Consultation with Dr. Claes von Wachenfeldt

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Background & What we learned

In order to deepen our knowledge of utilising Bacillus subtilis as a protein expression host, we consulted Dr. Claes von Wachenfeldt – Senior researcher in Molecular Biology at Lund University. With high level expertise in Bacillus subtilis, he made us aware of differences in protein expression compared to commonly used E. coli. The potential of protein secretion, chromosomal integration as well the organisms spore forming ability were pointed out by him and discussed together for possible implementation within our project. Furthermore, he shared hand-on-hand experience including protocols for different molecular methods. Additionally, biosafety concerns around this class of organisms were evaluated. From April 2022 and onwards we have been in contact for his help.

How did it influence our project?

After discussing important properties required on a good expression or integration vector, Dr. von Wachenfeldt provided us with his optimised integrative pCW101-vector and low-copy shuttle vector pCW7.

From our literature search, different constitutive promoters were identified for high-level protein expression in Bacillus subtilis. Few examples are P₄₃⁶ and P_veg⁷ , the former being highly considered for our project. Within his own research, ladder promoter however has been proven to result in higher levels of protein production compared to P₄₃. Until now, no direct comparison of promoter strength has been examined. Therefore we based our decision on Dr. von Wachenfeldt's practical experience leading us to introduce P_veg instead of P₄₃.

Restriction and ligation was the method of choice for our cloning approach. The choice of restriction enzyme followed random selection of restriction sites based on presence inside the vector. Here, 5’-HindIII and 3’-XbalI were considered for cloning into pCW7. After consultation with Dr. von Wachenfeldt, he pointed out that digestion with XbalI strongly reduces restriction efficiency of the second restriction enzyme and therefore should be avoided. Based on his experience, we introduced SalI at the insert C-terminus instead.

The provided vectors carried the ability to replicate in E. coli. Pointing out the limited transformation efficiency by direct introduction of target plasmids into Bacillus, we adapted our strategy by introducing an additional replication step in E. coli.

The choice of transformation method can strongly influence the overall efficiency. Heat-shock and electroporation are commonly used in E. coli to introduce foreign DNA into the cells. However, B. subtilis being a gram-positive bacteria might not respond to the same methods. Transformation based on natural induced competence can be used as an alternative strategy. Dr. von Wachenfeldt confirmed with us that using Bacillus natural competence carries the highest potential for our own transformation success. Therefore, we decided to focus on this approach. Further, he provided us with his own established protocols.

Wiki Help from iGEM SDU 2020

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Background & What we learned

In April of 2022, Before the wiki work began, our wikimaster, Nils, did a bit of research on how successful iGEM wikis have been made in the past. A team that stood out was the Danish Southern University of Denmark (SDU) team from 2021. Nils E-mailed them asking for advice and a very productive dialogue followed from the 2020 team. The SDU team wiki responsible also sent a detailed list of instructions and tips for managing the wiki work. This helped us to be structured and efficient in our wiki-work.

The document they sent, and the conversations had with the team truly helped us to be structured and efficient in our wiki-work. More specifically, we received tips on how to distribute work for the wiki, how to design the wiki to be accessible and organise a team, and coding advice.

How did it influence our project?

With the wiki being such an essential aspect of an iGEM project, having proper guidance from a former team provided us with an essential overview of what we needed to consider when creating our own wiki. Inspiration from what we learned from the SDU team gave us a basis from which we could adapt to our own style in our work.

Consultation with Jose Alfredo Zambrano Rodriguez

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Background & What we learned

Mr. Zambrano Rodriguez is a researcher at the Pure and Applied Biochemistry department, the same department the lab our iGEM team is located at, and a senior molecular biologist at OlsAro.

We gained useful insights on designing parts of our lab work, such as gene design, molecular insert design. We also talked about possible cloning strategies with him.

How did it influence our project?

Through these discussions we were better able to decide on what we wanted to prioritise and design in our lab. Any discussion regarding our lab design was considered and helped to hone our options and final plan.

