Team:NTNU Trondheim/Demonstrate

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We were aiming to reduce the production of biofilm by silencing one significant gene for communication and signaling. To get a realistic result that would be of relevance in different industries we chose media conditions that would be similar to that in nature. By our Integrated Human Practices work we learned that different strains vary in the amount of biofilm produced, and that a strain that should be a good biofilm producer not necessarily proves to be in the laboratory. In addition we learned that the biofilm production is based on different parameters like pH, temperature and the presence of sugar. Therefore, we tested 18 different media with different glucose concentrations and pH. In nature the resources are often limited, which gave us an indication on what conditions we should try to imitate. Based on the results, we chose to do measurements with the minimal medium M63B1 with 0.4 and 0.8% glucose and pH 7.2. We also chose the E.Coli strain TG1, a strain that should produce biofilm in nature.

We grew biofilm in 96 well plates before biofilm fixation and staining. After staining with Crystal Violet we were able to measure the density of cells in the wells and therefore quantify the amount of biofilm produced. Our results showed a significant decrease in biofilm formation grown in the wells that included the active CRISPRi-system induced by the antibiotic Tetracyclin, as shown in the picture. We used DH5a as a negative control since it is known that the strain contains a defect luxS-gene, which also the results clearly indicate. There is a distinct visual difference in color, due to the staining of cells. From figure 1, one can clearly see that in the wells with the activated CRISPRi-system there is less biofilm. The difference is also significant in the actual absorbance-values that shows low cell density for the wells with the active system.The graphs in figure 2 and 3 show absorbance as a function of time, and one can clearly see that there is higher levels of absorbance for the cells where the CRISPRi-system were not activated.

Figure 1: Picture from the crystal violet assay. One can clearly see a difference in color between the wells where CRISPRi were not activated, and where it was not.