iGEM 2021 — Team Aix-Marseille

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# Team:Aix-Marseille/Notebook

# Notebook

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

### What should this page have?

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

### Inspiration

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

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Aix-Marseille/Engineering

[![logo](https://static.igem.org/mediawiki/2021/9/90/T--Aix-Marseille--ARBO-BLOCK-logo.png)](https://2021.igem.org/Team%3AAix-Marseille)

* [Home](https://2021.igem.org/Team%3AAix-Marseille)
* Team
  + [Team members](https://2021.igem.org/Team%3AAix-Marseille/team-members)
  + [Advisors](https://2021.igem.org/Team%3AAix-Marseille/advisors)
  + [Instructors](https://2021.igem.org/Team%3AAix-Marseille/instructors)
* Project
  + [Inspirations](https://2021.igem.org/Team%3AAix-Marseille/Description)
  + [Design](https://2021.igem.org/Team%3AAix-Marseille/design)
  + [Experiments](https://2021.igem.org/Team%3AAix-Marseille/experiments)
  + [Engineering](https://2021.igem.org/Team%3AAix-Marseille/Engineering)
  + [Measurements](https://2021.igem.org/Team%3AAix-Marseille/Measurement)
  + [Implementation](https://2021.igem.org/Team%3AAix-Marseille/Implementation)
  + [Protocols](https://2021.igem.org/Team%3AAix-Marseille/protocols)
  + [Notebook](https://2021.igem.org/Team%3AAix-Marseille/notebook)
* Parts
  + [Primers](https://2021.igem.org/Team%3AAix-Marseille/primers)
  + [Part collection](https://2021.igem.org/Team%3AAix-Marseille/part-collection)
  + [Contribution](https://2021.igem.org/Team%3AAix-Marseille/Contribution)
* Human practices
  + [Human practice](https://2021.igem.org/Team%3AAix-Marseille/Human_Practices)
  + [Integrated Human Practices](https://2021.igem.org/Team%3AAix-Marseille/integrated-human-practices)
  + [Collaborations](https://2021.igem.org/Team%3AAix-Marseille/Collaborations)
  + [Events](https://2021.igem.org/Team%3AAix-Marseille/events)
  + [Safety](https://2021.igem.org/Team%3AAix-Marseille/Safety)
* Acknowledgments
  + [Attribution](https://2021.igem.org/Team%3AAix-Marseille/Attributions)
  + [Sponsors](https://2021.igem.org/Team%3AAix-Marseille/sponsors)

<!DOCTYPE html>

Site Igem

# Engineering

---

During our project, we fully experienced the concept of the engineering cycle.

In order to make our **design** and the entire ARBO-BLOCK project evolve, we **tested** each step of our thinking and **analysing** our results was necessary to **rethink** our first idea.

 Here, we will describe an example of how we worked with this concept. Indeed, it was especially applied during the **construction of our bacterial timer lysis device** (permitting Cry11Aa et Cyt1Aa toxins production, toxins release through cell lysis using Cal).

## **Design 1**

The pRL1 plasmid used to design our lysis device already contained the *lexA* promoter and coding sequences for ColA toxin, Cai immunity protein and Cal lysis protein (Figure 1).

 First, to assess the correct construction of the plasmid, the coding sequence for a GFP protein was inserted in the *caa* sequence (encoding for ColA). This allowed us to measure GFP fluorescence to quantify the toxin expression, as *gfp* gene is under the control of *caa* promoter. If the toxin is released in the extracellular medium after lysis of the bacteria by Cal, it will be possible to detect it and to validate the asynchronous lysis/toxin system. Theoretically, the lysis protein is supposed to be produced exponentially 7 hours after induction, permitting cell membrane breakage and release of the cytosolic compounds into the extracellular medium.

Three constructions were tested in this experiment: pRL1 wild-type (WT) (positive control), a mutant version of pRL1 containing the *gfp* sequence (pRL1-GFP) and a mutant version of pRL1 containing the *rfp* coding sequence fused to the *cal* sequence (negative control, pRL1-RFP) (Figure 2). With the latter construction no cell lysis should occur, and the GFP fluorescence should be present in the bacterium. For each experiment, increasing doses of mitomycin C were used.