Lund Sustainability Week Presentation

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Background & What we learned

Sustainability week is an annual event that was held in Lund in the first week of May 2022. It focused on bringing new sustainable ideas to light and sharing them with the general public to raise awareness and inspire people to create a better future. We participated on the 5th of May 2022.

The aim of this project was to raise public understanding of the bee decline and how it affects the human population in many aspects. One of the major takeaways from this presentation was to simplify the concept of synthetic biology for the general public. We also learned about the people’s concerns and how willing everyone can be to prevent bee decline. Along with this, we gained presentation skills as this was one of our very first presentations. To make the presentation and discussions more interactive, we distributed seed bombs that were filled with seeds of wildflowers from the region of Skåne. This made us more familiar with local flora and helped bees by planting them!

How did it influence our project?

This event helped us collect information from discussions about synthetic biology and how revolutionary its applications can be. We also understood public views on bee decline and the common mitigation methods that people use/would like to use.

Discussion with Hobbyist Beekeeper, Konrad Thomas

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Background & What we learned

Konrad is a private beekeeper who has been working on his family business for around 15 years. His hives are located in a rural area in the Czech Republic where farmers do not apply any form of pesticides. We spoke with him in May 2022.

Konrad introduced us to the bee system based on winter and summer bees. And talked about how they are fed twice after autumn. We discussed the risks threatening bee survival, such as the American foulbrood, or toxins related to agriculture. His bees do not normally encounter pesticides due to the rural location of the hives and the lack of agricultural pesticide use.

How did it influence our project?

Through this discussion, we had further insights into what concerns beekeepers, which is important for us in assessing whether or not our probiotic may be adopted. Talking with the beekeepers first-hand helped us in realising the other problems they may face that were previously unknown to us.

Stockholm iGEM Conference (SiC)

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Background & What we learned

From the 14th to the 16th of June 2022, iGEM Stockholm organised the very first Swedish iGEM Conference. The conference saw participation from iGEM teams Chalmers-Gothenburg, Uppsala, and us. The conference was a 2-day event that included presentations from each team, project discussions, workshops on entrepreneurship and honing presentation skills.

The conference provided us with a great platform for scientific discussion. We learnt about the recent advancements in the field of vaccinations and the role of synthetic biology in expediting drug discovery by Dr. Andreas Lundquist. Furthermore, we had a workshop from Mr. Pontus F. Christoffersen on the importance of body language and the use of PowerPoint tools to enhance presentations and their impact on our audience. His workshop introduced us to science-based techniques like positive pessimism and body scanning that helps boost confidence. These skill sets mitigate any risks posed by stress during the delivery of presentations.

How did it influence our project?

The workshops helped shape and enhance our presentations for later. The skills learnt from Mr Pontus were transferred to our social media posts to enhance outreach and engagement.

SynBio Dictionary, Partnership with iGEM GO Paris-Saclay

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Background & What we learned

The beginning of June 2022 marked the beginning of a partnership with iGEM GO Paris-Saclay. After last year’s successful collaboration between iGEM Lund 2021 and iGEM Paris-Saclay 2021 with iGETHICS, we thought it would be beneficial to continue the collaboration between our two teams this year with the creation of a ‘Synthetic Biology Dictionary’ as mentioned in the Education and Partnerships pages. The idea for the development of this handbook came to us after noticing one of our team members, who wasn’t pursuing a biology-related degree, struggling to understand what the rest of us were discussing during meetings regarding the lab and project brainstorming. We felt that future teams would benefit from our handbook designed to explain biological concepts and common terms in a way that can be understood by all. We also invited more igem teams to contribute terms to the dictionary and add their project descriptions to the book. We are grateful for the participation of iGEM Stockholm 2022, iGEM Aalto-Helsinki 2022, iGEM Chalmers-Gothenburg 2022, and iGEM Uppsala 2022. At the begining of October 2022, we printed our final copies of the book.

