Team:FSU/Design
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 transmission of our zinc ion signal to flow into the cell, activating our genetic response system. Particularly, zinc will enter the cytosol and bind to ZntR, a transcriptional activator protein for the gene zntA, which encodes a zinc exporter protein channel, ZntA.
The second system involves a protein-based stress response to sound. The protein BamE is responsible for outer membrane protein assembly, and is shown to be activated by RpoE, the sigma 24 factor of RNA polymerase, as RNA polymerase binds to the bamE promoter for the gene's transcription. RpoE is known for its response to stress that affects outer membrane proteins and membranous lipopolysaccharides.
The Rcs "Regulator Capsule Synthesis" signal transduction system is a two-component stress response that maintains the outer surface of the E. Coli cell. Proteins RscC and RcsD are inner membrane proteins that direct a signal to RcsB, a transcription factor that activates a number of genes that synthesize membrane proteins and capsules. More specifically, RcsC is a sensory histidine kinase that autophosphorylates in response to an environmental signal, and then transfers the phosphate group to RcsD, the response regulator, and then to RcsB.
The Psp "Phage Shock Protein" system involves an inner membrane protein pspA, which responds to membrane stresses and regulates the pspABCDE operon. The pspA protein suppresses the proton motive foce from extruding hydrogen protons through a damaged/leaky cell membrane due to cellular stress, and also have a likely role in protein export when conditions arise that block such activity.
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- Explanation of the engineering principles your team used in your design
- Sound as Stress The plasma membrane is the platform 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, 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 plasma membrane of E. Coli cells exist various mechanosensitive protein channels, whose primary role is to protect the cell integrity from osmolarity transition. The most common "Msc" channels in E. Coli are MscM (mini-conductance), MscS (small conductance), and MscL (large conductance). 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 changes in the tension of the lipid bilayer of the plasma membrane, created by a deformation of the lipid membrane that affects the membrane curvature and induces a bilayer-protein hydrophobic mismatch. We hypothesize that sound exertion can create such a tension, forcing the channel from a closed state to an open state. The general structure of the homoheptameric MscS channel consists of: three transmembrane helices, a C-terminus facing the selective and stable cytosolic domain and an N-terminus facing the periplasmic domain. Transmembrane 1 (TM1) and TM2 form a flexible paddle for tension-sensing, while the conserved TM3 forms a linked amphipathic helical pore structure. A channel pore connects the periplasm to the cytosolic vestibule, and a selectivity filter exists in the middle of the channel. The pore has a diameter for conductance values ranging from 14-16 amps, and opens and closes in a force-activated gating manner.
- 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. Bacteria are generally non-selective between the signaling molecules they select, so long as they are within a certain atomic radius. 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. Zinc was an ideal choice for this criteria, in addition to the fact that it has a slow exchange between its extracellular pool and intracellular binding. Once zinc enters the cytosol, the zinc-binding transcription factor ZntR binds it and the complex then binds to the promoter of gene zntA whose expression is induced by zinc. Once zntA is transcribed and translated, ZntA forms and exports zinc out of the cell. This will demonstrate that zinc successfully entered the cell via sound and activated a genetic response. Our second system involved testing the response of stress-induced protein systems to sound. The functions of these proteins, such as BamE, PspA, and RcsB, are to protect the cell from damage by the stress. BamE is located on the outer membrane and is responsible for outer membrane protein (OMP) aggregation and membrane permeability; and since it is activated by RpoE, it 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 membrane for it's repaired structure after a period of time. RcsB, located in the cytosol, is activated by the two-component sensor kinase/response regulator signaling system of RcsC and RcsD. RcsC is located on the inner membrane and is said to respond to osmotic shock and membrane perturbations. Once the phosphorelay signal transduction phosphorylates RcsB, it can transcriptionally activate genes that regulate the synthesis of periplasmic proteins and colanic acid capsules in response to changes in the lipopolysaccharide structure. If this system successfully responds to sound, the presence of colanic acid capsules and functional periplasmic proteins should be measurable. PspA is located in the inner membrane and responds to stress, including osmotic shock, by protecting membrane integrity, preventing proton leakage from damaged membranes, and assisting in protein export when it is inhibited. The lack of extracellular hydrogen protons is a direct method of measuring this system's response to sound.
- Discussion of the design iterations your team went through
- Experimental plan to test your designs