Contents 1 Main Results 1.1 Aerobic Expression 1.2 CEST Agent Testing 1.3 Anaerobic FNR Testing 1.4 General Result

Main Results

Aerobic Expression

In the experiments for the aerobic expression of the proteins, we have seen that we have produced functional Biobricks. The proteins 1ETF, 1PJN, 1G70 and EGFP were produced aerobically and purified by their His-Tag or exclusion body extraction, so that we can say that the following Biobricks were succesfull: [http://parts.igem.org/Part:BBa_K1123015 BBa_K11230014], [http://parts.igem.org/Part:BBa_K1123015 BBa_K1123015], [http://parts.igem.org/Part:BBa_K1123016 BBa_K1123016] and [http://parts.igem.org/Part:BBa_K1123015 BBa_K1123017]. For more detailed information and further results obtained by these experiments, see CEST Agent Testing.

CEST Agent Testing

To evaluate all the potential CEST contrast agents, MRI measurements were carried out and analyzed as described in MRI Data Processing. The most promising agents seemed to be the lysine based contrast agents. All compounds that have a high lysine percentage did actually express lysine based CEST contrast. The best distinguishable agent was 1PJN ([http://parts.igem.org/Part:BBa_K1123015 BBa_K1123015]), based on the graph below ().

Anaerobic FNR Testing

After analyzing the samples taken during anaerobically induced expression of EGFP, we can conclude that the FNR promoter is a functional Biobrick. We can conclude that the EGFP Protein Production Construct is functional too since EGFP was produced.

We performed Fluorescence measurments of the samples taken at different time points during the anaerobically induced expression of EGFP.

These experiments resulted in the observation that we have two more functional Biobricks: [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1123000 BBa_K1123001] and [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1123005 BBa_K1123005]. To see detailed information and results, see Anaerobic FNR Testing.

General Result

As a general result of all the experiments we have a promising CEST contrast Agent and we have a functional FNR promoter, which can be induced anaerobically at 37°C and produces more protein when it is longer exposed to the hypoxic conditions. Overall, we showed that all the seperate mechanisms are working and that the combined functions are functional too. The proof of concept seems to be promising, but further experiments, such as in vivo experiments and experiments to ensure the killing mechanism is working well, need to be performed to say whether this could really be used for tumor targetting.