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 28-38 kHz 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 promoters 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 promoters 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 bacterial cell. While there are few studies that focus on sound as an environmental stimulus on cells, 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 a mechanical force that can create a local stress. Sound may possibly 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 the 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 (MS) 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 channels will open (Booth, 2014).
Our Sound-Induced Systems
Two general systems are employed in our experiments.
The first system utilizes ions as ligands to activate a sensing mechanism within the cytoplasm. When an ion's intracellular concentration increases, its bacterial pathway can activate. We searched for ions that tend to be exported from the cell and that have a relatively low intracellular concentration. When an MS channel (Msc) 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 can 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 its proteins.
OUR SOUND INDUCIBLE SYSTEMS
zntA System
Our first design uses sound as a mechanical force to open an mechanosensitive channel within the E. Coli membrane, allowing the diffusion of ions into the cell. We utilize ZntR, a zinc-dependent transcription factor for E. Coli that will detect the influx of zinc ions into the cell cytoplasm. Zinc is an ideal choice for our mechanosensitive system due to its slow exchange between its intracellular and extracellular pools, its low cytosolic concentration, and its tendency to be extruded from the cell (F C Kung, 2018). Our plasmid incorporates the promoter for zntA, the E. Coli gene for an ATPase zinc exporter, which keeps intracellular zinc at a minimal concentration of 0.2 mM (Ammendola et al., 2007). For our system, MS channel opening due to sound pressure will be indicated using an mRFP signal. mRFP is incorporated into our plasmid downstream of the zntA promoter.
zntA Pathway Mechanism
The opening of an MS channel by sound will allow in surrounding molecules including zinc ions. 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 promoter zntAp.
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, allowing 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. We formulate that zinc will also travel through the MscS and/or other Msc channels. Compared to other ions, zinc exhibits an ideal behavior 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, having 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 the gene for BamE, 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. The 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, while BamE is a byproduct of our system. 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 independent transcription factors: 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 MscS 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 a force such as sound causes this disruption, PspA can restore the PMF 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, 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.
BIOFAB Collection
We understood that sound-inducible promoters had a high risk of not working. We pursued a lower risk project in parallel in order to have parts that had a higher probability of working. We decided to add constitutive promoters from the BIOFAB project to the iGEM Parts Registry. The BIOFAB project’s goal was to create libraries of engineered promoters, ribosome binding sites, and terminators that were well-characterized and freely available. We selected apFAB46, apFAB82, and apFAB90 from the BIOFAB Modular Promoters library and designed, built, and tested three new generators. The three promoters were expected to have high, medium, and low strength.
The design of the generators developed to test the BIOFAB promoters are described below.
Figure 1 - The design diagram describes the apFAB46 Test Cells that contain the apFAB46 Test Device (BBa_K2832014). The apFAB46 Test Device contains the apFAB46 promoter (BBa_K2832100), ribosome binding site BBa_B0034, coding sequence BBa_E1010, and the terminator BBa_B0015. The device was inserted into the plasmid backbone pSB1C3. The map of the plasmid pSB1C3-BBa_K2832014 is below the design diagram. pSB1C3-BBa_K2832014 was used to transform the E. coli NEB 5-alpha and NEB Express chassis.
Figure 2 - Design diagram of the apFAB82 Test Cells and the map of the plasmid pSB1C3-BBa_K2832015. The design pattern is the same as the apFAB46 Test Cells depicted in Figure 1.
Figure 3 - Design diagram of the apFAB90 Test Cells and the map of the plasmid pSB1C3-BBa_K2832016. The design pattern is the same as the apFAB46 Test Cells depicted in Figure 1.