Simon van der Els works for NIZO. NIZO is the Dutch institute for dairy research. Simon's research focuses on bacteriophages and CRISPR-Cas. We send him our project proposal and called to discuss the details. He was able to give us a better insight into the growing realization of the problem of bacteriophages in the dairy industry. We also talked about the feasibility of the project, about which steps should be working relatively straight out of the box and which steps would be really challenging to accomplish. It was pleasant to have someone specialized in the subject talking with us and giving us an idea of where we should start. At the 12th LAB (Lactic acid bacteria) symposium in Egmond aan Zee, we also met with Simon. We were able to talk about our research and ask technical questions, such as choosing suitable promoters for our system.

DSM cultures is one of the main providers of dairy starter cultures worldwide and has more than 100 years of expertise in the dairy industry. As they could potentially benefit from our detection device and have knowledge about bacteriophages we decided to get in contact with them. During a phone conversation we spoke with Thijs Kouwen, senior scientist and expert on bacteriophages and starter cultures. We learned that bacteriophage detection is performed by both major dairy factories and companies that provide starter cultures, such as DSM. There is a need for a fast bacteriophage detection system in the dairy industry and that, indeed, bacteriophages cause a significant disruption to fermentation processes. DSM provides a free service of bacteriophage testing for their customers, for which they mainly employ plaque assays and at times qPCR. Thijs mentioned that these tests take quite a lot of time. According to Thijs an ideal detector would have the following characteristics:

• Can differentiate up to 700 species of phages.
• Has a detection limit of 100 phages per ml.
• Has a detection time of 30 minutes to one hour.
• A detection limit of 10.000 – 100.000 would be useful as well, because the bacteriophage level will get problematic.

We are currently working very hard to ensure that our product meets these specifications as much as possible.

We had a phone call with Jan Willem Sanders, a science leader in Microbiology at Unilever. Unilever does not primarily focus on dairy products, but Jan gave us some good starting points for our project with questions such as:

• What is the detection limit of the method? What sample size is needed?
• Can different species (or strains) be detected in parallel?
• To what level of identification can the method be used: genus-species-strain?
• Can the method quantify live cells in the presence of dead cells of the same genus-species-strain?
• Is the method reliable to detect microbes in a complex food matrix (without enrichment), such as cheese, margarine, soups, powders containing spices, etc. and their ingredients?
• How would you validate the method? How does it compare to existing methods?
• How could you modify the method to allow for immediate readout (current methods take half a day up to multiple days)?
• How could you modify the method to allow for readout on a factory floor (current methods require a micro/molecular lab)?
• How could you modify the method to allow for read out by non-trained people (current methods require experience in microbiology/molecular biology)?

After this conversation and asking ourselves these critical questions, we decided to focus on an easy-to-use device that could possibly be used on the factory floor by someone without a synthetic biology background, hence we proposed a cartridge which would be doubly contained and very easy-to-use. Of course, such an application gives rise to other questions, such as safety and regulation. We discussed this thoroughly on our safety page.

When designing a product targeted towards the dairy industry, what better thing to do than visiting a dairy factory ourselves? As committed IGEM-team members, we wanted to experience dairy processes at work, on site. To this end, we were kindly invited by a major dairy company's technologist to get a tour of a cheese factory. Prior to our tour, we received safety instructions. Afterwards, we were given the opportunity to ask questions about the effect bacteriophages have on starter cultures. We learned how a bacteriophage infection is measured and how they proceed once a detection occurs. When the fermentation process is severely disrupted, either by bacteriophages or other factors, the cheese will be sliced into blocks and used for other purposes. She informed us on the occurrence of bacteriophage infections, but due to strict cleaning requirements, the impact has been greatly reduced. Through this excursion, we were able to envision the sort of environment where our final product could potentially be applied and directly contact the people who could potentially work with it. As we learned later in their innovation center that SK1 bacteriophages are especially resistant to cleaning we decided on using this bacteriophages for our proof of concept.

We talked to Arla in Denmark via Skype and got some advice from Harry Barraza on how to communicate our project to the general public. For instance using the word ‘virus’ on the homepage of our WIKI could immediately scare people and that is definitely something you do not want. So it is best to use the word phage or bacteriophage and explain what is meant. We implemented this, and some other suggestions they made on our wiki. Two researchers from Arla, Sander Sieuwerts and Valery Gutsal, who also joined the conversation, were really interested in our project and had some questions prepared. Besides that, Arla also decided to sponsor our project! We got to know which are the most often occurring phage infections and how they currently detect them.

