The BioBrick nominated for best part is MscS (BBa_K1965000) from our Mechanosensing Collection. It contains the coding sequence for the E.coli small-conductance mechanosensitive channel, MscS. Its role is to mediate turgor regulation in bacteria and it is activated by changes in the osmotic pressure Perozo2003. It has been previously shown that MscS forms a homoheptamer. Each subunit is 31kDa in size and contains three transmembrane helices 2 with the N-terminus facing the periplasm and the C-terminus embedded in the cytoplasm Pivetti2003.

MscS can be described as an important receptor, involved in the response to ultrasound stimulation. We used the MscS channel as a source of Ca²⁺ influx when stimulated with ultrasound 1.

Characterization

Expression and subcellular localization of MscS channels in HEK293T cells was inspected. HEK293T cells were transfected with plasmids encoding HA-tagged MscS channel and protein expression was confirmed by western blot analysis, while protein localization was investigated by confocal microscopy.

After confirming MscS expression in HEK293 cells, we stimulated the transfected cells with ultrasound to verify and characterize channel activity. Our experimental setup included an in-house built hardware MODUSON connected to unfocused transducer Olympus V318-SU. To monitor cell response in situ and in real time we used standard ratiometric fluorescent calcium indicators Fura Red, AM and Fluo-4, AM, which can be easily detected with confocal microscopy. When activated, mechanosensitive channels open, leading to calcium influx, which in turn binds the fluorescent calcium indicators. The indicator conformation changes upon calcium binding, resulting in an increase or a decrease of fluorescence.

When cells transfected with a mock plasmid were stimulated with ultrasound, we did not observe calcium influx. On the contrary, when cells transfected with the plasmid, encoding the MscS channel were stimulated with ultrasound, we detected a significant increase in calcium influx 3.

In an attempt to improve calcium influx, we co-transfected HEK293 cells with the MscS channel and gas vesicle-forming proteins GvpC (BBa_K1965003) and GvpA (BBa_K1965004). The voltage of ultrasound stimulation was decreased to 450 Vpp as higher voltage also causes calcium influx in cells expressing only gas vesicle-forming proteins Read more) . By decreasing the voltage of ultrasound stimulation we successfully showed that only cells expressing both the MscS channel and the gas vesicle-forming proteins were activated as a result of ultrasound stimulation 4.

To verify that calcium influx was indeed a result of mechanosensitive channel activity , we used gadolinium (Gd3+), an inhibitor of ion channels, which is a trivalent ion of the lanthanide series. Due to its high charge density and similar ionic radius to Ca2+ Bourne1982 it blocks the pore of the channel and therefore acts as an inhibitor of calcium ion channels. As shown in 5, the addition of the inhibitor prevented calcium influx after ultrasound stimulation, confirming that cell response was indeed dependent on the activity of mechanosensitive channels.

The construct was further caracterized under Mechanosensitive channels, Gas vesicles and Touch painitng.