Team:FSU/Design
OVERVIEW
**ZntA System** 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 its slow exchange between its extracellular pool and low cytosolic concentration, with a tendency to be extruded from the cell (F C Kung, 2018) . Our plasmid incorporated the gene for ZntR, a zinc-dependent Mer-type transcription factor that can bind to the promoter upstream of zntA in the E. Coli genome. ZntA is an ATPase zinc exporter, keeping intracellular zinc at a minimal concentration of 0.2 mM (Outten, Outten and O'Halloran, 1999). In our plasmid construct we also incorporated the DNA sequence for mRFP (monomeric red fluorescent protein). Zinc-bound zntR will bind to zntAp upstream of the mRFP promoter, and ZntR then will transcribe zntAp to export mRFP out of the cell. This will demonstrate that sound stress successfully generated a zinc transduction signal to activate a genetic response. Ideally, the importation of one zinc ion will result in the expression of one hundred mRFPs. Furthermore, zinc-bound ZntR may also activate the ZntA exporter in the E. Coli genome, causing zinc to be exported.
**ZntA Pathway Mechanism** For sound stress to activate 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 (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 most likely bind in our selected zntAp region within -127 b.p. before the transcription start site (T.S.S.) of zntA. In natural systems, ZntR binds to the zntAP spacer region of -35 and -10 b.p. 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, ZntR may also transcribe the gene zntA to express the ZntA zinc-exporter protein, which locates itself on the inner PM. ZntA will then draw cytosolic Zn(II) and couple its export with ATP hydrolysis, for which each exportation of a singular zinc ion will result ADP, inorganic phosphate, and water (Rensing, Mitra and Rosen, 1997).
*Learn More About Mechanosensitive Channels* Other ionic mechanotransduction systems exist in E. Coli that are similar to zinc and can be linked to gene expression. Calcium, zinc, and magnesium are interactive divalent signaling molecules in eukaryotic immune cells and exhibit similar traits in E. Coli. And 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). From this, we infer that a variety of signaling ions can be transduced through mechanosensitive channels. The most common "Msc" channels in E. Coli are MscM (mini-conductance), MscS (small conductance), and MscL (large conductance). Calcium is known to travel through eukaryotic mechanosensitive channels Piezo1 and TRPC5 (2016.igem.org, 2018), as well as the MscS channel(2016.igem.org, 2018). It is therefore very likely that magnesium and zinc also travel through the MscS and/or MscL channels. The MscK (potassium-dependent) MS channel is uniquely specific for potassium.
There is much context for the performance of other ions in comparison to zinc. Calcium is 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 often 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).
**RpoE System** Our second system involves the protein-directed detection of sound stress. RpoE, the sigma 24 factor of RNA polymerase, is known for its response to stress on outer membrane proteins (OMPs) and membranous lipopolysaccharides (LPS). Such stresses include misfolding of OMPs, heat shock, osmotic shock, and change in membranous LPS structure (Rouvière et al., 1995). A protein-based signal cascade activates RpoE's transcription, allowing it to activate a number of stress-response genes. For our system a sound-activated RpoE will bind to bamEp within a region of -41 b.p. before the T.S.S. to upregulate the upstream mRFP sequence. Additionally, RpoE can increase expression of bamE in the E. Coli genome, which produces an OMP, BamE, that's responsible for maintenance of OMP aggregation and membrane permeability (Rigel et al., 2011).
**RpoE Pathway Mechanism**To activate RpoE in our system, sound stress must strike the E. Coli cell culture and denature the OMPs, causing them to bind to protease DegS in the periplasmic space. DegS then cleaves RseA (regulator of RpoE), which tethered RpoE to the inner plasma membrane. Once RpoE is released, it is free to bind to RNAP and transcribe its own gene and many other genes, such as bamE. Once bamEp is activated by RpoE, mRFP can be activated and translated, and a byproduct of BamE may also be produced, which can now repair the outer membrane. A positive feedback loop increases the concentration of RpoE until the stress is reduced and degradation of RseA slows down (Ades, Grigorova and Gross, 2003).
**OsmC System**Our particularly advantageous third system involves the promoter OsmCp1 of the cytosolic peroxiredoxin, OsmC, that is stress-activated by two different transcription factors that are independent of each other: RcsB and NhaR. OsmC expression is induced under osmotic pressure (Toesca et al., 2001), a mechanical force similar to sound; its function is to defend against the oxidative stress of organic hydroperoxides (Lesniak, Barton and Nikolov, 2003). OsmCp2 is another promoter of the OsmC system and is also induced by osmolarity, but it is activated upon entry into the stationary phase and transcribed by RpoS. We have incorporated the mRFP DNA sequence upstream of OsmCp1 in our plasmid, hoping for a twice as likely chance of activation, as well as the possible byproduct of OsmC from the E. Coli genome.
