Results

Overview

Built a hybrid promoter that implements AND-gate logic evaluation of L-lactate and AHL and leads to expression of a gene under its control.

Tuned this AND-gate such that it is capable of distinguishing lactate levels associated with healthy and tumor tissue as well as taking into account the bacterial population density.

Characterized and tuned expression of a genetically encoded MRI contrast agent, Bfr, in E. coli.

Verified that this contrast agent leads to a marked decrease in T2 signal when expression is induced.

Developed an assay to characterize killing of cells in a cell line when supplemented with supernatant of lysed bacteria expressing Azurin.

Built and characterized a thermo-sensitve system that induces expression of a controlled gene at 45 °C but not at 37 °C.

Built an RBS library for the thermosensing system, whereby we reduced leakiness far enough to transform the potent lysis-inducing protein E under its control.

Showed that we can induce lysis of the bacteria when transformed with protein E under the thermosensitive promoter and induced at 45° C.

Future Perspectives

DIY FUS Transducer

To test the Effector module as described in the project description, we had initially thought of building a Magnetic-resonance guided Focused Ultra-sound transducer (MRgFUS) to test the functioning of the thermal bio-switch with FUS.

This was motivated by some recent articles like [1] by the FUS Foundation that talks about an open source DIY – MRIgFUS device proposed by Vanderbilt University [2], and shares access to detailed instructions on the hardware and software requirements for building it. Compared to commercial options, this is much cheaper although less powerful in terms of precision and less sensitive in terms of control. The idea was to demonstrate a proof-of-concept that would contribute to the cost-effectiveness and feasibility of the bacterial cancer therapy CATE.

However, as advised by our very own `FUS-man´, Beat Werner, a FUS-compatible test-bench requires proper material (acoustic impedance, thickness, etc.) and appropriate-containment for the test-sample. Also, adding MRI steerability increases the cost exponentially; typical MRI compatible FUS machines cost around $200,000. Due to lack of dedicated expertise in the team in this field, this idea was kept very low priority as far as iGEM was concerned. Instead, the FUS test conditions were emulated using an incubator at 45°C.

Moreover, due to requirement of an MRI compatible transducer, amplifier and waveform generator, the Vanderbilt University open-source system costs around $10,000 [3], which was not feasible in the scope of the funding we received, but more importantly not critical to the demonstration of the idea of CATE, as a bacterial cancer therapy, in the time scope of this competition.

CATE, FUS and Beyond

For future development of a bacterial cancer therapy such as CATE, it would be logical to develop a high intensity FUS curved-transducer phased-array, for triggering the thermal switch that lyses the cells and releases the cancer-toxin. Popular FUS transducers (like from Sonic Concepts Co.) cost around $4500 and could be used with front-end sensing solutions such as from Texas Instruments (for eg. TDC 1000), that would overall be much cheaper than an MRIgFUS.

Moreover, according to recent developments in MRI science [4], [5], the near-future will very likely see cheaper and better accessible MRIgFUS devices. Recent research for gadolinium replacement for MRI [6] will also possibly lead to cheaper MRI solutions in the future, and thus could potentially lead to more economically-feasible MRIgFUS technology.

As per Beat Werner, radio frequency (RF) is better than FUS for heating a larger volume and thus a better solution for us, to heat the whole bacteria layer while maintaining a temperature of 45°C for 3 hours to trigger expression of protein E. However, currently RF allows lesser control than FUS. But with rapid progress in this field, using sound/RF as a non-invasive tool used in medicine would become better, cheaper and more accessible with time.

References