Team:KU Leuven/Project/DataPage

Welcome to our data page! Here we will summarise everything we achieved this summer. Of course, if you want a more extensive explanation, please check out the corresponding wiki page.
Since the European Jamboree, we characterized our bricks further via Mass Spectrometry studies of MeS and quantitated the aphid response to the EBF producing BanAphids.

Our project aims to reduce aphid infestations and thus improve crop yields for the industrial end-user and the private customer. With an environmental project like ours, the importance of the computer and the feedback from our future end-users should not be underestimated. We adapted our project according to survey information and modelling results. Ultimately, we hope to reduce the costs of field tests via our in silico work.

First, we started our modelling on the cellular level. We must figure out the impact of E-β-farnesene and methyl salicylate production on E. coli. Thus, we performed a Flux Balance Analysis. Results were compared with wetlab data such as growth curves and GC-MS data. We also aimed to predict the exact amounts produced and find the rate limiting steps. Here we fed wetlab data into our algorithms. The outcome will define/defined our choice of promoters, plasmid copy number and additive requirements.

Secondly we did a lot of modelling on the colony level. We designed an oscillating transcription factor network to regulate pheromone production for the "sticker enclosed" BanAphids. This oscillator network allows communication between cells, enforcing a synchronised but oscillating production rhythm onto the whole colony. This will optimise the impact of our BanAphids on aphids and ladybugs even though a direct contact cue between aphids and BanAphids is prevented. We designed this model to answer the concerns of the private end-users regarding the spray (or honeydew) system.

Finally, we must know the effect of our pheromones on the ecosystem. We performed a series of modelling steps which you can find in our ecological model page. This information is essential in several ways :
It will define the choice of pheromone production rate, which we can regulate through eg. promoter ranges.
Dispersion data will indicate the optimal spacing of the BanAphid stickers, key information for the end-user.

Summarised, these algorithms allow us to model our system from the cellular metabolism throughout to the environmental impact. Based on our models, we continuously adapted the actual building of the system towards the most effective circuit. This will reduce costs and save time when our BanAphids are ready for field tests, and later for actual use.

Our wetlab work consists of 3 experimental parts :

1. The production and testing of the methyl salicylate bricks
2. The production and testing of the E-beta farnesene bricks
3. Ecological work, testing pheromone impact on the ecosystem (i.e. plants, ladybugs, ...). Here, we found industrial partners in the companies Biobest and pcfruit.

Methyl Salicylate Experiments

Throughout the summer, we made 4 different BioBricks involved in the production of methyl salicylate. After finding out that the MIT 2006 brick (BBa_J45700) only produced weak amounts of the wintergreen scent (MeS), we dove into the literature and discovered a possible lack of chorismate present in the bacteria. We tried to overcome this problem by overexpressing aroG in E. coli. In the literature, we found two mutations that could make DAHP synthase insensitive to allosteric inhibition. We succeeded in biobricking the normal aroG gene, which gives future teams the opportunity to introduce mutations themselves to overcome the chorismate problem. We characterised our bricks with a renewed smell test, GC and an SDS-PAGE analysis.

(E)-β-farnesene Experiments

After the whole summer's work, we finally made 5 BioBricks for this section. Our favourites are:

1. BBa_K1060002 contains an open reading frame that codes for (E)-β-farnesene synthase from Artemisia annua. The enzyme converts farnesyl diphosphate into E-β-farnesene. It was a milestone in our project work. We succeeded to remove an EcoRI site in the gene (AY835398.1). This gave us one of the basic parts we needed to create our system.
2. BBa_K1060009 is a construct that constitutively expresses β-farnesene synthase. This was the final device used for our Aphid experiments.
3. BBa_K1060011 is similar to BBa_K1060009. However, in this biobrick we added a lac operator in front of the β-farnesene synthase. This makes it possible to switch of (E)-β-farnesene production by using biosensors expressing LacI.

Our pilot studies with these biobricks and the aphids are promising and we now backed it up with quantitative data. Apart from this in vivo characterisation, we also initiated an SDS-PAGE analysis. Taken together this indicates that the EBF synthase is produced.

Ecosystem Experiments

Two companies (Biobest and pcfruit) specialised in biological pest management, were very interested in our project and invited us to perform experiments at their facilities. These experiments demonstrated the effect of MeS in inducing plant defence mechanisms and that this has an effect on the aphid population.

As a result of our unique collaboration between philosophers and scientists within our team, we formulated a new additional approach: Bottom-up or ethics from within. This approach improves the communication between different stakeholders and achieves reciprocal understanding in early stages of the project. From this approach the government, scientists, the public and even the industry can benefit. This new approach is bottom-up structured where the dialogue between philosophers, scientists and the general public is central. We have developed a new and strong framework for this approach in which we also explained why we should inform the general public using the ideas of philosopher Hannah Arendt and we examined the responsibility of synthetic biologists using the ideas of philosopher Hans Jonas. This is not only a first time in the iGEM competition that these well-known philosophers are read to provide a strong foundation, but has also never been done in the scientific literature!
In short, we consider this bottom-up collaboration between students a new starting point for approaching human practices within the iGEM competition.

Thanks to extensive dialogue we designed a product that interests both relevant industries like Biobest & pcfruit and the public. Furthermore, we also asked end users (farmers) if they would use our BanAphids and the results were very positive!

Our symposium for the general public was another opportunity for interaction with the public. We invited the other Benelux (Belgium, The Netherlands, Luxembourg) teams to present their project. We hope that this symposium will become a yearly tradition in the iGEM competition.

We also go the youth interested in synthetic biology! We went to high schools with our very own Plexiglas "biobricks" which the students can use to work get familiar with the BioBrick system and we made a 3D-bacterial model, which gives the students an idea of what a bacterium looks like.