Our first system involves using sound as a mechanical force to open a mechanosensitive channel within the E. Coli membrane. This opening allows the diffusion of ions into the cell. Zinc was an ideal choice for our mechanosensitive system due to it slow exchange between its extracellular pool and low cytosolic concentration, with a tendency to be extruded from the cell. ZntR is a zinc-dependent Mer-type transcription factor that binds to the promoter zntAp upstream of zntA in the E. Coli genome. ZntA is an ATPase zinc exporter, keeping intracellular zinc at optimal concentration . Zinc-bound zntR will also bind upstream of mRFP, and ZntR then transribes zntAp to export zinc and RFP (red fluorescent protein) out of the cell. This will demonstrate that zinc successfully entered the cell via sound and activated a genetic response.

For sound to induce the response of zntAp (and mRFP), zinc ions must be placed in the liquid culture outside of the cell's plasma membrane (PM), in which sound will force open the gate of the MscS channel (a protein embedded in the PM), into which zinc ions will travel. Once zinc ions traverse the PM and reach the cytosol, Zn(II) will bind to the ZntR protein, and this complex will

Other ion signal transduction systems exist in E. Coli that are similar to zinc and can be linked to gene expression, as well as play a role in mechanotransduction. Calcium, zinc, and magnesium are interactive divalent signaling molecules in eukaryotic immune cells and exhibit similar traits in E. Coli. For example, calcium is known to travel through eukaryotic mechanosensitive channels Piezo1 and TRPC5 (2016.igem.org, 2018), and is also suspected to be a tightly regulated signaling molecule in E. Coli (Dominguez, 2004). Calcium signaling is often inhibited by zinc and magnesium ions due to binding site interferences or blocking of mobilization (Chaigne-Delalande and Lenardo, 2014). Magnesium is also tightly regulated in eukaryotes and prokaryotes; however, partially due to its better binding affinity for oxygen, it is more often bound as a cofactor (Chaigne-Delalande and Lenardo, 2014). Unlike calcium and zinc, magnesium also exhibits an smaller chemical gradient for mobilization across the plasma membrane (inward gradient), as does potassium (although it is an essential mineral and less regulated) (F C Kung, 2018). Sodium also participates in mechanotransduction through osmosis, with an inward gradient in E. Coli due to the sodium/hydrogen antiporter, NhaA.

Transition metals such as copper, nickel, and iron exist in the E. Coli cell, but like zinc, they exist in very low concentrations due to toxicity. They also tend to form complexes with nitrogen donors, typically existing as cofactors with a decreased regulatory role. Zinc is the exception, and is typically ionized in physiological solutions, implying a regulatory role in ion channels, leading us to believe that it may facilitate a genetic response to sound (Elinder and rhem, 2003).

Our second system is the direct protein-directed detection of sound stress. RpoE, the sigma 24 factor of RNA polymerase, is known for its response to stress that affects outer membrane proteins and membranous lipopolysaccharides. While the signal cascade to activate RpoE is induced by many stress-sensing proteins, we only require one such protein to "detect" sound. An activated RpoE will bind to bamEp to upregulate expression of BamE, which is responsible for outer membrane protein aggregation and membrane permeability.

BamE is located on the outer membrane and once bamEp is activated by RpoE, BamE very likely responds to stress on the membrane by repairing damaged proteins. The viability of this system can be evaluated by striking it with sound, and then examining the presence of RFP as a result of activation of mRFP by the upstream binding of RpoE to bamEp.

Our third system involves the promoter OsmCp1 of the osmotically-inducible peroxiredoxin, OsmC, that is stress-activated by two different transcription factors that are independent of each other: RcsB and NhaR. The Rcs "Regulator Capsule Synthesis" system is a two-component signal transduction system that regulates critical cellular functions in response to environmental effects on the cell envelope. The regulated functions include cell division, activity of periplasmic proteins, motility, biofilm formation, etc. Protein RcsC acts as a sensor kinase that autophosphorylates in response to an environmental signal such as the alteration of the outer membrane proteins, and then transfers the phosphate group to inner membrane protein RcsD, and then to the cytosolic response regulator RcsB.

NhaR, while independent of RcsB, is necessary for OsmCp1's activation in response to osmotic shock . More specifically, NhaR transcribes OsmCp1 in the presence of NaCl, LiCl, and sucrose (Toesca et al., 2001). If either NhaR or Rcs activate OsmCp1 in response to sound, measurable expression of mRFP should result.

The pspABCE operon of the "Phage Shock Protein" represents our fourth system, which responds to membrane stresses. Under normal cell conditions PspA suppresses PspF. During membrane damage and loss of proton motive force the pspBCE cascade suppresses pspA thereby freeing PspF. PspF is then free to bind upregulate several genes including the pspABCE operon and the promoter, pspAp. Sound stress may be able to damage the bacterial membrane or disrupt the proton motive force.

PspA, while serving as a proteinaceous response to membrane damage, may have a unique response to sound that involves the activity of mechanosensitive channels. If sound disrupts the membrane and thereby damages the proton motive force, simultaneous Msc channel activation may re-allow the protons back in, as PspA functions to restore the proton motive force and expel the protons.