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Part:BBa_K737005:Experience

Designed by: Li Kang   Group: iGEM12_OUC-China   (2012-09-18)


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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 [2] formed by GvpA (BBa_K1965004) and strengthens its structure [1]..

We used the gas vesicle forming proteins to improve the sensitivity of HEK293 cells to ultrasound stimulation, as shown in (1).

Scheme of ultrasound stimulation.
Schematic presentation of a cell expressing mechanosensitive channels and gas vesicle-forming proteins. When exposed to mechanical stimuli, the gas vesicles interact with the cytoskeleton, resulting in activation of mechanosensitive channels on the cell membrane.

Characterisation

Expression of both GvpA and GvpC was determined by western blotting (2) and co-localization was observed by confocal microscopy (3)

Expression of gas vesicle forming proteins.
Expression of GvpC (A) and GvpA (B) protein was determined by Western blot using anti-FLAG (A) and anti-AU1 (B) antibodies. Expected sizes (marked with *) are 22,5 kDa for GvpC and 8,5 kDa for GvpA. HEK293T cells were transfected with plasmids, encoding gas vesicle-forming proteins GvpA and GvpC. 48 h after transfection cells were collected, lysed and total protein concentration was measured. 50ng of proteins were loaded on SDS page gels. After separation by size, proteins were transferred to nitrocellulose membrane by western blot. Membranes were then immunoblotted with anti-FLAG or anti-AU1 antibodies.
Localization of GvpA and GvpC
HEK293T cells were transfected with plasmids, encoding gas vesicle-forming proteins GvpA and GvpC. 24 h after transfection cells were fixed, permeabilized and immuno-stained with anti-FLAG (upper row) and anti-AU1 (bottom row). Both GvpA and GvpC are localized in cytosol. Co-localization is depicted in the overlay picture (far right, upper row).

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.

Gas vesicle forming proteins GvpA and GvpC are not toxic to mammalian cells.
HEK293 cells were transfected with GvpA and/or GvpC. 24 h after the transfection the cells were stained with the trypan blue dye and their viability was determined using the Countess™ automated cell counter.

Ultrasound stimulation

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.

Gas protein vesicles improve sensitivity of cells to ultrasound.
(A) Presentation of stimulation sequences for 900 Vpp (red) and 450 Vpp (grey) Vpp and (B) signal parameters used for stimulation.
(C) Genetically encoded gas vesicle-forming proteins increased cell response at high-power (900 Vpp) in comparison to low-power ultrasound (450 Vpp). HEK293 cells expressing Gvp proteins were stimulated with ultrasound for 10 s and calcium influx was recorded in real time . Fluo-4 (green line) and Fura Red dyes (red line) were used for ratiometric calcium imaging.

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).

MscS with Gvps improve sensitivity of cells to ultrasound even at lower voltages
(A) Schematic presentation of the stimulation sequence and (B) signal parameters used for stimulation.
(C) Co-expression of mechanosensitive channels and gas vesicle-forming proteins increased sensitivity to ultrasound stimulation in comparison to the cells without exogenous mechanosensitive channels. HEK293 cells expressing gas vesicle-forming proteins GvpA and GvpC with or without MscS were stimulated with ultrasound for 10 s and calcium influx was recorded in real time

References

[1]Hayes, 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
[2]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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