Difference between revisions of "Team:Aix-Marseille/Results"

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{{Aix-Marseille|title=Results}} __NOTOC__
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Positive thoughts, positive actions and positive results. As a hard working team, we aimed high this year. We have generated results that met with the bronze, silver and gold medals requiremements. We were able to produce a functional endochitinase (Gold medal criterion), metabolic intermediates from the benzaldehyde/benzylalcohol biosynthesis pathway, and the DMTS/DMDS pheromones (Silver medal criterion) . We have successfully modeled the trap (Gold medal criterion) and tested it in infested premises. Furthermore, we have made collaborations (Silver medal criterion) with the iGEM committee, as well as with private companies. This sets a solid ground for further developments around the project. Last but not least, a nationwide advertisement campaign engaged the public in our adventure. We had a mutual interest: all bed bugs must come to an end.
  
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=Wetlab=
<h1>Results</h1>
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==[https://2018.igem.org/Team:Aix-Marseille/Experiments Endochitinase]==
<p>Here you can describe the results of your project and your future plans. </p>
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[[File:T--Aix-Marseille--(fourmis).jpg|250 px|right]]
</div>
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The endochitinase is a protein naturally produced by ''Beauveria bassiana'' an enthomopathogenic fungus. We wanted to overproduce it (in ''E. coli'') and extract it to enhance the killing efficieny and speed of the fungus propagation.
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We succesfully produced, and purified the functional endochitinase ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K2718022 BBa_K2718022]).
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This part is an improvement of the part [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1913000 BBa_K1913000] '''Gold medal criteria'''.
  
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==Pheromone production==
  
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The aim was to produce two types of pheromone: DMTS/DMDS and benzaldehyde/benzylalcohol. We did some  research on the two molecules to get the biosynthesis pathways to produce them in ''E. coli''. First, we needed to add the three enzymes implicated in the biosynthesis of benzaldehyde/benzylalcohol: MdlB, MdlC and Hmas. We successfully cloned the Hmas gene ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K2718011 BBa_K2718011]) and produced the equivalent protein. This enabled [https://2018.igem.org/Team:Aix-Marseille/Experiments the production of the first metabolic intermediate] (S-Mandelate) of the pathway.
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Second, we were able to clone the methionine-γ-lyase gene ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K2718006 BBa_K2718006]) and produce the equivalent protein. This enabled the achievement of a first goal: the production of the DMTS/DMDS pheromone. This part [http://parts.igem.org/wiki/index.php?title=Part:BBa_K2718006 BBa_K2718006] is our validated part '''Silver medal criteria'''
  
<h3>What should this page contain?</h3>
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==Trap tests==
<ul>
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A few weeks before the giant jamboree we have decided to go all the way with the breaking bugs project. We took a preliminary [https://2018.igem.org/Team:Aix-Marseille/Design trap design] to the field to test the efficiency of both units: ''Beauveria bassiana'' combined with the adjuvants, and the pheromones efficiency. In collaboration with local infested residences, we had the chance to demonstrate some functional aspects about the prototype.
<li> Clearly and objectively describe the results of your work.</li>
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<li> Future plans for the project. </li>
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<li> Considerations for replicating the experiments. </li>
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</ul>
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</div>
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==[https://2018.igem.org/Team:Aix-Marseille/Model Model]==
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[[File:T--Aix-Marseille--run bedbug 1.png|350 px|right]]
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We decided to model the deployment of our bed bug trap in a realistic environment, to understand how it was likely to work and what parameters might be important. This task involved a number of different modeling tasks, that posed different problems: modelling the diffusion of pheromones in the room coming both from a natural bed bug nest and the traps that we plan to deploy; modelling the movement of bed bugs influenced both by the pheromone field and their nest; and modeling the fungal epidemic that we plan to induce in the bed bug population.  Though the model is complex and includes many parameters, it has already allowed us to draw several conclusions, and as the model is improved and the parameters are refined other conclusions will follow.
  
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For instance, using realistic diffusion parameters it is clear that the pheromone field is relatively rapidly established and so the precise nature and concentration of pheromones is probably not critical. In contrast, the delay between infection and death of bed bugs is critical for ensuring the eradication of the nest. Our modeling thus helps understand critical aspects of the proposed design. This is a '''Gold medal criteria'''.
  
