Global level contribution

We as a group were particularly troubled by the issue of global inequity in the distribution of resources. We thus felt the need to initiate bridging the enormous gap between developing and developed nations. More precisely, we learned from the literature that resistant tomato varieties, that are immune to TSWV, are imported, particularly in nations with powerful economies. Unfortunately, the virus has a high spreading rate which enables it to be transmitted, endangering even wild ecosystems, while the destruction of crops by viral phytopathogens plagues nations that are strongly affected by poverty. All this could mean detrimental results for biodiversity. Our diagnostic tool might thus rescue these crops and spare millions of people from the tragedy of the food crisis in regions where farmers are unable to afford to sustain resistant crops.

Local level contribution

Greece, a nation that relies heavily on tourism, has suffered severe economic setbacks in recent years, particularly after the awful blow of Covid-19, which had a huge impact on the general economy and also on the agriculture industry. What’s more, tomatoes are widely consumed in Greece as they are a main ingredient of many foods of the Mediterranean diet, consequently, they are cultivated in many fields, gardens and greenhouses across the country, and especially in Crete. Thus, even if the introduction of resistant variants has been relatively active up until now, the financial recession prevented an increasing number of farmers from raising such varieties.

This is where our project provides a solution for farmers who have suffered a financial setback and are unable to invest in resistant varieties, allowing them to grow wild type tomatoes while still being able to identify an infection efficiently and precisely. Its low cost makes it appealing to even the smallest, part time cultivator, who grows tomatoes for his family in his garden.

As we mention in our implementation proposal, the time-consuming and expensive procedure of transporting the sample to the laboratory is also avoided. Reducing the time needed to diagnose an infected tomato can be vital for the rest of the field and neighboring fields. The more tomatoes we save, the better the production and the profit will be. In order to create breeding ground and make the above happen, we directed our efforts in educating local farmers about agrodiagnostics and raising awareness about tospoviruses and the food crisis. They were amazed by how the utilization of our project will shield them from the economic detrimmentation that TSWV and economic crises co-created. We sincerely hope that we have actively contributed in paving the way for a new era of diagnostics utilizing LRRs. More details about our wet lab work are analyzed at the last paragraph.

iGEM community contribution

Overall we wanted to embrace the goals of the competition and make them our own.

We encouraged not only high school students but also university students and teachers to familiarize themselves with the spirit of the competition and to reflect on what an extraordinary life experience their participation in the competition would be. In addition, as we were incredibly excited to interact with so many other iGEMTeams, we participated in a great number of collaborations, which excited us so much that we decided to establish a long-term and meaningful collaboration, which later evolved into a partnership, with iGEM Thessaloniki. In this way, we aspire to have done our bit to promote the spirit of mutual support which is one of the main aims of the competition. Also, through our constant communication with the public, we tried to make synthetic biology the property of all and to spread the message that scientific progress is not dangerous when it is accompanied by pure purpose.

Moving on our team faced a plethora of problems during the initiation and development of our project. Therefore we will summarize some key problems we faced, that may potentially help future iGEM teams overcome them.

  • During the initial brainstorming sessions we considered using LRR domains from VLR-B molecules, which originated from lampreys. VLR-B molecules are responsible for the identification of epitopes and antigens in lamprey's innate immunity. This idea brought some obstacles to the surface, given the fact that we wanted to keep our project bioethical and simultaneously free of any safety concerns.

    • Lampreys are critically endangered in Greece, so we decided to find an alternative to avoid immunizing lampreys and extract the lymphocytes to obtain the VLR-B molecules. We searched in bibliography and we found some articles that referred to experiments with a VLR library including all of lampreys’s VLR-B molecules. Unfortunately we couldn’t find a way to obtain this library.
    • Furthermore there was a lack of resources in our workplace to accomplish FACS and MACS experiments which were necessary for the experimental procedures.

  • To overcome these difficulties we concluded using LRR domains isolated from Solanum Peruvianum and specifically the LRR and the NB-LRR from the Sw-5b resistance gene. This gene confers resistance against some tospoviruses such as Tomato Spotted Wilt Virus (TSWV).

  • The first idea was to surface display the LRR from Sw-5b on E.coli cells by fusing it with the Fc domain of an IgG antibody. Sadly we couldn’t obtain a vector containing this construct in time. So the concept of the project ended up into using a surface display protein included in iGEM 2022 Distribution Kit.

    • The first idea was to surface display the LRR from Sw-5b on E.coli cells by fusing it with the Fc domain of an IgG antibody. Sadly we couldn’t obtain a vector containing this construct in time. So the concept of the project ended up into using a surface display protein included in iGEM 2022 Distribution Kit.

