The E. coli Promoter collection [5] is delivered in 21 separate 96 well plates. Each well contains a strain stored in glycerol with a different promoter upstream of a Green Fluorecent Protein(GFP) coding sequence.

We used our custom 96 Well Plate Replicator to replicate the plates AZ_01 to AZ_05 of the promoter collection (a complete list of the library as well as the content of individual wells can be found here). We also prepared a plate with curated candidate promoters known to be implicated in the stress response, Redox sensing, Amino Acid synthesis and Ion Channel production in E. coli.

Cells were grown overnight in M9 minimal media (0.4% Glucose, no amino acids). this media was used to limit intefering substances found in rich media. In the morning the cells were diluted 1:100 in 96well plates with fresh media

We based our chosen electroshock amplitudes on those of a precedent paper [13] which produced different dynamics of membrane potential changes. 1 volt showed a strong un-sustained hyperpolarisation of the membrane, while 3 volts produced a less strong, sustained hyperpolarisation of the membrane.

Initial Screening

We used the High throughput Electro Actuator (HTEA) in conjunction with the AC Dispatcher (ACD) and the Electro Planner to shock the bacteria with AC current in 3 different conditions:

• Condition A is a negative control where cells were grown without electrical shock exposure.
• Condition B cells were exposed to an AC current of 1 volt, 100HZ, sine wave for 5seconds.
• Condition C cells were exposed to an AC current of 3 volts, 100HZ, sine wave for 20seconds.

After exposure to AC, cells from the 3 condition were left to grow for 2 hours before measuring GFP expression with a Flow cytometer.

For each well, we looked at the mean fluorescence of the cells, compared the expression of each electrical shock condition (B & C) to the control condition (A), and measured the fold change in fluorescence.

We discarded wells where fold change was found in between 0.6 and 1.4 as we considered these less significant.

This fold change threshold yielded 31 promoters with significant fold change.

To verify the ability of these promoters to respond to electrical signals as well as to characterise their activity, the previous 31 candidates were grown overnight in triplicates in a single 96-well plate in M9 minimal media (0.4% Glucose, no amino acids).

Cells were diluted 1:100 in 96-well plates with fresh media.

We repeated the 3 conditions used in the previous experiment and added 2 new conditions:

• Condition B cells were exposed to an AC current of 1 volt, 100HZ, sine wave for 5seconds.
• Condition C cells were exposed to an AC current of 3 volts, 100HZ, sine wave for 20seconds.
• Condition D cells were exposed to an AC current of 1 volt, 100HZ, sine wave for 5 seconds every 15 minutes for 2 hours (cells were put back in the incubator in-between the shocks).
• Condition E cells were exposed to an AC current of 3 volts, 100HZ, sine wave for 20 seconds every 15 minutes for 2 hours (cells were put back in the incubator in-between the shocks).

After exposure to AC , cells from conditions A, B and C, were left to grow for 2 hours before measuring GFP expression for all conditions with a Flow cytometer.

We used 2 different selection methods to validate positive electrical response of promoters:

In a second selection, the mean fluorescence of the triplicates was measured and filtered through the same threshold values to be validated as positive results.

This screening approach yielded 8 promoters with inducible activity and 11 promoters with repressive activity.

A complete list of these promoters can be found in our Part Collection

Our electronic screening test is high-throughput, but not exhaustive. More plates from the promoter library remain to be screened. Further electroshock conditions should be tested.