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==[https://2018.igem.org/Team:Aix-Marseille/Interlab Interlab]==
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The InterLab study has been developing a robust measurement procedure for green fluorescent protein (GFP) over the last several years. GFP was chosen as the measurement marker for this study since it's one of the most used markers in synthetic biology and, as a result, most laboratories are equipped to measure this protein. This year, we helped in answering the following question: Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?
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We participated in this '''Bronze medal criteria'''
  
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==[https://2018.igem.org/Team:Aix-Marseille/Perspectives Perspectives]==
<h3>Describe what your results mean </h3>
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The nationwide success of our approach to treat bed bugs has encouraged us to elaborate perspectives for long term development. We are collaborating with multiple potential investors we have met at the [https://2018.igem.org/Team:Aix-Marseille/SHUC social housing union convention] and [https://contact.rentokil.fr/fr-deratisation-v3/?utm_term=rentokil&gclid=CjwKCAjwu5veBRBBEiwAFTqDwYY8qKmbHDOWKutmcTcQWmW9qgyysHT0DWQj_fK3BRepTRwrgH8MIBoCjJQQAvD_BwE Rentokil] for further research and development around our plan.
<ul>
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<li> 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. </li>
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<li> Show data, but remember all measurement and characterization data must be on part pages in the Registry. </li>
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<li> 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. </li>
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</ul>
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</div>
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==[https://2018.igem.org/Team:Aix-Marseille/Collaborations Collaborations]==
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Connecting with other iGEM teams from all around the world was the power engine of our project. We managed to make collaborations on many levels. We helped the Mexican team with their project's advancement, the German team with their awareness campaign and the Chinese team with their international language project. Additionnally, we organized the 2nd edition of the Mediterranean meetup to help the fellow iGEMers prepare for the giant jamboree. This is a '''Silver Medal criteria'''
  
<div class="clear extra_space"></div>
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==[https://2018.igem.org/Team:Aix-Marseille/Human_Practices Human Practices]==
  
 
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The public engagement was the pillar of the breaking bugs project. After a succesfull nationwide adverstisement campaign, we were able to engage the french and international commitees in our plan. We had a solid validation by a massive waves of donations for our crowfunding campaign.
 