Click here to read more about this project:
https://2022.igem.wiki/lund/partnership
https://2022.igem.wiki/lund/communication

Through this partnership we learned a great deal about simplifying and communicating science, which directly benefits our presentation and outreach skills. We also better learned how to work with other teams on an international-level.

How did it influence our project?

A significant aspect of iGEM is the communication and presentation of ideas and products. In the creation of this book, we learned how to better present our project in a way that can be understood by a wider audience. This skill will be crucial in the case of the potential distribution of our probiotics to beekeepers, as noted from some of our interviews with beekeepers. This partnership resulted in a tangible educational product that can be printed, downloaded, and adapted into new versions.

Interview with Bee Researcher, Dr. Laura James

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Background & What we learned

On 14 July 2022, we met with Dr. Laura James who currently works as a technical expert and ecotoxicology risk assessor at Syngenta. We met with her as, during her dissertation, she specialised in bumblebee behaviour and how exposure to different pesticides affects bee landing and memory.

In our conversation with Dr. James, we first discussed the delivery of our probiotics to the bee gut. We learned that winter feeding could be a problem due to the loss of foragers and gained insight into bee vector technologies as a method for delivery. Another point discussed was how pre-emergence application of glyphosate influences the glyphosate concentration on pollen. In this context, we also talked about data on the degradation rate. She confirmed that glyphosate isn’t directly integrated into the pollen nectar of the crop but can instead accumulate as drift residue over longer periods.

Furthermore, we learned that honey is stored in the honey stomach which is separate from the gut. Dr. James informed us about how anaerobic conditions within the bee gut could influence probiotic delivery. We also discussed our probiotic’s potential to upregulate certain P450⁸enzymes within the bees’ immune system, which naturally detoxify insecticides and certain metabolites.

Finally, an important part of the talk was the current reapproval process of glyphosate by EFSA and how that could impact our project as a whole idea.

How did it influence our project?

After our talk with Dr. James, we reevaluated several aspects of our project, such as the method of delivery for our probiotic. We also found different challenges related to the initially planned winter feeding including for instance the early loss of foragers.

Beehive Visit and Interview with Local Beekeeper, Beewild / Gunn Baustad

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Background & What we learned

On 22 July 2022, we went on a ‘Bee Safari’ at Beewild located in the nearby town of Svedala, Sweden. Beewild is a honey farm managed by Gunn Baustad, who produces honey and provides services such as hive leasing and tours. After getting a first-hand glimpse at the farm, we had an interview with Gunn.

As our project focuses on honeybee health and conservation, it was important to speak with local beekeepers to hear their perspectives on the current state of honey bee health. We jumped into our bee suits to see and touch our ‘subjects’ of study. While talking with Gunn, we also learned that current issues such as disease require bee-keepers to treat their hives with supplements and that food supplementation is important during rough winters, however, she thinks it is important to keep the bees as self-regulating when possible. The seasonal cycles of the hives are likely to affect administration of our Beecilli probiotic in the future.

How did it influence our project?

During the safari, we learned more about the potential future uses of our probiotic. We got a realistic insight into when beekeepers feed their bees and whether or not they are already using supplements and medicines that serve a similar purpose as our product. However, explaining new technologies (i.e. GMOs) to small beekeepers may be difficult according to Gunn. This further highlighted the need for more science communication tools like our ‘synthetic biology dictionary’.

European Commission Meeting

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Background & What we learned

On 28 July 2022, we met with members of Unit E4 ‘Pesticides and Biocides’ of the European Commission’s Directorate-General Health & Food Safety⁹. Unit members present in this meeting included Dr. Klaus Berend, Dr. Karin Nienstedt, Ms.Sofie Hofkens, Mr.Mark Williams, and Mr.Nikolay Tzvetkov. The purpose of this meeting was to talk about the pesticide authorisation system, glyphosate and its ongoing renewal process, as well as GMO regulation in the EU.

