The aim of our project was to make biosensors applicable in everyday life. Therefore, we developed two cell-free systems and tested several biosensors in vivo and in vitro.

A central aspect was the establishment of a cell-free protein synthesis system. An ideal reporter protein is superfolder GFP (sfGFP). Consequently, we used sfGFP as a reporter gene and as a positive control for all our experiments. We started with the BioBrick BBa_I746909 by team Cambridge 2008 and observed that it was expressed in our CFPS. However, the expression strength was not sufficient for our biosensors. Therefore, we inserted a translation enhancing untranslated region (UTR) between the T7 promoter and the coding sequence, creating Part:BBa_K1758102. We observed a drastic increase in fluorescence when testing this modified construct and performed our further experiments with this optimized sfGFP. We also tested whether the optimized UTR has an effect in vivo as well. We observed that the fluorescence of E. coli expressing the optimized sfGFP was significantly higher than the fluorescence of cultures expressing BBa_I746909. The difference could even be seen with the naked eye.

The next step was to test biosensors for their functionality in our cell-free systems. Therefore, we used the following biosensors by previous iGEM teams:

• arsenic (Edinburgh 2006, Buenos Aires 2013)
• mercury (Peking 2010, BBa_K346002)
• nickel (LMU Munich 2011, BBa_K549001)
• lead (Brown 2007, BBa_I721003)
• chromium (BIT 2013, BBa_K1058007)

We improved the characterization of these biosensors by performing tests in vivo as well as in our CFPS. In particular, we improved the chromium sensor by codon-optimization.

Both of our biosensor designs make use of fluorescence as an output signal because this requires no cofactors or subtrates and offers a good sensitivity. However, fluorescence can usually not be seen with the naked eye. Nevertheless, it is possible to easily visualize fluorescence using a smartphone and filters. In 2014, iGEM Aachen have demonstrated this possibility using a filter for excitation. We improved this idea with a second filter in front of the camera, which enables everyone to specifally detect fluorescence by taking an image with their smartphone.

In summary, our project builds upon the projects of several previous iGEM teams. We managed to improve the function and characterization of several of their BioBricks.

Let´s take a closer look at CFPS!