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Our human practices and public engagement was very strong, completing a '''Silver medal criteria''', and we integrated our human practices work into the project design a '''Gold medal criteria'''.
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<h3> Project Achievements </h3>
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<p>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.</p>
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<li>A list of linked bullet points of the successful results during your project</li>
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<li>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.</li>
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<div class="highlight decoration_A_full">
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<h3>Inspiration</h3>
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<p>See how other teams presented their results.</p>
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<ul>
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<li><a href="https://2014.igem.org/Team:TU_Darmstadt/Results/Pathway">2014 TU Darmstadt </a></li>
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<li><a href="https://2014.igem.org/Team:Imperial/Results">2014 Imperial </a></li>
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<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Results">2014 Paris Bettencourt </a></li>
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</ul>
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</div>
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</html>
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= Results =
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==CHITINASE==
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=== Chitinase production:===
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We achieve to produce chitinase thanks to our construction. An inducible overproduction by pLac promoter was used to study chitinase production, induction or none-induction experiments by IPTG can show the presence of the protein. We choose to produce chitinase by E. coli K-12 DH5-alpha strain '''''(and by BL21 strain ?)'''''
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For this two strains two differents recombinated plasmids with chit1 were tested, plasmid N°10 and N°11.
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[[File:T--Aix-Marseille--ChitinaseResults1.jpg|600px|center|]]
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Figure 1: Figure 1: SDS-PAGE of chitinase produced by E. coli K-12 DH5-alpha.
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As seen in Figure 1, a protein at  the expected lenght (30 kDa) was overproduced only at 30°C after IPTG induction in DH5alpha. The non-induction by IPTG show an low band of the protein overproduced. This overproduction should be the chitinase, but it isn't normal that clone N°11 wasn't able to produced at least a significant chitinase quantity at 16°C and 37°C. We clarified that thanks to a western blot, to be sure of chitinase expression by clone N°11.
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Chitinase output are better after IPTG induction at 30°C. This condition of culture will be used to produce in large quantity chitinase for purification and activity tests.
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'''''(put file of western and blue of BL21 or not?)'''''
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=== Chitinase purification:===
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Chitinase designed had a polyhistidine-tag which allows it to be specifically separated among total proteins. A Akta purification was realized for purify Chit1. Akta Pure is a automated chromatography system for quick and easy purification. A nickel-nitrilotriacetic acid column was necessary to allow specifical interaction between histidines residues and metal ions  from the nickel-nitrilotriacetic acid matrix.
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[[File:T--Aix-Marseille--ChitinaseResults2.jpg|600px|center|]]
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Figure 2: SDS-PAGE of  polyhistidine-tagged chitinase purification produced by E. coli K-12 DH5-alpha, using a nickel-nitrilotriacetic acid matrix.
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Showed in figure 2, a protein at 30 kDa corresponding at the chitinase is obtained on bacteria lysate, on the non-purified fraction and on fraction N°6 to fraction N°10. This fractions can be pooled and used for activity tests.
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Numerous controls has been done,chitinase was produced without its peptide signal of secretion, so it will be contained in the cytoplasm,  the absence of the band at 30 kDa in the pellet verify that chitinase wasn't contained in the membrane.
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Bacteria lysate confirmed the presence of chitinase at 30kDa; non-purified migration shows a low quantity of chitinase with all proteins contaminants; break-though sample didn't contain the protein target demonstrating that all chitinase was fixed on nickel-nitrilotriacetic acid matrix.
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The results of chitinase purification prove that chitinase can be purified thanks to its polyhistidine-tag. But purification wasn't optimal and can be ameliorated because a lot of contaminants are isolated with chitinase. The nickel-nitrilotriacetic acid matrix column used was well-worn, that can reduced its efficiency. For an optimal separation of chitinase, we should pool eluate fractions containing chitinase and purify until reduce significantly protein contaminants. But without time we continued with this purification to do activity tests.
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=== Chitinase activity test:===
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Schales' procedure was used to test the chitinase activity. Schales' procedure is a colorimetric method commoly used for measuring the reducing sugars.
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Chitin is composed of sugar units of either N-acetyl-G-glucosamine. Chitinase degrade chitin producing N-acetyl-G-glucosamine. To monitor chitinase activity, the reducing sugars released after chitin hydrolyse are detected by a color diminution of the yellow Shales' reagent. This color diminution is translated by a absorbance diminution.
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To analyse activity test results, an etalon curve have been done using the final product of chitinase reaction (N-acetyl-G-glucosamine), to know in which concentration of final product must be obtained after reaction to be detected by the method of Schales.
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[[File:T--Aix-Marseille--ChitinaseResults3.jpg|600px|center|]]
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Figure 3: Etalon curve of N-acetyl-G-glucosamine after Schales's activity test.
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A final curve has been obtain showing a absorbance diminution in function of increasing N-acetyl-G-glucosamine concentration. With this etalon curve, concentration of final product obtain after chitinase reaction can be determined. This results revealed the efficiency of Schales'procedure to detected N-acetyl-G-glucosamine to 0 at 200 uM.
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[[File:T--Aix-Marseille--ChitinaseResults9.jpg|600px|center|]]
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Figure 4: Table of absorbance measurements of chitinase activity test
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[[File:T--Aix-Marseille--ChitinaseResults4.jpg|600px|center|]]
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Figure 5: Chitinase activity test by Schales' procedure
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According to figure 4, after 30 minutes of reaction a significant activity was mesured. But after 1 hour of reaction the standart deviation put in doubt the significant result at 30 minutes. One of the difficulties encountered was the chitin sample. Chitin sample wasn't completely dissolved in water, it was precipited and a lot of big fragments af shells of shrimps was contained making difficult the pippetage and with difficulty reproducibility. That's why an centrifugation step was added to avoid precipitated chitin to asborbe and affects the results. After centrifugation chitin wasn't really pelleted, it was easily removed. Chitin pellet should be removed in the duplicated sample of 1 hour after reaction, that can be the cause of the high standart deviation in the results. Due to limited time, we weren't able to do another activity test to improve the resutls and be the most careful at removing and may be we should centrigutated a longer time (not 1 minute but 5 minutes maybe).
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Thanks to etalon curve if we consider only the significant results at 30 minutes, we can conclude that after 30 minutes of enzymatic reaction, our chitinase can released 125 uM of N-acetyl-G-glucosamine.
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[[File:T--Aix-Marseille--ChitinaseResults10.jpg|600px|center|]]
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Figure 6: Table of measurements of controls for chitinase activity test
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[[File:T--Aix-Marseille--ChitinaseResults5.jpg|600px|center|]]
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Figure 7: Controls for chitinase activity test
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==HMAS==
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=== 4-hydroxyphenylpyruvate synthase activity test ===
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After inserting the p_lac-RBS-hmaS construct into the psb1c3 plasmid [http://parts.igem.org/wiki/index.php?title=Part:BBa_K2718011] and getting our 4-hydroxyphenylpyruvate synthase production and purification results, we were wondered if our protein was functional. In order to test the 4-hydroxyphenylpyruvate synthase activity in our E.coli strain, we have made an HPLC test on supernatent cells as described in our protocols tab : [https://2018.igem.org/Team:Aix-Marseille/Protocols]
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Latest revision as of 02:00, 18 October 2018