Through this meeting, we gained insights into the legislative aspects encompassing our project. We learned about the process of active substance approval and renewal in the EU, as well as criteria that would lead to revocation of the approval of a substance. If a decision not to renew the approval of glyphosate were to be taken at the end of the process, it would include the delisting of the substance from the list of approved substances and a subsequent grace period for marketing and use of plant protection products containing it, which would drastically alter the course/utility of our own project. A very important aspect concerning our project are the regulations surrounding GMO use in the EU. We learned that there is no blanket-ban of GMO use and that individual countries are responsible for opting out of the use of authorised GMO. The public opinion can influence regulation and the sentiment regarding GMOs is variable between countries.

How did it influence our project?

Realising the risk of non-renewal of glyphosate in Europe was important for our team to understand, as this risk determines the usability of the probiotic within the whole of Europe and would therefore shift the scope of stakeholder localisation. This allowed us to have a greater understanding of the obstacles our project would face outside of the lab. Before this meeting, our team was less confident about our ability to someday use the probiotic in the field due to EU regulation regarding GMOs, but the clarification of this legislation sparked our motivation in the sense that the project has a better chance at realisation than previously thought. Of course, all requirements in the GMO directives would need to be fulfilled before any potential commercial use. For the future state of GMO use in the EU, it will be important for us to continue to destigmatize GMOs on the societal level, which is achieved through our goals in education and public outreach.

Nordic iGEM Conference (NiC)

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Background & What we learned

Nordic iGEM Conference (NiC) is an annual conference hosted by the Nordic teams. This year the NiC was hosted by the iGEM Linköping team from the 5th to 7th of August. Ten iGEM teams from four Nordic countries attended the conference along with representatives from the iGEM community and iGEM ambassadors from Europe. Two of our team members, Harshal and Leon, attended the conference and presented our project with a focus on the project motivation, methods, and human practice efforts.

All ten teams presented in the Jamboree format and received feedback on their presentations. We got to hear interesting lectures from experts and entrepreneurs and got to discuss case studies with other teams about the general iGEM experience or biosafety and biosecurity issues.

How did it influence our project?

It was a rewarding experience to hear other iGEM teams present and get valuable feedback on our presentation. We were also able to discuss certain challenges we experienced with our project and how they could be solved.

Beekeeper Questionnaire, Lena H.

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Background & What we learned

The first talk with Lena was on 7th August 2022 and was followed by a questionnaire on her experience with her hives. She has been keeping her hive of Carnica honey bees for several months in the countryside of Southern Germany.

Through the questionnaire, we learned about her experience with beekeeping through winter and that she uses nutritional supplements (apifonda and apiinvert). Within a 15km radius of the hive, only organic farming practices are applied. She tries to keep her bees away from monocultures and closer to more diverse flora. In her opinion, the most prominent stressors to her bees are pesticides, parasites, and climate change, but is most concerned about climate and the varroa mite in her region. Against the latter, she already uses formic acid treatments.

How did it influence our project?

Through this questionnaire, we realised that beekeepers are concerned about the stressors affecting their hives and that they are willing to treat and supplement the hives when needed. Many beekeepers are cautious about what they supplement their hives with and proper communication about our probiotic would be necessary if it were to be used. Because of this, we continued to focus on making our project and science more communicable to those outside of the scientific community.

Interview with Bee Microbiome Researcher, Dr. Eva Forsgren

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Background & What we learned

On 11 August 2022, we met with Dr. Eva Forsgren who works in the Honeybee Research Group at the Swedish University of Agricultural Sciences (SLU). We were interested in meeting with her due to her research on the honeybee gut microbiome and her experience with honeybee pathology.

Dr. Forsgren clarified that honeybee decline affects only certain regions of the world and that the honeybee is unique due to its domestication. We learned to view the honeybee colony as more of a ‘super’ organism. This presents a challenge because as such it is resilient to change like the introduction of a probiotic strain. Because of this, scaling probiotic adoption to the microbiomes of honeybees from the individual to the colony level is difficult. We also learned about how we might feed the probiotic to the bees and at what life stages. She mentioned that it may be difficult to sell the probiotic to beekeepers – something we also came across in our interviews with beekeepers. Colonising the gut of a bee during the winter would also differ from that of a bee in the summer. Gut colonisation is likely not possible in a whole honeybee colony. Instead, we would need to focus on individual bees.

