is not an optimized strain for protein production, unlike BL21 which may
explain the lack of expression difference between the two UTR sequences.

All the data obtained suggested that the system is functioning, we have a trigger for the expression
of P*lsr* depending on the *E. coli* population but it is necessary to control the
environment of the
culture. Perhaps conducting the culture in bioreactors could permit us to identify other parameters
that influence this mechanism. It is important to notice that the fluorescence monitoring was
stopped
after 10 hours as we could not do more but it would be interesting to see if the difference
between high and low becomes more pronounced over time.

### 2. *E. coli* lysis

For this part, which concerns the lysis itself, we were able to obtain only one among the ones we
had planned to do: construction with P*lsr*100%-*E7*
[BBa\_K3463025](http://parts.igem.org/Part%3ABBa_K3463025).
We succeeded in cloning the lysis gene with the *lsr*A promoter (results not shown).
Unfortunately, we couldn’t optimize the experiment with [BBa\_K3463025](http://parts.igem.org/Part%3ABBa_K3463025), but we were ready to change
strategies.
It is possible to boost the expression of P*lsrA* by different methods, in particular by
expressing the phosphorylated AI-2 degradation proteins as shown by the work of the [Diaz Z](https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0006820) team
[1].

[GO TO DESIGN](https://2020.igem.org/Team%3AGrenoble_Alpes/Design#deliverySystem)

[GO TO EXPERIMENTS](https://2020.igem.org/Team%3AGrenoble_Alpes/Experiments#deliverySystem)

![incubator schema](https://static.igem.org/mediawiki/2020/c/c6/T--Grenoble_Alpes--incubatorSchema.png)

![](https://static.igem.org/mediawiki/2020/c/c6/T--Grenoble_Alpes--incubatorSchema.png)

# Dry Lab Results

PyoBusters’ goal is to destroy the biofilm of *P. aeruginosa* which acts in
different environments. As engineers we wanted to provide the experimenters a way to
grow their biofilms and insert the modified *E. coli* in an automated experience. Hence
we
built an incubator called the Automated Measurement Incubator (AMI) which is built and
implemented in 4 parts:

* The environment module that controls and monitor the atmosphere inside the box.
* The agitation module that operates the plate movements.
* The fluorescent module that makes the analysis during the whole experiment.
* The software that controls the 3 previous modules and allows a Human Computer Interface.

# DryLab Introduction

The results presented here are associated with the incubator functions: atmosphere
controlling and growth bacteria inside our incubator. You will see that our device is able to
stabilise at extreme temperature and humidity values which means it is able to reach lower values.
Our detection module was finished when the laboratory time was over which means that no results can
be shown.

# Environment module

### 1. Monitoring of the temperature and humidity module

We did many experiments to understand and calibrate this module. Our module works on adapting the
heating power thanks to monitoring. To understand how temperature and humidity were evolving inside
our box we run different experiments. We first calibrated the PID coefficient to obtain a smooth
control, then we checked what was happening with disruptions and lastly we tried to see for how long
it could be working.

All the results on the environment module are done on the prototype that you can see on figure 21.
Explanations around our prototype are developed in the [engineering page](https://2020.igem.org/Team%3AGrenoble_Alpes/Engineering).

![box plot 1](https://static.igem.org/mediawiki/2020/c/c1/T--Grenoble_Alpes--firstPrototype.jpeg)

Figure 21: prototype for the
environment module experiences

#### Calibration of the PID coefficient

The PID coefficients are in total 3: Kp, Ki and Kd. Basically, the time to reach the desired
temperature depends on Kp but a final error and an overshoot are created, Ki lowers the final error
and Kp lowers the overshoot. Hence, you have to find which triplet gives you the evolution you want.
In our case, we want low overshoot and low final error because we precisely need to reach the
desired temperature.

To do so, we look at the datas of sensors 0 to 4 during 30 minutes. The desired temperature is 37°C
and the desired relative humidity is 100%. On the next graphes, you will see the typical shape of
our curves. Temperature rises sharply and stabilises most of the time with an overshoot. Humidity
rises and encounters a drop for a small time.

On the sensor number 0, you may see one or two vertical lines, this means that an error of lecture
has been detected and the software puts the value at 0. We had trouble with this one sensor and
later we replaced this sensor by a new one.

![graph with Kp=50, Ki=0, Kd=0](https://static.igem.org/mediawiki/2020/c/c7/T--Grenoble_Alpes--graph1_Kp50_Ki0_Kd0.png)

This experience tells us that it is possible to stabilize our box
at the
desired temperature and desired humidity with only Kp=50. It needs about 15 minutes to reach the
desired atmosphere and then it is stable. When you look around the plate (sensors number 0 and
1), you see a nice overshoot of about 1°K and after this overshoot there is a little slamp. It
is a secondary small oscillation that can be clearly checked on raw datas.

