Main Results

Aerobic Expression

In the experiments for the aerobic expression of the proteins, we saw that we were able to produce a number of proteins correctly. The proteins 1ETF, 1PJN, 1G70 and EGFP were visibly produced and purified by means of their His-Tag and/or exclusion body extraction. The other proteins we had designed were not seen as clearly, many not all, after expression, however, for most of these proteins, we did end up seeing CEST contrast. We can conclude that the following Biobricks were expressed most succesfully: BBa_K11230014, BBa_K1123015, BBa_K1123016 and BBa_K1123017. For more detailed information and further results obtained during 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 expressed lysine based CEST contrast. The best distinguishable agent was 1PJN (BBa_K1123015), based on the graph below ().

TU-Eindhoven Images 1PJNMRIPlot.png
1PJNMRIPlot Contrast plots of the agent 1PJN, for explanation see Team:TU-Eindhoven/MRIProcessing MRI Data Processing.

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. This conclusion was made after viewing the understanding gels and noticing the increased intensity of the bands related to the EGFP protein.

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SDSgelXL1blue 12% SDS gel with the bugbustered samples of the XL1-Blue anaerobic expressions of EGFP.
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SDSgelXL1bluepur 12% SDS gel with the purified samples of the XL1-Blue anaerobic expressions of EGFP

To strengthen this argument a Western Blot was performed allowing us to bind antibodies directly to EGFP. By performing the necessary washing steps and staining the following blot membrane was found.

TU-Eindhoven Images WesternBlot.png
WesternBlot Western Blot performed on purified samples of EGFP expressed anaerobically. C- is a negative control, a purified sample of culture medium allowed to house EGFP expressing bacteria in an aerobic environment. C+ is a positive control of EGFP bound to a CNA35 protein. The first 6 samples t1 to t6 were expressed anaerobically at 0% Oxygen whereas the second set of 6 samples t1 to t6 were expressed at 5% Oxygen

In it becomes clear that EGFP is being produced as only EGFP proteins will bind to the EGFP specific antibodies. In we also note the disappearance of all other bands on the Western Blot.

Furthermore we also performed fluorescence measurements of the samples taken at different time points during the anaerobically induced expression of EGFP. These can be viewed below.

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Fluorescencemeasurements Emission spectra of EGFP from the XL1-Blue anaerobic expression samples
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Emissionpeaks Values of the peaks of the emission spectra of EGFP per sample
What becomes clear from the fluorescence measurements is that the amount of EGFP is indeed increasing in accordance with increases in the time. (Which also confirms that the low intensity bands seen in the gels above were in fact EGFP). These experiments resulted in the conclusion that we have two more functional Biobricks: BBa_K1123001 and BBa_K1123005. To see detailed information and results, see Anaerobic FNR Testing.

General Result

As a general result of all the experiments, we have obtained a promising CEST contrast agent alongside a functional FNR promoter, which can be induced anaerobically at 37°C. When exposed to hypoxic conditions for longer periods of time we also notice an increase in the amount of protein being produced, as we would expect. Overall, we showed that all the separate mechanisms are working correctly and that a combined construct is still functional. This proof of concept is promising, but further experiments, such as in vivo experiments and experiments to ensure the killing mechanism works, need to be performed to say whether this could really be used for tumor targeting within the human body.