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iGEM Team Slovenia 2016 improved this part.
We improved it’s sequence by adding a 3xFLAG tag to the N-terminus and further characterized it by cloning it under the CMV promotor and expressing it in mammalian HEK293 cells. The improved part is deposited in the Registry under the name BBa_K1965003.
This part contains the coding sequence for the GvpC protein from cyanobacteria Planktotrix rubescens. GvpC is 22,5 kDa hydrophilic protein which attaches to the outer surface of the gas vesicle  formed by GvpA (BBa_K1965004) and strengthens its structure ..
We used the gas vesicle forming proteins to improve the sensitivity of HEK293 cells to ultrasound stimulation, as shown in (1).
In order to ensure that gas vesicles were not toxic to mammalian cells, a viability assay was performed. Viability of transfected cells overexpressing GvpA and GvpC was determined using the trypan blue dye, which selectively stains only dead cells. 4 shows that the viability of cells was not altered when expressing the proteins for formation of gas vesicles.
After confirming GvpA and GvpC expression in HEK293 cells, we stimulated the transfected cells with ultrasound. 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.
The HEK293 cells, expressing gas vesicle-forming proteins, were not responsive to ultrasound stimulation at mild ultrasound conditions (voltage 450Vpp), but exhibited increased sensitivity to ultrasound stimulation at higher voltages (900Vpp) even in the absence of exogenous mechanosensitive channels 5 .This phenomenon is most likely due to activation of endogenous mechanosensitive channels in mammalian cells.
In an attempt to improve calcium influx, we co-transfected HEK293 cells with plasmids encoding the MscS channel (BBa_K1965000) and gas vesicle-forming proteins. 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 (5). 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. (6).
ReferencesHayes, P. K., Buchholz, B., & Walsby, a E. (1992). Gas vesicles are strengthened by the outer-surface protein, GvpC. Archives of Microbiology, 157(3), 229–234. https://doi.org/10.1007/BF00245155
Walsby, a E., & Hayes, P. K. (1988). The minor cyanobacterial gas vesicle protein, GvpC, is attached to the outer surface of the gas vesicle. Journal of General Microbiology, 134(10), 2647–2657. https://doi.org/10.1099/00221287-134-10-2647
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