**Rcs Pathway Mechanism**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. Originally known for regulating capsular polysaccharide synthesis, other regulated functions include cell division, activity of periplasmic proteins (i.e. OsmC), motility, biofilm formation, etc. Protein RcsC acts as a sensor kinase whose histidine domain autophosphorylates in response to an environmental signal such as the alteration of the outer membrane proteins, and then transfers the phosphate group in a conserved manner to inner membrane phosphotransferprotein RcsD, and then to the aspartate residue of the cytosolic response regulator RcsB (Davalos-Garcia et al., 2001). Once RcsB is phosphorylated, its transcriptional capability is enhanced and it can bind to OsmCp1.
**NhaR Pathway Mechanism** NhaR, while independent of RcsB, is necessary and powerful inducer for OsmCp1's activation in response to osmotic shock. More specifically, NhaR transcribes OsmCp1 in the presence NaCl, LiCl, and sucrose by binding to one of these solutes in the cytosol and then to the promoter sequence (Toesca et al., 2001). Furthermore, increased ionic osmolyte concentration has been shown to stimulate induction of OsmCp1 (Toesca et al., 2001). If either NhaR or Rcs activate OsmCp1 in response to sound, measurable expression of mRFP should result. We selected a promoter region of -191 b.p. before the T.S.S. of the osmC gene for our system; and in the E. Coli genome, the sequence that's necessary for NhaR activation is adjacent to the -35 OsmCp1 region.
**PspA System** The pspABCE operon of the "Phage Shock Protein" represents our fourth system, which responds to membrane stresses that deplete energy. This includes phage infection, osmotic shock, exposure to hydrophobic organic solvents, blockage of Sec machinery for protein export, etc.(Brissette et al., 1991). PspA of the inner membrane specifically maintains the membrane potnetial by suppressing proton leakage of damaged membranous phospholipids and of damaged liposomes, and it's also unique: while serving as a proteinaceous response to membrane damage, it may respond to sound by involving the activity of mechanosensitive channels. If sound disrupts the membrane and thereby damages the proton motive force (PMF), simultaneous Msc channel activation may re-allow the protons back in, as PspA functions to restore the proton motive force and expel the protons. Therefore for our system, we selected promoter sequence pspAp within -142 b.p. before the T.S.S. for pspA; naturally, bound PspF (the transcriptional activator for pspABCE) binds between -127 and -111 b.p. before the T.S.S. Our mRFP sequence will be located upstream of pspAp.
**PspA Pathway Mechanism**Under normal cell conditions PspA suppresses its own expression by inhibiting PspF. During membrane damage and loss of PMF, 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 (Jovanovic et al., 2006). Sound stress may be able to damage the bacterial membrane or disrupt the proton motive force, to which PspA may respond to, if transcribed successfully for the E. Coli genome.
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. We hypothesize sound may cause membrane perturbations, altered membrane tension, protein misfolding, and increased growth rates. Membrane perturbations are tested using the pspA system. Increased membrane tension is tested using the zntA system. Protein misfolding is explored with bamE design. The osmC system tests the upregulation of cell surface proteins, cell growth pathways, and protection proteins against a changing environment.
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 channels respond to mechanical stimuli by transmitting ions and electric flux via a gate-opening mechanism, changing the cell's membrane potential(Martinac, Adler and Kung, 1990). 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 (Malcolm, Blount and Maurer, 2015). 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, RcsB, and NhaR, are to protect the cell from damage. NhaR, for example, binds to an intracellular ionic solute to stimulate transcription of stress-response gene OsmC; and PspA is foreseen to rely on the hydrogen protons that have entered through mechanotransduction, in order to fulfill its role of restoring the proton motive force after sound stress damage. These combinations of response mechanisms further leads us to believe that sound stress can induce a genetic response in the E. Coli cell.
BIOFAB Collection
In order to mitigate the risk of the sound inducible project we decided to add constitutive promoters from the BIOFAB project. This project’s goal was to create a library of promoters that would allow for consistency in the synthetic biology community, we took these parts and made them available to the iGEM community. We then tested three promoters apFAB46, apFAB82, and apFAB90 from this collection with high, medium and low activities, respectively. The plasmid maps shown describe the design of our plasmids for the BIOFAB test devices. Testing these promoters consisted of adding the BIOFAB promoter, shown in bright green, to RFP, shown in blue, to a chloramphenicol resistant plasmid. These plasmids were transformed to the NEB-5-alpha and grown to stationary phase. We used a spectrophotometer to test fluorescence at OD/700 and compared them to a negative control with no RFP and a positive control with the iGEM RFP control BBa_J04450.
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