How did it influence our project?

After this meeting, we knew that we had to focus on the individual honeybees before expanding our horizons to the colony level. She also confirmed our previous assumption that Lactobacillus would be a better host organism than B. subtilis. In terms of selling the probiotic, we also realised that we need to focus more on education and communication, which would help in destigmatizing GMOs as well as give exposure to the probiotic.

Mass Spectrometry Consultation with Dr. Katja Bernfur

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Background & What we learned

Through August and September 2022, Dr. Bernfur helped us in analysing our protein samples. Dr. Bernfur is a research engineer at the Department of Biochemistry and Structural Biology at Lund University who operates the mass spectrometry lab. She performed the MS analysis of samples from SDS PAGE after protein expression experiments.

How did it influence our project?

Dr. Bernfur was essential to the efforts of our lab team in achieving proof of concept, which sadly could not be achieved. Showing the successful expression of the protein of interest is a crucial checkpoint and verifying the respective protein bands from the SDS-PAGE via MS would provide more reliable results than visual expectation of the gel.

Wiki Collaboration, iGEM Rochester 2022

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Background & What we learned

Our webmaster, Nils, first came in contact with the 2022 Rochester team in August 2022 when asking about the struggles of integrating R-shiny apps into gitlab pages. This turned into a broader wiki-collaboration, discussions about how to arrange the wiki-work, create a user-friendly wiki, technical problems such as how to display some forms of content like, PDF:s, images, and downloadable content. We let ourselves inspire each of our wikis and communication. A large part of this partnership was focused on integrating our models into our wikis through a wiki model integration tool.

Through this partnership we learned about how to create a user-friendly wiki built into gitlab and how different aspects of the wiki influence 2 different projects.

How did it influence our project?

With the wiki being such an important way to display our achievements throughout the iGEM cycle, we knew we would have to put a lot of effort into designing it to be both aesthetically pleasing and intuitive. The integrated model into the wiki is the crossroad between our model and its implementation on our wiki.

Meeting with Senior Regulatory Issues Manager at Bayer, Dr. Steven Levine

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Background & What we learned

We spoke with the Senior Regulatory Issues Manager and Distinguished Fellow at Bayer Crop Science, Dr. Steven Levine, on two separate occasions: 21 September 2022 and 27 September 2022. Dr. Levine has an extensive background in toxicology within an academic, governmental, and industrial context. We reached out to Dr. Levine in order to have a greater understanding of how research on pesticides is analysed and how risk assessments and guidance in relation to bee health has been evolving in the regulatory landscape globally. His experience of over 20 years with glyphosate provided a clear context to our meetings.

During our meetings, Dr. Levine shared his extensive background working as an ecotoxicologist within the government and industry, giving us insights into how one navigates the topic of regulation and risk assessment. He discussed the evolution of bee testing and assessments, providing context to research papers. We learned a lot about the process of testing and what a risk assessment is comprised of. Dr. Levine also explained to us more details about glyphosate’s properties and how honeybees are exposed. We also talked about different ways in which acute toxic studies are performed for glyphosate and certain Good Lab Practices being essential to bringing a substance to market. We spoke about the effects glyphosate might have on the honeybee gut microbiome and were told that effects are transient. We were pleased to hear that scientists try hard to create systematic reviews and that they do not just read supporting evidence as a confirmation bias. We had a long discussion about modelling as well, which is important for predicting long term effects on bees from pesticides.

How did it influence our project?

By learning more about EPA risk assessments and the certain regulatory processes, we were better able to envision what future steps might look like in terms of getting approval for our probiotic on the use of bees, with differences from the pesticide approval path of course. We also gained insight into how to more critically examine research articles, which is an all-applicable skill. The discussion about modelling is kind of a stepping stone towards making our existing model more efficient.