![graph with Kp=50, Ki=0.01, Kd=10](https://static.igem.org/mediawiki/2020/2/22/T--Grenoble_Alpes--graph2_Kp50_Ki001_Kd10.png)

This experience shows the impact of the three coefficients
together: oscillations are erased, the overshoot is lowered and temperature is stabilized
sooner than the previous experience. Both curves have the same shape, but the evolution is
smoothed. This triplet of coefficients are fitted for our prototype and now we will make
experiences with this PID coefficients. We calibrated our PID corrector.

#### Evolution inside the covered multiwell plate

The next step is to understand how temperature and humidity evolve inside the covered plate. Hence,
we inserted the sensor number 5 inside a plate and put the cover on. Sensors number 0, 1, 2, 3 and 4
are at the same position, so the sensors 0 and 1 are above the cover.

![graph with Kp=40, Ki=0.005, Kd=10](https://static.igem.org/mediawiki/2020/4/4b/T--Grenoble_Alpes--graph3_covered_plate.png)

Figure 22: results of the automation
of
the
environment module

On the figure 22, we used the sensor number 5 to monitor what is happening inside the
covered plate. Sensors number 0 to 4 measure the atmosphere in the very same way and you have the
same results even though we slightly changed the PID coefficients. This slight change shows that
there is a wide range of coefficient values for having a nice calibration.

Sensors number 5 measures the parameters inside the covered plate. It shows that
temperature and humidity slowly catch back the parameters of the atmosphere. It needs 30 minutes for
the wells to reach the temperature desired value and humidity was about 90%.
Thanks to this experiment, we can validate that our incubator fulfills its first goal, which is to
create the bacteria growth conditions even with the cover on.

#### Disruption experiments

The goal of this experience is to validate that our control works even with disruptions. It lasted 1
hour and 30 minutes (5400 seconds), the desired temperature was 37°C and the disruption was to open
the cover for 10 seconds. We did this disruption at 30 minutes, 40 minutes, 70 minutes and 80
minutes. Sensor number 5 is still inside the covered plate. The figure 23 shows the result of the
monitoring of this experience.
Sensor number 0 has been removed because it broke and so we adapted the software to do without its
datas.

![temperature evolution with perturbation](https://static.igem.org/mediawiki/2020/3/38/T--Grenoble_Alpes--graph4_disruptions.png)

Figure 23: Experiments with
disruptions

The first result is that temperature is nicely stabilised at 37°C and sometimes you see a sudden
drop that matches the time when the box was opened: the heated atmosphere inside the box was leaking
out and mixing with the external atmosphere at 26°C, which means our atmosphere was cooling. The
consequence is the presence of variations in the temperature curves. These variations are also
visible on the PWM graph (which is the heating resistance supply in percent). When temperature goes
down, the PWM goes up to compensate, this is clearly shown by the sharp peaks on this graph. It
takes around 5 minutes to our box to re-stabilise perfectly at the desired temperature.
You can see that the sensor number 5 inside the covered plate also receives the same variations and
stabilises the same way.

#### Discussions

An incubator should provide a closed and controlled atmosphere, work as long as you want and
stabilise quickly after you open the door for a little time. We made long experiences during which
we created disruptions at different moments. Results are that our prototype stabilises at the
desired temperature within 20 minutes, it adapts against disruptions and it can work for 12hours
long and even more.

You have to know that the perfect calibration depends on the atmosphere around the box and this
variable moves by only heating or refreshing around the box. It won’t have a significant impact on
the results of our prototype if it is a small variation around the overshoot.

To solve this issue, we thought of building a black box in wood around our final hardware. It has
both advantages of isolating from atmosphere variations for the environment results and isolating
from light for the fluorescent results.

You may have released that sometimes, humidity is not reaching 100% and is not homogeneous even
though every sensor has datas higher than 90%. More time or more atomizers are needed to achieve the
100% relative humidity. For our prototype, it can reach in 30 minutes a desired relative humidity of
95%. That means that it can reach any value between the initial value and 95%. To solve issues such
as failures or volume increasing, 3 atomizers have been prepared
for our hardware of 36L.

### 2. Temperature control and bacteria growth

The chamber where the biofilms and the bacteria have to be grown is heated by resistors and
monitored by microcontrollers. In order to validate this module and our construction, we carried out
three experiments with the assumption that both the prototype and the industrial incubator
ThermoFisher MaxQ 6000 present in the laboratory will induce the same bacteria growth.