Results

Positive thoughts, positive actions and positive results. As a hard working team, we aimed high this year. We have generated results that met with the bronze, silver and gold medals requiremements. We were able to produce a functional endochitinase (Gold medal criterion), metabolic intermediates from the benzaldehyde/benzylalcohol biosynthesis pathway, and the DMTS/DMDS pheromones (Silver medal criterion) . We have successfully modeled the trap (Gold medal criterion) and tested it in infested premises. Furthermore, we have made collaborations (Silver medal criterion) with the iGEM committee, as well as with private companies. This sets a solid ground for further developments around the project. Last but not least, a nationwide advertisement campaign engaged the public in our adventure. We had a mutual interest: all bed bugs must come to an end.

Wetlab

Endochitinase

T--Aix-Marseille--(fourmis).jpg

The endochitinase is a protein naturally produced by Beauveria bassiana an enthomopathogenic fungus. We wanted to overproduce it (in E. coli) and extract it to enhance the killing efficieny and speed of the fungus propagation. We succesfully produced, and purified the functional endochitinase (BBa_K2718022). This part is an improvement of the part BBa_K1913000 Gold medal criteria.

Pheromone production

The aim was to produce two types of pheromone: DMTS/DMDS and benzaldehyde/benzylalcohol. We did some research on the two molecules to get the biosynthesis pathways to produce them in E. coli. First, we needed to add the three enzymes implicated in the biosynthesis of benzaldehyde/benzylalcohol: MdlB, MdlC and Hmas. We successfully cloned the Hmas gene (BBa_K2718011) and produced the equivalent protein. This enabled the production of the first metabolic intermediate (S-Mandelate) of the pathway. Second, we were able to clone the methionine-γ-lyase gene (BBa_K2718006) and produce the equivalent protein. This enabled the achievement of a first goal: the production of the DMTS/DMDS pheromone. This part BBa_K2718006 is our validated part Silver medal criteria

Trap tests

A few weeks before the giant jamboree we have decided to go all the way with the breaking bugs project. We took a preliminary trap design to the field to test the efficiency of both units: Beauveria bassiana combined with the adjuvants, and the pheromones efficiency. In collaboration with local infested residences, we had the chance to demonstrate some functional aspects about the prototype.

Model

T--Aix-Marseille--run bedbug 1.png

We decided to model the deployment of our bed bug trap in a realistic environment, to understand how it was likely to work and what parameters might be important. This task involved a number of different modeling tasks, that posed different problems: modelling the diffusion of pheromones in the room coming both from a natural bed bug nest and the traps that we plan to deploy; modelling the movement of bed bugs influenced both by the pheromone field and their nest; and modeling the fungal epidemic that we plan to induce in the bed bug population. Though the model is complex and includes many parameters, it has already allowed us to draw several conclusions, and as the model is improved and the parameters are refined other conclusions will follow.

For instance, using realistic diffusion parameters it is clear that the pheromone field is relatively rapidly established and so the precise nature and concentration of pheromones is probably not critical. In contrast, the delay between infection and death of bed bugs is critical for ensuring the eradication of the nest. Our modeling thus helps understand critical aspects of the proposed design. This is a Gold medal criteria.

Interlab

The InterLab study has been developing a robust measurement procedure for green fluorescent protein (GFP) over the last several years. GFP was chosen as the measurement marker for this study since it's one of the most used markers in synthetic biology and, as a result, most laboratories are equipped to measure this protein. This year, we helped in answering the following question: Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD? We participated in this Bronze medal criteria

Perspectives

The nationwide success of our approach to treat bed bugs has encouraged us to elaborate perspectives for long term development. We are collaborating with multiple potential investors we have met at the social housing union convention and Rentokil for further research and development around our plan.

Collaborations

Connecting with other iGEM teams from all around the world was the power engine of our project. We managed to make collaborations on many levels. We helped the Mexican team with their project's advancement, the German team with their awareness campaign and the Chinese team with their international language project. Additionnally, we organized the 2nd edition of the Mediterranean meetup to help the fellow iGEMers prepare for the giant jamboree. This is a Silver Medal criteria

Human Practices

The public engagement was the pillar of the breaking bugs project. After a succesfull nationwide adverstisement campaign, we were able to engage the french and international commitees in our plan. We had a solid validation by a massive waves of donations for our crowfunding campaign. Our human practices and public engagement was very strong, completing a Silver medal criteria, and we integrated our human practices work into the project design a Gold medal criteria.