Presentation Workshop with Olle Bergman

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Background & What we learned

On 26 September 2022 we hosted a presentation workshop with a free-lance science communication educator, Olle Bergman. Mr. Bergman has run his communications consultancy, Bergman Bokstäver AB, for nearly 25 years. We invited multiple teams such as iGEM Stockholm, iGEM Aalto-Helsinki, and a highschool team from Sydney, Australia, The Kings School, to participate in this collaborative workshop in order to guide each other improve and their own presentations in preparation for the Jamboree in Paris.

From Mr. Bergman, we learned both essentials involved in project presentation and communication, as well as specific tips given to the various presenting teams as well as to our own presentation. We learned that presenting is not only about showing off what we have done as a whole, but to share the deeper message of our project and why it is so important to our society. From each individual team, we learned that there is no best standard presentation style and that the qualities of each style adapt to who is presenting, and what their message is. The process of criticising and receiving constructive criticism after every presentation was an integral aspect of this workshop.

How did it influence our project?

Improvement of our presentation skills has been of great emphasis throughout our time in iGEM. This experience was very important for our development, as it was strategically planned to be closer to the Jamboree, which allowed us to better integrate a more developed and realistic version of our project into our presentation. Through this workshop, we have focused our presentation to be more of a multi-path human practices experience where multiple parties benefit, rather than a one-way distribution of information related to our project. We have also guided our Jamboree presenters, Chantal and Leon, to speak in a way to hone their individual presentation styles and complement the content on the slides.

Survey to Save Bees

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Background & What we learned

We made a survey in order to spread awareness about the survivability and importance of bees and have a better understanding from the public about their opinions about GMOs. We also tried to promote our team and spread the message that bees are important for our ecosystem. The survey closed on October 1, 2022.

From the survey, we found that people are concerned about the well-being and protection of honeybees in Sweden, along with important threats to honeybees which are mostly due to the use of pesticides, climate change and habitat loss. Through our survey we found out that people think the federal/provincial government should be the most responsible for the protection of wild native bees and their populations. A majority of the people don't use any sort of pesticides in their garden and they think the application of pesticides in agriculture should be reduced. When it comes to health, people are concerned about the effects of glyphosate on themselves as well as on insects and animals.It was also observed that the regulations in the EU on GMOs should be changed and that they would be willing to consume GMO based products in the future. Honey products from genetically modified bees are something the majority would be willing to consume with a few concerns regarding any side effects post consumption. With limited bee farming enthusiasts, we couldn't get a concrete response regarding the use of our product on field. Home remedies are usually considered to be safe especially when it comes to using them as an alternative for pesticides, we however found out that the majority wasn't 100% sure if it was the best option and that use of pesticides/insecticides is still on their minds.

How did it influence our project?

The survey was very useful for our team in terms of education of GMOs, bees, and pesticides. People seem more interested in the potential use of GMOs and especially with our product in the field. It was interesting to see what the survey submitters thought about GMOs, especially since it seems to be such a polarised topic in society. Overall, from this survey we realised that external opinions concerning our problem and solution will be very important to the development of our product, as public approval is crucial for acceptance of GMOs and their products in society.

Social Media

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Background & How it influenced our project

We used our social media handles as a platform to showcase our journey to the jamboree and bridge the gap between the general public and our science communities. We tried to educate our audience on the importance of honey bees as pollinators by posting weekly fun facts on bees named: ‘Bee-informed’. We also made use of our Instagram account to increase public outreach and connect with other igem teams. This helped us connect with iGEM ASIJ_TOKYO and iGEM Waterloo who participated in our presentation series wherein teams would present and discuss their projects and ongoing progress on them.

Furthermore, social media was crucial in organising and contacting teams iGEM GO Paris-Saclay and iGEM Chalmers-Gothenburg who helped contribute in making the SynBio Dictionary, a simplified dictionary that defines complex biological terms in simple plain english. We at Beecilli aimed to expand scientific communication by engaging with people across the globe and social media played a humongous role in achieving that aim.