Team:Penn/LightActivatedLysis

We then wanted to prove that our pDawn-ClyA construct was able to lyse mammalian cells in a light-dependent manner. To assess this, we plated BL21 bacteria transformed with pDawn-ClyA or pDawn-mCherry on Columbia Agar plates supplemented with 5% sheep blood (BD). These plates are used to qualitatively detect hemolytic activity in bacteria by visually confirming lysis through a color change in the media as the blood cells are lysed. After plating the bacteria, cultures were grown in non-inducing conditions at 37°C until visible colonies were present (~12 hours). Plates were then grown at 25°C under either inducing or non-inducing conditions for 24 hours and imaged. These results are visible in Figure 1. The plates with pDawn-mCherry exposed to both dark and light conditions show no evidence of cell lysis. Similarly, the pDawn-His-ClyA plate exposed to dark conditions shows no cell lysis. However, in the pDawn-His-ClyA exposed to light conditions, there is significant cell lysis throughout the plate.

This cell lysis demonstrates that ClyA is being secreted from the bacteria in a light-dependent manner

pDawn-mCherry Dark

pDawn-mCherry Light

pDawn-His-ClyA Dark

pDawn-His-ClyA Light

Since our goal is a system for drug delivery, it is especially important to show spatial control of lysis. The advantage of our light-based system is the precision gained through using light to selectively target regions of the body to kill cells. We tested this concept on our blood agar plates by selectively exposing half of the plates to light conditions and the other half to dark conditions. Shown below in Figure 2, only the region exposed to light shows cell lysis. Similarly, when we plated a fun pattern in Figure 3, only the bottom half exposed to light shows cell lysis. There is also no leakage evident in these plates indicating the high degree of control which our system provides

Figure 2
Figure 2: Colony Spatial Control.

Figure 3
Figure 3: Penn iGEM spatial control

Having proven above in Figure 4 that ClyA was being secreted, our next step was to ensure that ClyA was being properly expressed and was the factor causing the cell lysis we observed on the blood agar plates. To test this, we conducted an affinity protein purifaction and the results of the protein gel are shown below in Figure 4. It is evident that there is a 34 kDA band in both the lysate and culture medium confirming that the protein was being expressed. Furthermore, this band is only evident in the pDawn-ClyA Light column. The fact that there is a band only on the light column as well as no evidence of a band in the pDawn-ClyA Dark column shows the strong on/off expression of the system as well as confirms the light-dependent expression of ClyA.

Thus, through the combination of our blood agar experiments and protein purification, we were able to show light-dependent expression and secretion of our cytolytic protein, ClyA

Figure 4
Figure 4: The production of clyA-his in BL21 in both bacteral lysate and culture medium. Production was light-dependent and showed the correct 34kDa band in pDawn-ClyA (right two lanes). The correct band was also seen in the IPTG induced control plasmid pET26b-ClyA (left two lanes).

Having demonstrated the expression and secretion of ClyA in a light-dependent manner, our next step was to determine whether it would be able to kill cancer cells and also better characterize its level of toxicity. We used a commerical Promega CytoTox-Fluor cytotoxicity assay kit which qualifies cell lysis at a fluorescence emission of 520 nm. The assay was run on two important cell types: the first is SKBR3 cells, which are derived from breast cancer cells and over-express the Human Epidermal Growth Receptor (HER2), and the second are HEK293T cells, commonly used mammalian cells that express basal levels of HER2. We used a negative control (water), positive control(toxic detergent, digitonin) and our cytolytic protein, ClyA. In both plots, it is evident that ClyA had a significantly higher level of cytotoxicity compared to our negative control. Furthermore, ClyA cytotoxicity is shown to be so potent that it also is more effective than the positive control provided by the assay.

Figure 5

Figure 5: Cytotoxicity results performed on SKBR3 cells (high levels of HER2 expression). Comparison of the ClyA cytotoxicity with the negative control shows over a 7-fold increase and the effect is statistically significant by t-test with a p-value less than 0.0001.

Figure 6

Figure 6: Cytotoxicity results performed on HEK293T cells (basal levels of HER2 expression). Comparison of the ClyA cytotoxicity with the negative control shows over a 8-fold increase and the effect is statistically significant by t-test with a p-value less than 0.0001.