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).

The channels respond to mechanical stimuli by conformational change; a gate-opening mechanism then allows the transmitting ions and electric flux, 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). Previous iGEM experiments with eukaryotic mechanosensitive channels, TRPC5 and Piezo1, have shown sound-mediated opening (Fan, J et al., 2016).

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.

OUR SOUND INDUCIBLE SYSTEMS

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.

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.