Team:FSU/Design
Introduction
The Effects of Sound on Bacteria
Investigators report that the growth rate of bacteria increases when exposed to audible sound (Lee Ying, Dayou and Phin, 2009). Increased E. Coli growth rates were observed when cultures were exposed to 5 kHz sound (Lee Ying, Dayou and Phin, 2009). Other studies indicate that E. Coli exposed to a wide range of frequencies, 500 Hz to 16 kHz, generally showed an increased biomass, in particular, 8 kHz was found to increase protein and RNA concentration (Gu, Zhang and Wu, 2016). The investigators reported a positive correlation between sound power, measured in decibels, and bacterial biomass (Gu, Zhang and Wu, 2016). Other studies report that bacteria generate sounds that may enhance the growth rates of neighboring bacteria (Matsuhashi et al., 1998). The investigators found that frequencies of 6-10, 18-22, and 28-38kHz promoted B. Carboniphilus growth rates (Matsuhashi et al., 1998). They also found that B. subtilis emits sound between 8 kHz and 43 kHz, with the most intense waves at 8.5, 19, 29, and 37 kHz. The studies we reviewed point to the possibility that E. coli could be induced to express a gene using sound. Our project pursues this possibility.
2008 Berkeley iGEM Team
The 2008 U.C. Berkeley iGEM team uploaded several sequences expected to contain promotors activated when an Escherichia Coli cell was exposed to sound. For characterization of previous iGEM parts, the FSU 2018 iGEM team further investigated Berkeley’s parts to test these sequences, which are found naturally in E. coli. We designed five devices which include the Berkeley putative promotors and the red fluorescent protein coding sequence. The designs of the "Berkeley Promoter Test Cells" will be shown during the presentation and poster at the iGEM Giant Jamboree. .
Design Principles
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. Since sound is ultimately a wave of pressure, it can be considered to be a mechanical force that can create a local stress, such as membrane tension. 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
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. For example, if there are too many solutes in the cell's cytoplasm, water will flow into the cytoplasm to lower the osmolarity, creating a turgor pressure that pushes outward against the inside of the plasma membrane; once a certain pressure is reached, the MS channels will open (Booth, 2014).
Our Sound-Induced Systems
Two general systems were employed in our experiments.
The first utilized ions as ligands activating a sensing mechanism within the cytoplasm. When the ions' intracellular concentration increased, so would the bacterial pathway. We searched for ions that tend to be exported from the cell and that have a relatively low intracellular concentration. When a mechanosensitive channel opens the ions are able to diffuse quickly down their concentration gradient. Two component systems, designed to detect a certain ion, are frequently utilized to detect this influx.
The second system involves membrane stress response pathways. Sound stress could trigger pathways important for adapting to the environment and protecting membrane integrity. This system does not rely on the addition of any substances to a bacteria culture, only sound frequencies. Sound can potentially cause significant stress to the E. Coli membrane and membrane proteins.
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.
OUR SOUND INDUCIBLE SYSTEMS
Our first design 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. zntR is a zinc-dependent transcription factor and will detect the influx of zinc ions into the cell cytoplasm. 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 promoter for zntA, an ATPase zinc exporter, keeping intracellular zinc at a minimal concentration of 0.2 mM (Outten, Outten and O'Halloran, 1999). msc opening due to sound pressure will be indicated using an mRFP signal. mRFP is incorperated into our plasmid downstream of the zntA promoter.
zntA Pathway Mechanism
Sound, acting as a mechanical pressure wave, has the capacity to open mechanosensitive channels. The opening of an msc will allow in surrounding molecules including zinc ions. zntR is an E Coli zinc detection protein. zntR requires the binding of 4 zinc ions before it can be an active transcription factor. The zinc-zntR complex has an affinity for the binding site at -35 and -10 upstream of the zntA promoter.
Mechanosensitive Channels
Other ionic mechanotransduction systems exist in E. Coli that are similar to our zntA design. Unlike most eukaryotic channels, bacterial MscS and MscL are unbiased in their selection of molecules and ions. MscS and MscL allow in molecules up to 1,000 M.W. (Peyronnet et al., 2014). This implies that a variety of signaling ions and macromolecules 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 iGEM to travel through eukaryotic mechanosensitive channels: Piezo1 and TRPC5 (Fan et al., 2016), and E. Coli MscS (Leben et al., 2016) upon opening with sound pressure. It is therefore very likely that zinc will also travel through the MscS and/or other msc channels. Other E. Coli mechanosensitive channels include MscK, MscM, YnaI, YbiO, YbdG.