We visited the Research facility of the same major dairy company as the factory in the Netherlands to discuss our project with a Senior Scientist Nutritional Sciences, who specializes in bacteriophages. Since our project focuses on detecting bacteriophages which can negatively impact various dairy production lines, we were excited to talk to a bacteriophage research expert in the dairy industry such as him. He kindly discussed the issues he faces in his research concerning bacteriophages and provided us with helpful advice for our project. He pointed out that the use of GMOs for the dairy industry is very tricky. Even though our detection device will not get in contact with the product, the factory still needs a permit to use it. This permit is accessible by the public and as the use of GMOs in Europe is still very controversial, they do not want the risk of a NGO getting hold of it. For these reasons we were not allowed to name the company or the person we spoke to on this day. Our detection device could however be useful in their research laboratory. Current detection techniques are not able to detect new bacteriophages. If we could implement a way that new bacteriophage sequences can be obtained, this would give new opportunities for bacteriophage research. This is not implemented in our current design due to the strict timeline which we are working with, however we have taken this into consideration in designing the future scientific outlook of our project. This conversation let us to thinking about how we could achieve food industries to use our product and started looking into legislation about the use of GMOs outside of a lab. On our future outlook page we came up with a solution for this problem.

During our skype call with Wouter Ghering (RIVM), who deals with biosensors and its effect on the environment, we came across a certain German scientist and the company ARSOlux that was able to work with GMO’s in a moving truck. During our talks with dairy companies, we learned that getting a permit in order to work with GMO’s in factories is a big threshold limiting innovate new Biotech approaches for companies, primarily due to their public perception. To this end, we were interested if it would possible for a biosensor to be used without the dairy company having to request a permit for working with GMOs. Therefore, the informaton that Wouter gave us was extremely helpful. In this scenario, we envision a dairy company that will have their samples picked up by a mobile element where our product, IMPACT, is used to detect whether bacteriophages are present. In this way, the dairy company is not associated with the use of GMO’s. Wouter also suggested that we talk to Rob Duba, a senior policy officer on biotechnology at the Department of I&E. We contacted him as part of our Human practices. We also established communications with ARSOlux in order to analyze the logistics of our product potentially being used in a moving truck, you can read about that on the future outlook page.

As part of considering the implications of our product and finding a possible potential market niche for it, we contacted Thomas Janzen from Christian Hansen and had a skype call with him on the 13th of September. The company mostly deals with starter cultures and tests for the presence of bacterial phages. From our talk with him, we discovered that the current go-to method for their company is doing a plaque assay and that when required, they also resorted to using qPCR. Their test takes 2-3 days if everything works properly. Thomas mentioned three points that were particularly relevant to us. First, that our product would be useful if it takes a few hours and able to detect similar phage concentrations as current technologies do. We took this into consideration and are designing a final product which will (hopefully) only take a few hours. Secondly, the device will be more useful if it can detect a specific viral strain. This is something we’ve taken into consideration in building our final product. By integrating different plasmids into our system or adjusting the plasmid for different strains, it will be able to detect specific strains. Thirdly, that our device can also be used on the factory floor. Currently, European regulations prevent our device from being used on the factory floor as it contains GMO's. We however still took this into consideration by designing an on-site detection system which could potentially be used in a factory. We designed our product as safe as possible as if it would be allowed. Based on this conversation, together with the impressions we received from conversations with other factory specialists and our factory visits, we decided to broaden our horizons and look beyond the dairy industry in Europe. We tried to contact companies in some other continents which have less strict GMO standards, however, these companies never responded to our inquiries.

After we were informed in the cheese factory that whey samples were sent to an external laboratory facility for phage analysis, we definitely needed to visit that as well! The technician of the factory kindly gave us contact information from an analyst working there. During the day we met multiple persons working at the laboratory. They do weekly bacteriophage testing for dairy factories located in the Netherlands, Belgium and Germany. Mostly they resorted to using either spot assays or PCR detection technologies. We presented our project, which made them very interested, but also hesitant. "Why do we want to know exactly which bacteriophage is in the milk? We only care about the fermentation, did the phages influence that or not." And also an impotant question we got was: "Is it really possible to make your product cheaper than the current tests?" We explained the benefits of having more information about the phages in a factory. For example, you can make different choices in the starter cultures you use. We took all of their questions into account in developing our final product.