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OVERVIEW

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 cross the PM and reach the cytosol, Zn(II) will bind to the ZntR protein, and this complex will bind most likely in the zntAP spacer region of -35 and -10 n.t. Here, ZntR will alter the zntAp DNA structure to make it a better substrate for RNA Polymerase (RNAP), thus activating transcription of zntAp (Outten, Outten and O'Halloran, 1999). If zntAp is successfully activated, mRFP will be transcribed and produce a red fluorescent protein that will be released by the cell and visible in solution. Furthermore, the gene zntA will be transcribed and will express the ZntA zinc-exporter protein, which locates itself on the inner PM. ZntA will then draw cytosolic Zn(II) and couple it's efflux with ATP hydrolysis, for which each exportation of a singular zinc ion will result ADP, inorganic phosphate, and water.

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.

Design Principles

Bacteria Sensing of Sound

Bacteria growth rate increases when exposed to audible sound. Increased E. Coli culture growth rates were observed optimally under 5kHz (Lee Ying, Dayou and Phin, 2009). E. Coli exposed to a wide range of frequencies, 500Hz to 16kHz, generally showed an increased biomass. 8kHz was found to increase protein concentration and RNA concentration. The same paper found a positive relationship between sound power, measured in decibels, and bacterial biomass (Gu, Zhang and Wu, 2016). Bacteria produce sound frequencies that may support the growth rates of neighboring bacteria. Matsuhashi et al., 1998 found that frequencies of 6-10, 18-22, and 28-38kHz promoted B. Carboniphilus growth rates. More interestingly, Matsuhashi et al. also found that B. Subtilis emits sound waves between 8 and 43kHz, with the most intense waves at 8.5, 19, 29, and 37kHz.

Sound as Stress

The plasma membrane is the interface for conversion of environmental stimuli into biochemical signals that induce a genetic response within the cell. While there are few studies that focus on sound as an environmental stimulus on bacteria, there is a plethora of evidence for similar stimuli, such as osmotic shock, heat shock, and metal ion exposure. We hypothesize that sound may induce a similar "shock" response on the membrane. Osmotic shock, for example, can create turgor pressure upon the plasma membrane when extracellular solute concentration is low, so we hypothesize that sound can cause a similar pressure on the plasma membrane, thus resulting in stress.

Mechanosensitive Channels for Transduction of Sound

Since sound is ultimately a wave of pressure, it can be considered to be a mechanical stress. Within the double membrane of E. Coli cells exist various mechanosensitive protein channels, whose primary role are to protect the cell's integrity from shifts in osmolarity. The most common "Msc" channels in E. Coli are MscM (mini-conductance), MscS (small conductance), and MscL (large conductance). Unlike eukaryotic channels, bacterial MscS and MscL channels are non-selective in their charged signaling molecules, as long as they are under 1,000 M.W. (Peyronnet et al., 2014). They respond to mechanical stimuli by transmitting ions and electric flux via a gate-opening mechanism, changing the cell's membrane potential. Mechanical stimuli include forces that create changes in the tension of the lipid bilayer of the plasma membrane, created by a deformation affecting the membrane curvature and inducing a bilayer-protein hydrophobic mismatch. It has been shown in previous iGEM experiments that the MscS can be opened using ultrasound (Leben et al., 2016). Using sound to open a mechanosensitive channel will allow cations into the cell.

Our Sound-Induced Systems

Two types of systems were tested in our experiments. The first type of system utilized ions as ligands whose signaling was activated by sound; and the second system involved a membrane stress response, in which the cell contains pre-existing protein systems that respond to external stress on the outer membrane. In this case, the stress is sound. For our ion ligand system, we were able to select ions based on factors that would induce a mechanoreceptive response. Therefore, we searched for ions that tend to be exported from the cell and that have a relatively low intracellular concentration. With this tendency, sound could be utilized to open a channel and "pull" the ions back into the cell. Our second system involved testing the response of stress-induced protein systems to sound. The functions of these proteins, such as RpoE, PspA, and RcsB, are to protect the cell from damage by the stress.

Bibliography

Gu, S., Zhang, Y. and Wu, Y. (2016). Effects of sound exposure on the growth and intracellular macromolecular synthesis of E. colik-12. PeerJ, 4, p.e1920. Leben, K., Krese, R., Plaper, T., Franko, N., Magdevska, L., Gradisek, M., Merljak, E., Roskar, S., Jerala, N., Cerovic, K., Praznik, A. and Pusnik, Z. (2016). Team:Slovenia. [online] 2016.igem.org. Available at: https://2016.igem.org/Team:Slovenia/Mechanosensing/Mechanosensitive_channels [Accessed 15 Oct. 2018]. Lee Ying, J., Dayou, J. and Phin, C. (2009). Experimental Investigation on the Effects of Audible Sound to the Growth of Escherichia coli. Modern Applied Science, 3(3). Matsuhashi, M., Pankrushina, A., Takeuchi, S., Ohshima, H., Miyoi, H., Endoh, K., Murayama, K., Watanabe, H., Endo, S., Tobi, M., Mano, Y., Hyodo, M., Kobayashi, T., Kaneko, T., Otani, S., Yoshimura, S., Harata, A. and Sawada, T. (1998). Production of sound waves by bacterial cells and the response of bacterial cells to sound. The Journal of General and Applied Microbiology, 44(1), pp.49-55. Shah, A., Raval, A. and Kothari, V. (2016). Sound stimulation can influence microbial growth and production of certain key metabolites. Journal of Microbiology, Biotechnology and Food Sciences, 05(04), pp.330-334.

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 Our <b>first system</b> 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.
 <p>
-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 cross the PM and reach the cytosol, Zn(II) will bind to the ZntR protein, and this complex will bind most likely in the zntAP spacer region of -35 and -10 n.t. Here, ZntR will alter the zntAp DNA structure to make it a better substrate for RNA Polymerase (RNAP), thus activating transcription of zntAp (Outten, Outten and O'Halloran, 1999).
+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 cross the PM and reach the cytosol, Zn(II) will bind to the ZntR protein, and this complex will bind most likely in the zntAP spacer region of -35 and -10 n.t. Here, ZntR will alter the zntAp DNA structure to make it a better substrate for RNA Polymerase (RNAP), thus activating transcription of zntAp (Outten, Outten and O'Halloran, 1999). If zntAp is successfully activated, mRFP will be transcribed and produce a red fluorescent protein that will be released by the cell and visible in solution. Furthermore, the gene zntA will be transcribed and will express the ZntA zinc-exporter protein, which locates itself on the inner PM. ZntA will then draw cytosolic Zn(II) and couple it's efflux with ATP hydrolysis, for which each exportation of a singular zinc ion will result ADP, inorganic phosphate, and water.
 <p>
 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.

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