Compared to other ions, zinc exhibits an ideal physiological nature for our system. Calcium and magnesium are well-known signaling ions; however, calcium signaling is often inhibited by zinc and magnesium ions. Furthermore, magnesium has a better binding affinity for oxygen and is more often bound as a cofactor (Chaigne-Delalande and Lenardo, 2014). Magnesium, potassium, and sodium also exhibit a smaller chemical gradient across the plasma membrane than zinc does, and therefore they have a tendency to be imported into the cell (F C Kung, 2018). And unlike other transition metals that are bound as cofactors, zinc is often ionized in physiological solutions, implying a regulatory role in ion channels (Elinder and rhem, 2003).
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. RpoE can upregulate BamE, which is an OMP that is responsible for maintenance of OMP aggregation and membrane permeability (Rigel et al., 2011). For our system a sound-activated RpoE will bind to bamEp within a region of -41 b.p. before the transcription start site to upregulate mRFP.
RpoE Pathway Mechanism
To activate RpoE in our system, sound stress must strike the E. Coli cell culture and denature an OMP. A misfolded OMP binds to the protease DegS in the periplasmic space. DegS is then able to cleave RseA/B, which physically tethers RpoE to the inner plasma membrane. Once RpoE is released, it is free to bind RNA Polymerase and form the RNA Polymerase Holoenzyme. The addition of RpoE to RNA Polymerase is essential for the upregulation many genes, including its own and bamE. In our design we use the bamE promoter to upregulate mRFP. A byproduct of our system is BamE, which is a repair enzyme for 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).
Our third system involves the promoter OsmCp1 of the cytosolic peroxiredoxin, OsmC. This promoter is particularly advantageous because it is stress-activated by two different transcription factors that are independent of each other: RcsB and NhaR. Expression of OsmC occurs under osmotic pressure (Toesca et al., 2001) and 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. To investigate this system, we have incorporated the mRFP DNA sequence upstream of OsmCp1 in our plasmid, expecting a high chance of activation of OsmCp1. Also possible is the 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 was originally known for regulating capsular polysaccharide synthesis in response to external effects on the plasma membrane. Studies now show that it regulates critical cellular functions. These include cell division, activity of periplasmic proteins (i.e. OsmC), motility, biofilm formation, etc. Looking at the mechanism: RcsF is an OMP that activates RcsC for the signal transduction by reducing the inhibitory effect of IgaA on the Rcs system. 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 to inner membrane phosphotransferprotein RcsD in a conserved manner, and then to the aspartate residue of the cytosolic response regulator RcsB (Davalos-Garcia et al., 2001). Once RcsB is phosphorylated, its enhanced transcriptional capability allows it to bind to OsmCp1.
NhaR Pathway Mechanism
NhaR is a powerful inducer for OsmCp1's activation in response to osmotic shock. 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). 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 have 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.
The pspABCE operon of the "Phage Shock Protein" represents our fourth system, which responds to membrane stresses that deplete energy. This system utilizes a membrane stress response as well as an ion signal. Membrane stresses include phage infection, osmotic shock, exposure to hydrophobic organic solvents, blockage of Sec machinery for protein export, etc.(Brissette et al., 1991). The PspA system also responds to the presence of holes in the membrane (i.e. an open Msc channel) as well as loss of the proton motive force (PMF). It aids the damaged PMF by blocking proton leakage of damaged membranous phospholipids and of damaged liposomes (Jovanovic et al., 2010) . Loss of the PMF can be caused by a force opening an MscS channel, resulting in an influx of protons. If Msc channel activation by sound disrupts the PMF and allows the protons in, PspA can restore it by expelling 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 (the pspABCE operon) by inhibiting PspF ATPase activity. The image shows that during membrane damage (including loss of PMF), the pspBCE cascade involves PspB and PspC transducing the signal to PspA, thereby suppressing it and relieving its inhibitory effect on PspF (Jovanovic et al., 2010). PspF is then free to upregulate several genes including the pspABCE operon and the promoter, pspAp (Jovanovic et al., 2006). PspA and PspD are also known to regulate the redox (metabolic) state of the cell (Jovanovic et al., 2006). And while it is unknown how the periplasmic PspE protein specifically contributes to the operon's function, it is established as a periplasmic sulfurtransferase for the system (Adams et al., 2002). If pspABCE is transcribed successfully for the E. Coli genome, it may respond to sound damage on the PMF.