To do so, the experiments were based on the culture of three different *E. coli* strains:
BL21, Nissle
and DH5 alpha grown in a Luria Broth medium. Each strain was placed in 8 wells of a 96 well plate as
well as a control solution which contains only the LB medium. We have used these bacteria strains
because they are the ones used by the team of biologists. The DH5 alpha are used for cloning, the
BL21 used for the production of proteins and the Nissle is the bacteria strain which will be used at
the end and will contain the entire biological system.

We prepared two identical plates, one for our prototype and the other for the industrial incubator.
After starting the experiment, we followed for ten hours the optical density of each well. For
bacteria growth, after 5 hours the growth isn’t as strong as at the beginning but it allowed us to
validate the performances of our prototype over a long period of time. Also the bacterial growth
reaches a plateau at a certain point of the development of the colonies due to the limiting access
of the cells to the nutrients. In my opinion, there is nothing strange in the curves you present
(except the drop of the OD of the BL21 at 9h, which might be due to an unpredicted event that
disturbed the growth of the bacteria).

The optical density monitoring of the plates was carried out by FLUOstar Optima from BMG LABTECH.

![bacteria population growth graph](https://static.igem.org/mediawiki/2020/7/79/T--Grenoble_Alpes--growth_results.png)

Figure 24. Evolution of the OD of
the 3 strains for 10 hours the 02/09/2020

To verify the correlation between the different evolution, we measured the distances between two
curves with the spearman’s correlation coefficient. The spearman coefficient is an indicator which
allows to compute a correlation coefficient for non linear correlation.
The results of the calculations made with Python are as follows:

* BL21 pearson's correlation coefficient = 0.97
* DH5 alpha pearson's correlation coefficient = 1.0
* ECN pearson's correlation coefficient = 0.99
* LB pearson's correlation coefficient = 0.43

Each time, the correlation coefficients for the three strains are above 0.97 and prove that the
evolution of the optical density is correlated in both incubators.

There is an exception, the correlation coefficient for the evolution of the Luria Broth medium is a
bit lower, this is due to a small difference (always below 0.07) between 2 and 6 hours after the
beginning of the experiment in our prototype but at the end, the OD values are equal (difference of
0.008). Plus, as there is no bacteria in the LB medium, the OD shouldn’t evolve and the small
evolution can be due to some small amounts of contamination, but the evolution isn’t significant
(<0.12).

Furthermore, the difference of growth between the LB and and the three other strains is
significant.It can be concluded that our incubator allows us to cultivate bacteria in the same way
as the industrial incubator.

[GO TO DESIGN](https://2020.igem.org/Team%3AGrenoble_Alpes/Design#automatedEnvironmentControl)

# References

Diaz Z, Xavier KB, Miller ST. The crystal structure of the *Escherichia coli* autoinducer-2 processing
protein LsrF. PLoS One. 2009;4(8):e6820. Published 2009 Aug 28.
[doi:10.1371/journal.pone.0006820](https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0006820)

### Team Grenoble\_Alpes

![](https://static.igem.org/mediawiki/2020/e/ea/T--Grenoble_Alpes--Logo_final_Grenoble.png)
![](https://static.igem.org/mediawiki/2020/7/7e/T--Grenoble_Alpes--Logo_UGAINP.png)

![](https://static.igem.org/mediawiki/2020/7/7a/T--Grenoble_Alpes--PyoBustersText.png)

### Contact

* #### Igem grenoble Place du Commandant Nal 38700, La Tronche, France
* #### igem.grenoble.2020@gmail.com

[![](https://static.igem.org/mediawiki/2020/4/41/T--Grenoble_Alpes--Logo_facebook.png)](https://www.facebook.com/igemfrancegrenoble)
[![](https://static.igem.org/mediawiki/2020/4/4d/T--Grenoble_Alpes--Logo_instagram.png)](https://www.instagram.com/igem_grenoble/)
[![](https://static.igem.org/mediawiki/2020/3/3a/T--Grenoble_Alpes--Logo_linkedIn.png)](https://www.linkedin.com/in/igem-grenoble/)
[![](https://static.igem.org/mediawiki/2020/6/62/T--Grenoble_Alpes--Logo_twitter.png)](https://twitter.com/igem_grenoble)

This wiki was completely designed and coded by the team members of the 2020 Grenoble\_Alpes' Team, all content on this wiki is available under the Creative Commons [Attribution 4.0](https://creativecommons.org/licenses/by/4.0/) license (or any later version).