Throughout the policy and practice assessment, it became clear that the strong point of our printer was that it could be used for different types and strains of bacteria. We believed that it was important to test this hypothesis. At the RIVM/Rathenau discussion meeting we met all the different Dutch iGEM teams. After hearing what each team was planning on making, we found out that other teams were using biofilms as well, including Groningen.

We organised a skype meeting on the 17th of August to discuss the possibilities of a collaboration between us. The iGEM Groningen team are creating a biofilm that will be used to create energy out of salt and fresh water. Their plan is to create a selective biofilm that is placed as a barrier between the salt and fresh water, that causes a membrane potential difference. This membrane charge difference creates a current, which is called Blue Energy. But they had a couple of troubles with creating a stable biofilm containing their engineered selective bacteria. We believed that combining our projects could lead to a good result.

Our 3D-printer is able to print bacteria in a structured composition by printing with bacteria and alginate to form a gel. By using our 3D-printer to print their biofilms, we provide team Groningen a method to grow and develop the biofilm in the structure that is desired. The advantage for us is that we could be able to prove that our 3D-printer is suitable for other strains and bacteria. Therefore we traveled to Groningen from Delft on the 1st of september.

In Groningen we executed several experiments. By adding calcium chloride to the alginate, a gel forms with their bacteria encased. By using a plate filled with LB agar, the bacteria would be able to survive and create a biofilm.

The first experiment was printing with the bacteria in alginate in a certain structure. The team members of Groningen printed with their own bacteria, B.subtilis, in a form of their own choosing. The Groningen team prepared beforehand their own bioink (Sodium alginate+bacteria), calcium chloride and LB agar plates. Their plates did not require rhamnose as inducer, since their cells create biofilms by themselves.

They pasteurized 100mL of 1%(w/v) sodium alginate with appropriate antibiotic, and autoclaved 100mL of 0,1M calcium chloride to acquire sterile solutions. The LB agar plates were made using standard protocol. They had grown their bacteria overnight in 5mL LB with appropriate antibiotic. The overnight culture was then spun down, and the supernatant was discarded. The pellet was then resuspended in 100µL LB and mixed with 500µL sterile sodium alginate to make the bioink, and transferred into a 1mL syringe Before printing on the LB agar, 500µL calcium chloride was added to enable formation of the hydrogel.

The team then proceeded with printing with their self-made bioink. They were able to print on two agar plates, in the form of a house and a flower. They noted that the controls were not very user friendly, but after a few minutes they got the hang of it. In the end they agreed that our Biolinker was functioning as a 3D printer.

The second experiment, was measuring the selectivity of alginate. As mentioned above, the membrane should only let sodium ions (Na+) ions pass through to be a selective bio-membrane. Since the previous experiment proved that we were able to print the bacteria in the gel, we now wanted to print a biofilm made of alginate and see if it was selective. We drew a square on carrier material, on which we printed the alginate in “full color”. The alginate filter was then placed in a self-made voltmeter system, where the filter would separate the fresh from salt water. This separation of salt and fresh was not acquired. This was due to the salt water added on one side of the alginate filter, it leaked to the other side, which didn’t allow the possibility of adding fresh water. The voltmeter did measure a voltage of 10 mV, but the Groningen team stated that the this was caused by the carrier material, which produces a voltage of around 10-12 mV by itself. Therefore we could not conclude that our alginate filter is selective for sodium ions.

We stated that “our 3D-printer made out of K’NEX is able to print with our strains.”. We proved with the first experiment that the 3D-printer is an open platform that can be used for different strains of bacteria.

We would like to thank the iGEM Groningen team for their time, bacteria and for the great dinner we had afterwards.

We received a request from the iGEM team Aalto-Helsinki to fill in a questionnaire. This questionnaire was about modeling in combination with experiments. Because our modeling is quite extensive, this questions gave us a lot of insight in how we can best implement modeling with science. So we were pleased to help them.

On the 12th of June we had contact for the first time with a team outside the Netherlands. We arranged a Skype meeting with them for an hour. We explained our project, and they explained their project. We had just started in the lab, and it was very interesting to hear how far they were in the process and what their experience was so far. CGU team was further in the development of the plasmids. In their iGEM project they try to develop an organism that can detect Oral squamous cell carcinoma (OSCC). They use the idea of the TU Delft team of 2012, the snifferomyces project.

Unfortunately our projects are now too different, so there was no reason to exchange plasmids. They had some questions for us about the modeling, because our model was already quite developed at that time. It was a good experience, and nice to meet another team!

On the 4th of August we had a Skype conversation with the iGEM team of NCTU. After 15 minutes of getting the technology to work, we finally can talk. We started with sharing our project topics. Jenny Gee, one of the team members held a comprehensive presentation of their project. What we noticed and surprised us is that it seems that most of the teams have a lot of members. However, our team consist only of nine(9) members, we seem to be quite on track. We are all now working at least 40 hour a week on iGEM, and it was nice to hear that NCTU team also does. They are arranging a big event among the Asian iGEM teams. For us that’s maybe also an idea, to arrange one more time an event among the Dutch teams. It was a nice conversation, we had fun meeting them and seeing how far other teams are in their project.

Oh the 24th of June we received a message via Facebook from Kyle Sam Bennett of the Oxford iGEM Team. They contacted us because we were both working with biofilms. In their project they focus on the breakdown of biofilms in the urinary tract in order to treat Urinary Tract Infections. For testing the breakdown, they need biofilms with their modified bacteria. Because we also create biofilms with a modified E. Coli bacteria, with the help of a 3D printer, our projects fit really well.

Our project can be a good support for theirs. With the use of our printer in combination with the vector with the curli gene inserted in their modified bacteria, they can print biofilms in the structure. A second subject in which we can help each other is to discuss the safety of the projects. We finally decided to induce the biofilm formation with rhamnose. The last collaboration point with Oxford was about the modeling. We had in total 2 skype meetings, and a few times contact by email. We shared ideas.

Where most teams ask for the opinion of other iGEM teams, this team wants us to go and ask around for other people to answer the question. The guidelines for doing the survey are below. On the 26^th of August we heard that we provided enough answers to earn the Bronze medal. We are really glad to hear that we gave the team enough input and we are curious to see what they did with the answers for the progress of their project.