![](https://static.igem.org/mediawiki/2021/c/cc/T--Aix-Marseille--Engineering-Fig1.png)***Figure 1: Operonic structure and regulation of the colicin A system.** Caa:* colicinA *gene; Cai: immunity protein caI gene; Cal: lysis protein.*

![](https://static.igem.org/mediawiki/2021/c/cc/T--Aix-Marseille--Engineering-Fig2.jpg)

***Figure 2: Scheme of the first design of the pRL1 plasmid containing the gfp coding sequence and the design of the rfp-cal negative control.***

## **Testing 1**

This first designed pRL1 plasmid was tested in *E. coli* cells. As the *lexA* promoter is commonly activated by stress stimuli in nature, mitomycin C induction was used to mimic this phenomenon. Indeed, this chemical is known to induce DNA mutations, which triggered the *lexA* promoter. Due to bacterial lysis by Cal, the toxin is detectable in the extracellular medium by Optic Density measurement using TECAN methods. The three previously described construction were tested.

## **Analysis 1**

GFP is used in our design as a reporter gene, the production of ColA is therefore followed using fluorescence intensity measurements. The production of lysis proteins induce the lysis of the bacterium, which leads to release GFP in the extracellular medium.

After induction with mitomycin C, WT cultures show a decrease in OD600 (Optical Density at 600nm) signal when high concentrations of the inducer are added to the culture. Indeed, when *lexA* is induced in increasing levels of mitomycin C, cell lysis is observed. When the induction of cultures containing pRL1-RFP is done, the GFP signal does not decrease, which means that cell lysis does not occur, as we expected. For the engineered plasmid with the *gfp* sequence, the same trend is observed as the negative control. The result is not the one expected. Indeed, a decrease in the OD600 signal, reflecting an effective lysis of the bacterial cell, should have been detected. In all 3 cases, as GFP is detected, the toxin is produced.

**Hypothesis: While inserting *gfp* coding sequence in the system, the regulation of *cal* expression could have been altered.**

## **Design 2**

Given the previous results, another plasmid was constructed according to our hypothesis (Figure 3).

For this second design, a longer *caa* sequence was cloned in the pRL1 plasmid. An alternative *lexA* box was also added in this sequence. This box is naturally present within the *caa* sequence. However, when the *gfp* sequence was cloned in the *caa* gene, it seemed to alter the expression of the alternative *lexA* box, potentially leading to no transcription of the lysis gene, *cal*. In this new construct, *caa* sequence is still implemented with the *gfp* sequence as shown on the figure below, but the alternative *lexA* box is kept in the sequence encoding for the toxin.

With this second engineered plasmid, we expect to induce Cal production as well as ColA. The latter should be released into the extracellular medium.

![](https://static.igem.org/mediawiki/2021/6/60/T--Aix-Marseille--Engineering-Fig3.png)

***Figure 3: Scheme of the second design of the pRL1 plasmid containing the gfp coding sequence.***

## **Future experiments**

With this experiment, we have been able to execute the engineering cycle at least one time.

In future experiments, our 2nd design should be tested and analyzed. If the latter is successful, it would be more than interesting to replace the *colA* sequence by the sequences encoding for Cry11Aa, Cyt1Aa and P20. In order to observe and quantify if toxins are well-produced, fluorescent coding sequences will be fused to each toxin coding sequence. This will allow us to compare toxins production.

If this 3rd design is successful, reporter genes could be deleted and plasmids could be tested in vivo in *E. coli* cells or even in *Asaia sp* cells.

Finally, if the expected results are obtained, it means that the engineered system can be applied to the bacterium of interest, and it can be tested on mosquitoes.

[![](https://static.igem.org/mediawiki/2021/7/72/T--Aix-Marseille--Attribution4.png)](https://www.instagram.com/igemaixmarseille/)

[![](https://static.igem.org/mediawiki/2021/3/35/T--Aix-Marseille--Attribution5.png)](https://www.linkedin.com/in/igem-aix-marseille-a92480161/)

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[![](https://static.igem.org/mediawiki/2021/e/ef/T--Aix-Marseille--Attribution6.png)](https://twitter.com/iGEMteamAMU)

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IGEM Aix-Marseille 2021

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# Team:Aix-Marseille/Experiments

# Experiments

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

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

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Aix-Marseille/Results

# Results

You can describe the results of your project and your future plans here.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)