We collaborated with iGEM TUDelft. As both projects involve a diagnostic tool in live cells, it is important to correctly and easily preserve the cells for storage and shipping, ideally without the need of a cold-chain.

The iGEM team of TUDelft created BioBricks containing proteins from the Tardigrade, an animal known for its tolerance to extreme environments. These proteins can help preserve and stabilize enzymes, which can be greatly beneficial when drying E. coli cells. We tested these three BioBricks:

• BBa_K2306011 (T4)
• BBa_K2306010 (T6)
• BBa_K2306008 (T10)

We tested the cell stabilization properties of these BioBricks by drying E. coli BL21(DE3) cells that produce these proteins and measuring their viability. Apart from cell survival data, we also provided a possible improvement in their protocol. Instead of washing the cells with water, we proposed to wash with PBS. In our case, it provides a significant improvement in cell survival for 1 out of 3 BioBricks. The protocol can be found here and the results can be found in Figure 1.

In return, iGEM TUDelft tested another preservation method for us. We tested an easy method to dry and preserve cells: in kaolin clay. More about this experiment can be found on our demo page. TUDelft performed the same experiment for us using the same clay. This gives us more data on this procedure and feedback on the comprehensibility of the protocol we created. The protocol can be found here.

The results of this experiment performed by iGEM TUDelft were inconclusive. Either zero colonies were counted, or the plate was overgrown. However, we received valuable feedback on our cell preservation method. They used E. coli BL21(DE3) whereas we used DH5a. Since their results are inconclusive, the strain of E. coli might have an influence on the success rate of drying. Due to time constraints this could not be validated. Moreover, in their opinion this drying protocol was not easy nor practical to perform. Scraping the dried cells of the plate is a lengthy process and high concentrations of kaolin made it troublesome to distinguish colonies on the plates. From this, we conclude that our protocol is far from ideal. Although this preservation method is cheap and does not require expensive equipment, it may be more labor-intensive than expected and not as reproducible as we hoped. A next step would be to perform this experiments in different strains of E. coli and measure viability and reproducibility, as well as looking further into preservation methods. In this research, the use of Tardigrade proteins can also be taken into consideration.

We also performed a mutual collaboration with iGEM Utrecht. For us, they characterized three BioBricks involved in the fluorescent output of our device. In turn, we analyzed their samples on a western blot.

To verify the secretion of sCpf1 and sCas9 by the animal cells of iGEM Utrecht, we performed a western blot analysis. For this, samples of both the medium and the cells were prepared. We ran these samples on a SDS-gel in our laboratory, followed by western blotting. The full protocol can be found here and the results of the western blot can be found below.

iGEM Utrecht did measurements on our visualization module by testing five of our BioBricks: the CpxR construct (BBa_K2387032) and the BiFC system with both GFP and Venus ( BBa_K2387045, BBa_K2387046, BBa_K2387047 and BBa_K2387048). Read more about this on the specific visualization and fluorescent proteins pages. The protocol that is used in this collaboration can be found here.

To get a better insight into the situation in Brazil with regard to viral diseases like Zika and dengue, we contacted the iGEM team from Brazil. Apart from reading literature and talking to experts in the Western world, talking to locals can also be of great value. We got inside information from the iGEM team of Brazil and via them, we also got into contact with dr. Tatiana Mingote Ferreira de Azara, Health Surveillance Secretary of the Brazilian Ministry of Health.

Through a Skype meeting with iGEM USP-Brazil we really got to know the local situation in Brazil. We gained valuable insight into the measures being taken by the government and the population to prevent infection by mosquito-borne diseases. We learned that the government does not put enough effort into stopping the spread of the diseases. It is advised to wear long-sleeved shirts in the evening and at night, which is the time mosquitoes are most active. Furthermore, individuals should spray themselves with an anti-insect solution. Moreover, as mosquitoes are located close to water sources (e.g. ponds and lakes), the removal of still water would aid in reducing the mosquito population. However, in the rainy seasons, this is nearly impossible. The bad water distribution system makes people store their water in boxes that are breeding places of the mosquitoes. Waste is also a problem, as every bottle cap can be a breeding place after the rain.

We also learned that in Brazil, you can go to the doctor for free. However, most doctors do not have the equipment to perform diagnostic tests. Also the limited production capacity of tests is the main bottleneck. A diagnostic test for viral diseases that is cheap and can be used by these doctors would be beneficial, especially to test pregnant women or decide on further treatment options.

Current serological diagnostic tests and other point-of-care tests are not reliable enough. More tests, like PCR and ELISA-based tests are needed for confirmation.

Due to this Skype meeting and the information from the Brazilian Ministry of Health, we realized that many people are affected by these terrible diseases and everyone in Brazil knows someone who at least is, or has been, infected by viral diseases like dengue or Zika. These diseases have a great impact on the patient, the people close by and society in general.

The project of USP-Brazil covers fighting mosquito-borne diseases from within the mosquito. They engineer symbiotic bacteria living in the guts of mosquitoes that can fight the viruses when the mosquito is infected, during the so-called paratransgenesis. One issue is, however, that this symbiotic bacteria should only start to produce the toxins in infected mosquitoes. Our project can be a nice addition by combining our engineered bacteria with their toxin system of the symbiotic bacteria. The resulting fusion is then able to detect the virus with the affinity body on the surface, after which our downstream signaling pathway results in the activation of the symbiotic bacteria to fight the virus or kill the mosquito.