Experiments | Paris_Bettencourt

Impact on Growth of electroshock

Abstract

Membrane potential (Vmem) is known to take part in a wide variety of processes in bacteria (from powering the flagellar stator[1] to powering the activity of the ATP synthase). Recently, Vmem has been shown to play a role in the regulation of metaboollic processes through time and space in a growing biofilm of B. subtilis[2].

As more work tend to uncover the dynamical nature of Vmem in bacteria, the relationship between gene expression and the electrical state of the cells remain largely unexplored.

From a synthetic biology perspective this represent an opportunity to control of gene regulatory networks through control of membrane potential.

To explore this route, we conducted a large scale high throughput screening experiment where a Library of promoter fused to reporters is exposed to AC current dispatched by our custom Hardware toolkit.

The following experiment was conducted in preparation of the screening and with 2 objective in mind: 1) Validate the capacity of our hardware and methods to induce changes in membrane potential, and 2), to direct the screening experiment toward a small range of parameters.

We used the [High throughput Electro Actuator (HTEA)](https://www.notion.so/f0034928f1414de6a9a001c32dd2694f) in conjunction with the AC Dispatcher (ACD) and the Electro Planner to shock the bacteria with AC current of different amplitudes (from 0.5volts to 4.5volts) and with varied pulse lenght. Fluorescence of cells stained with a dye reporting membrane potential was measured using Flow cytometry at different time points.

The result shows that our harware and methods are capable of inducing a membrane potential change and that the dynamics of this change is dependant on the nature of the signal sent

Materials and methods

As previous experiments [3] failed in ruling out electrical signals (as none of the signals what the HTEA was capable of gating provoke complete or major growth arrest), we turned out to a range of voltages that have been shown capable of inducing changes in membrane potential[4].

We used the HTEA in conjunction with the AC Dispatcher (ACD) and the Electro Planner to shock the bacteria with AC current of different amplitudes (from 0.5volts to 4.5 volts). Frequency and pulse durations were always respectively 100Hz and 5 seconds. All AC current waveforms were sine waves.

We used the MHS2300A dual function generator to generate the sine waves and the MHS2300A Python library to communicate with the Electro Planner.

The day before conducting the experiments, the cells were grown to saturation overnight in M9 minimal media (0.4% Glucose, complete Amino Acids).0

On the morning of the experiement cultures were rediuluted in fresh medium to a ratio of 1:100. Cultures were grown for 4 hours to reach mid log phase accroding to growth curved obtained in a previous experiment[5].

Experiments were done in triplicates.

High throughput Electro Actuator (HTEA)

Results

The following figure show Thioflavin T fluorescence right after the electrical stimulation (t=0) with different applied voltages. Negative control are populations of bacteria where no electrical stimulation have been applied. Error bars are standard deviation.

ThT is a positively charged cationic dye. Its ability to permeate the bacterial membrane depend on the charge content of the cell. As a more negatively charged cell will collect more ThT, hyperpolarisation result in higher fluorescence signal

We can see that different applied AC current yield varied response with a clear specific increase at 1volt Peak to Peak (P.P.)

We looked at the dynamics of fluorescence of ThT and took samples at t=30minutes and t=90minutes

We observe different dynamics with a clear instantaneous (t=0) hyperpolarisation effect for stimulation at 1V. In this condition Vmem reach control level at t=30.

Interesting behaviour occurs for stimulation at 1v where a small hyperpolarisation visible at t=0 seems to be sustained at t=30 before reaching control levels at t=90.

Future Work

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References

1. Biquet-Bisquert, A. *et al.* (2021) ‘The Dynamic Ion Motive Force Powering the Bacterial Flagellar Motor’, *Frontiers in Microbiology*, 12, p. 659464. Available at: https://doi.org/10.3389/fmicb.2021.659464

2. Prindle, A. *et al.* (2015) ‘Ion channels enable electrical communication in bacterial communities’, *Nature*, 527(7576), pp. 59–63. Available at: (https://doi.org/10.1038/nature15709

3. Impact on Growth of electroshock

4. Stratford, J.P. *et al.* (2019) ‘Electrically induced bacterial membrane-potential dynamics correspond to cellular proliferation capacity’, *Proceedings of the National Academy of Sciences*, 116(19), pp. 9552–9557. Available at: https://doi.org/10.1073/pnas.1901788116.

4. Growth curve on M9

Buttress, J.A. et al. (2022) ‘A guide for membrane potential measurements in Gram-negative bacteria using voltage-sensitive dyes’, Microbiology, 168(9). Available at: https://doi.org/10.1099/mic.0.001227.