Team:Slovenia/Implementation/Touch painting
Touch painting
While ultrasound was used to stimulate mechanosensors in our project, since it is most appropriate for the stimulation of deep tissue, other mechanical stimuli
could also trigger activation of mechanosensors. Mechanical stimulation appears to be a critical modulator for many aspects of biology, both of living tissue and
cells
Results
Therefore our system might be also activated by other mechanical stimuli. One of them was stimulation by direct contact that underlies the sense of touch. Our designed mechano-responsive system is composed of two modules. First we included modules to provide and increase the sensitivity to mechanical stress by mechanoresponsive ion channels and gas vesicles and secondly, the influx of calcium is visualized by the dimerization of calmodulin (attached to the C terminus of split luciferase) and M13 (attached to the N terminus of split luciferase) which results in bioluminescence after the reconstitution of split luciferase. This means that this system is able to convert the mechanical stimulus into light signal, or to put it differently, with our system we are able to see when cells are touched.
Shear flow also exerts mechanical force on cells, which also has relevance for the endothelial cells within blood vessels, therefore also the flow of the liquid medium around cells might be sensed by cells. The experiment to test this ability was performed by agitating the petri dish with attached cells. In order to identify if the engineered cells are able to respond to the sheaf flow we included control cells that either constitutively express the luciferase and cells that harbour only the calcium-dependent reporter without constructs to increase the mechanosensing along with cells expressing the gas vesicles and reporters.
Firefly luciferase activity before (A) and immediately after (B) manual agitation is show. 24 hours after the transfection, luciferin and calcium were added to the cell medium. The cells were shaken a few times and the response was measured via bioluminescence imaging.
Results show that the light was emitted only in cells with the constitutive luciferase if the plate has not been moved (1). On the other hand shaking triggered activation of the luciferase, with significantly higher level in cells that harboured gas vesicles in addition to the reporter, demonstrating that activation does not require high pressure on cells.
Next we wondered if cells could respond to a gentle touch. We were excited to see that already in the first experiment we could clearly see where cells have been touched by a glass rod. The best paintbush to stimulate cells without removing them from the plate has yet to be identified but we found that glass rod functions quite well. We realized that we could use touch to illuminate cells, effectively painting or drawing on cells by recording the trace with emitted light, which we called Touchpaint. At that time an Argentinian visual artist Laura Olalde, PhD, interested in arts and science symbiosis, and interested in connections between art and science visited our lab. She was immediately eager to test the new artistic medium.
The immediate results were visible from drawing of symbols of our project on cells that were immediately (within 5 min) visualized by imager.
(A) Schematic of a cell with increased sensitivity to mechanical stimulation due to expression of mechanosensitive ion channels MscS and gas vesicle-forming proteins. Split calcium sensing system based on split firefly luciferase linked to M13 and calmodulin is shown.(B) Images of petri dishes seeded with HEK293 cells transfected with mechanosensing enhancers and luciferase reporters after stimulation of cells with glass rod. HEK293 cells were transfected with split calcium sensors. 24h after transfection cells were stimulated by touching with a glass rod. Afterwards, camera images were taken in darkness with exposure time 30 s.
Light emitted from cells was recorded by an imager by capturing light for with 30 sec. We observed that the lines that were drawn first were more fain that the those drawn later, which suggested that the response is quite fast. This was tested by sequentially drawing separate letters and recording the image after draing each letter.
Laura prepared several stamps and we experimented with different methods of cell immobilization on the plate, however due to the time constraints of the iGEM project we have yet to explore the intricacies of Touchpaint techniques. Response of the system is quite fast as the sequential drawing of letters of the iGEM acronym already started to fade by drawing the text letter few minutes after the first one, therefore real time monitoring of cell drawing is a very realistic proposition. While Touchpaint is just one artistic implementation of the technology, there are many other scientific and applied uses of this technology, which are discussed in the Impact section.
