Our project, NanoBlade, is an engineered E. coli capable of detecting cancer cells and eliminating them locally upon blue light irradiation. Accordingly, it comprises two elements: a detecting device called Nanobody Display System and a drug delivery device, the BLADE Expression System, which secretes toxins into the tumor environment upon blue-light induction.

When we designed NanoBlade, one of our main considerations was to make it highly modular because this way, we could evaluate the features of each system individually. (Fig. 1.)

During our work, we could characterize neither of our detection devices, as we could not assemble our Nanobody Display System. Fortunately, we could its successor, the Mininano System, but it did not arrive on time, therefore, we had no time left to study it in depth. (If you wonder what difficulties we have faced, please visit our Notebook page.)

Although by overcoming great difficulties, we were much more successful with BLADE Expression System, which has allowed us to carry out many experiments. In the following, we will concentrate on these results.

Assembly of the BLADE constructs

We ordered the sequences from iGEM sponsor IDT, and as they arrived, we digested their ends with XhoI and XbaI restriction endonucleases. This was done similarly to pETARA plasmid, which we chose as the plasmid for our constructs (Fig2/B, C). Ligation was done in a standard 3:1 insert:vector ratio with T4 ligase, then transformation followed into competent Xl1Blu cells. As colonies were visible, we isolated the plasmids and confirmed our results with diagnostic digestion, which gave us positive results (Fig2/D).

Modeling blue-light inducible production with mCherry

With mCherry production, we aimed to study light-inducible protein expression. This protein seemed to be ideal for this purpose since it was also used for BLADE characterization and has a similar size to Cytolysin A (34 kDa), which we have planned to deliver into the tumor environment. [1, 2]

Team ELTE has developed two types of illumination devices, both capable of emitting blue light around 450 nm. (To know about them, please refer to our Hardware page) The first is a quite simple device with whom we could monitor the mCherry production over time at a given light intensity. Fig. 3/A shows the results of this experiment. After 7.5 hours of irradiation (at 37°C), we observed an 8.25 fold increase in fluorescence which was significantly less than was described in the literature. (15x) However, this could be easily explained by the difference between light sources since Romano et al. used an illumination setup that produces an irradiance of 5 W·m⁻². [1] We have not obtained such information about our device yet, therefore, these results cannot be compared exactly.

Our other illumination device is capable of irradiating a 96-well plate and is highly programmable. With it, we could investigate the light intensity-dependent nature of our protein production. (Fig. 3/B) We could detect differences between illuminated and dark samples during this experiment, but significantly less than we previously did. This also could be reasoned by the different light sources or the different temperatures which we used for the protein expression. (37°C vs. 25°C) Moreover, we could not put our second device into an incubator, the culture was not as oxygenated as in the first case.

Later we hypothesized that the difference could be explained by the different bacterial culturing protocols, which could affect the number of plasmid copies. The previous measurement was conducted with a freshly transformed culture, but in the other case, we used frozen glycerol bacteria stock.

We observed a slight difference between protein productions at different light intensities; they leaned into saturation which met our expectations. It seemed, over 57% of power, that there was no substantial difference between samples.

Cytolysin A production of the BLADE Expression System

We aimed to characterize the Cytolysin A production BLADE Expression System using SDS-PAGE, and study its hemolytic activity on blood agar plates. (Fig 4.)

For the Cytolysin production characterization, we obtained the supernatant of the bacterial culture after 4 h of blue light illumination and precipitated its protein content. We observed a band indicating Cytolysin A production, where we expected, which was absent in the case of the dark control sample. (Fig. 4/B) We implemented the protocol of Chiang et al. for this aim, but it should be noted, that this experiment needs to be repeated with a construct to which an affinity tag, such as 6xHis-Tag, is added. [3] In this way, the protein of our interest could be purified, lessening the number of aspecific bands.

We also meant to assess the hemolytic activity of Cytolysin A, using the BLADE Expression System. We spread the overnight cultures on Blood Agar plates, grew them for 8 h in dark at 37°C, which was followed by a 4 h long illumination with blue light at 25°C. (The dark control plate was kept in dark at 25°C for that time.) For additional negative control, we also spread BLADE-mCherry sample on the plates.) As we expected, no hemolytic activity was observed in the case of mCherry. Regarding the BLADE-ClyA samples, the biological activity of Cytolysin A was undoubtful since the blood agar discolored where the samples were plated. Unfortunately, we could not observe a significant difference between dark and illuminated plates. This could also be explained by the considerations we beforehand mentioned (Temperature, frozen bacterial culture).

Preliminary experiment for ClyA cytotoxicity characterization on cancer cell culture

As part of our future experiments, we would like to study the cytotoxic effect of light-inducible ClyA production on cancer cells. For this aim, we did optimization measures of a widely used resazurin-based cell viability assay, where we assessed the optimal cell number (around 6000 cells/well) which should be plated on a 96-well plate for the experiment we would like to conduct in the future.

Future Experiments

We appended Fig 6. to demonstrate the future of NanoBlade; from this point to market approval.

[1] Romano, E.; Baumschlager, A.; Akmeriç, E. B.; Palanisamy, N.; Houmani, M.; Schmidt, G.; Öztürk, M. A.; Ernst, L.; Khammash, M.; Ventura, B. D. Engineering AraC to Make It Responsive to Light Instead of Arabinose. Nat. Chem. Biol. 2021, 17, 817–827. https://doi.org/10.1038/s41589-021-00787-6.

[2] http://parts.igem.org/Part:BBa_K811000

Chiang, C.-J.; Huang, P.-H. Metabolic Engineering of Probiotic Escherichia Coli for Cytolytic Therapy of Tumors. Sci. Rep. 2021, 11. https://doi.org/10.1038/s41598-021-85372-6.