    • The last thing we expected was for the Distribution Kit to be stopped at the customs office in Greece and returned back to iGEM HQ. In this rampage of bad luck we had to end up with a new alternative, so we decided to use an OmpA surface display protein for E.colicells included in the 2021 iGEM Distribution Kit.

    • Unfortunately, the 2021 iGEM Distribution Kit we had was stored for one year in room temperature as the DNA parts into the wells had creosol red dye which stabilized them in room temperature. To be completely sure that the transformation of our part of interest will be a success, we tried several other parts included in the kit first. Failure after failure while checking all of the parameters for the transformation we drew the conclusion that the problem was the DNA in the kit. We had only one chance for our part of interest, so we decided to isolate it using PCR, without trying to first transform competent E.coli cells and this was finally a huge success.

For this year’s iGEM competition we not only registered new parts, but also improved a part already registered from previous iGEMers. 

The basic parts we registered include:

  • LRR Sw-5b: This part is a gene which expresses the LRR domain of the Sw5b resistance gene of Solanum Peruvianum and naturally gives resistance against some tospoviruses.
  • NB-LRR Sw-5b: This is a part that is basically the LRR with both the NB domain. This part is enrolled in the same defense mechanism of Solanum Peruvianum as the previous part.
  • Nsm115-135: It’s a viral protein that during infection contributes to viral cell to cell movement and translocates to nearby cells before other viral components reach them. It is a gene sequence from several tospovirus species.

The part we modified is BBa_K1362060 registered by iGEM Heidelberg in iGEM 2014 competition and it is the following:

  • OmpA Surface Display Protein: It is a bacterial protein that is used to surface display any protein of interest when N-terminally fused in E.coli. Our protein is a modified version of BBa_K1362060 with an extra nucleotide that ensures that our fusion partner remains in frame.

Lastly we also registered a couple of composite parts:

  • OmpA:NB-LRR: Which is a fusion of OmpA surface display protein and the NB-LRR
  • OmpA:LRR: This also our OmpA surface displayed protein fused with the LRR
Type Part Name Kind Description Length
Basic BBa_K4495000 Coding NB-LRR Sw-5b 2190
Basic BBa_K4495001 Coding LRR Sw-5b 1089
Basic BBa_K4495002 Coding Nsm115-135 66
Basic BBa_K4495003 Coding OmpA Surface Display Protein 456
Composite BBa_K4495004 Coding OmpA:NB-LRR 2652
Composite BBa_K4495005 Coding OmpA:LRR 1553

Tips for future iGEM Teams

At the beginning when the group was created, we were all very happy that this year we would be able to spend quality time as a group, since most of the measures for the pandemic had already been lifted. Thus, we prioritized the solid foundation of our relationship, setting two weekly meetings. In these meetings we brainstormed for our future work, but we also exchanged our news and generally bonded as this is the most important stage for a successful and efficient teamwork. Specifically, at the HP level, we sought for an external unofficial ‘‘judge’’ to evaluate our work. Attending a series of workshops held by the iGEM community aided us to a great extent to achieve this goal. We were trained both in the approach to properly reach all age groups and in setting our future goals in order to assist the world overcome its fear towards an innovative, unknown method of diagnosis which does not use antibodies.

Regarding our fund team’s experience, we would advise future teams to start looking for sponsors early in the competition, offering them a simple but comprehensive update of the project so far. The consistent availability and immediate responses build a trustworthy image of the team to the outside world.

Regarding our wet lab’s experience, we acquired knowledge and a better understanding about the competition and the experimental procedures. Thus, we would like to advise future iGEM teams on some more of the issues we encountered. Firstly, it would be helpful to set a common course and goal that resonates with the whole team and try to stay on track throughout the competition. During brainstorming we found it useful to research separately different project ideas and present them to the team for critical suggestions and improvements.

After we concluded our final project, the wet lab members scheduled weekly meetings to assess and check on the progress on individual tasks assigned to each member. Before starting your project, be certain to obtain your iGEM distribution kits, even if you think that they contain parts that are not useful for your experiments. It is possible that you will need to adjust your protocols if something does not work. Overall the innovative idea of ​​introducing a new diagnostic method to the scientific community required a thorough literature search, as well as a long-term plan of the experimental procedure that we will pursue.

Finally, without a comprehensive and well-designed wiki, none of the aforementioned would be available to the public and future iGEM teams. According to the dry lab, the wiki page was implemented utilizing the new Astro javascript framework, which creates faster websites withnext-generation island architecture. The created wiki complies with the requirements of a well-designed responsive website for mobile and tablet devices. Lastly, the written code and design can be applied to the creation of the following wikis.

We hope that the future iGEM teams will benefit from our mishaps and proceed to a smoother project execution!