The Pixcell construct was designed to test whether we could control the expression of GFP by controlling the oxidation state of SoxR through our electrochemical set up.

Using the Golden Gate assembly method, with two inserts; the sensing portion of the soxRS regulon and GFP, which were originally amplified out from the E. coli genome and a storage vector respectively, the construct was assembled. Gel electrophoresis showed that a construct of approximately the right length was produced and the construct was then sequence verified.

In order to lower the basal level of GFP expression a novel degradation tag was added. Therefore, any increase in GFP expression induced by the oxidation of SoxR would be more predominant.

The PixCell construct enables us to test whether we can control gene expression by controlling the oxidation state of SoxR. The PixCell construct DegTag enables the system to be more dynamic in that seeing the system switch from on to off is quicker.

These results demonstrate that the assembled PixCell circuit can be switched on by oxidative signals and switched off by reductive signals. These experimental data were also handed over the dry lab, where information about fold induction and degradation rates supported and further implemented the theoretical models.

The construct was first tested with a range of pyocyanin concentrations (0-100μM) to measure the response of the system to the redox-cycling drug. High-concentrations of pyocyanin exert significant stress upon the cell leading to cell death. A working concentration of 2.5μM of pyocyanin is therefore recommended when using it as an inducer or within an electrogenetic device, as it provides an optimal trade-off between fold induction and cell viability.

A significant sixfold induction in fluorescence was seen in the device when comparing values at 0μM and 2.5μM of pyocyanin, as shown in Fig. 3.

Oxidisation of pyocyanin, without intentional induction, was observed in the aerobic environment, as evidenced by the response of the cells. To prevent this behaviour, sodium sulfite (an oxygen scavenger) was added to the medium to maintain an OFF state in the absence of deliberate induction. Without this innovation, the device could only work in anaerobic conditions, limiting the potential of electronic induction of gene expression. A final sodium sulfite concentration of 0.02% was selected for the electrogenetic system as it prevented GFP expression from an agar plate in aerobic conditions with 2.5μM pyocyanin and 2.5mM ferrocyanide.

We observed that the oxygen scavenger sodium sulfite reduces pyocyanyn in a linear relationship until a concentration of 2.5% sulfite added. At these concentrations, we observed electrochemical interference when perfoming square-wave voltammetry experiments. We hypothesised that sulfites could react with pyocyanin, producing pyocyanin sulfonate, which has an absorption peak at 400 nm

The results shown in figure 5 supported the hypothesis that pyocyanin sulfonate is produced at high sulfite concentration. Therefore, we performed the next experiments at sulfite concentrations between 0 and 0.03% (figure 6), which appeared to fall in the linear region of the graph.

Next the concentration of ferrocyanide was optimised, as shown in figure 7. A range of ferrocyanide and ferricyanide concentrations (0-100mM) were tested using 2.5μM pyocyanin and 0.02% sodium sulfite. This was done in order to find a single concentration at which ferrocyanide would provide no GFP expression whereas ferricyanide would allow for a large induction of GFP expression. The final condition of 10mM was selected for an optimal fold change; these concentrations also did not significantly impact cell growth. A 10mM concentration of ferricyanide provided at least an order of magnitudefold induction of GFP expression compared to ferrocyanide. Therefore if bulk oxidation of ferrocyanide to ferricyanide could be achieved then gene expression could be electronically induced in aerobic conditions.

In figure 8, we demonstrate that the chosen conditions are sufficient to produce high GFP expression when oxidised ferricyanide is added into the plate and low GFP when the same concentration of reduced ferrocyanide is added.

These experiments allowed us to demonstrate that our constructs are responsive to oxidising input and enabled us to identify the concentrations of redox-molecule. This allowed us to select a suitable chemical composition of 2.5uM Pyo, 0.02% sodium sulfite and 10mM ferrocyanide, which works both in liquid and solid cultures.

These results show that our set-up is able to selectively and reversibly oxidise and reduce our redox modulators. Furthermore , by performing these electrochemistry experiments, we proved that addition of the reducing agent sodium sulfite enables complete chemical reduction of the system, even in aerobic environment.

These results describe the first ever aerobic electrogenetic control system, that works both in liquid and solid E. coli cultures.

The results below serve as proof-of-concept for our electrode set-up

An electrode rig was set up to apply the potentials identified above to cells grown on an agar plate containing the final reaction condition of 2.5μM pyocyanin, 0.02% sodium sulfite and 10mM ferrocyanide. Redox reactions only occur at the electrode surface during an electrochemistry experiment, meaning that oxidised pyocyanin and ferrocyanide were only produced in close proximity to the working electrode upon the application of a +0.5V pulse. Fluorescence images of agar plates clearly show localised expression of GFP around the working electrode (W), as observed in fig. 4 and 5. This not only shows that the device allows for electronic control of gene expression, but also demonstrates high spatial control meaning it can be used for programmable spatial patterning of cell populations.

Figure 1. Setup of the electrode rig for the electronic control of spatial gene induction

Figure 2. Image of the electrode rig placed in the incubator.

Figures 1 and Image 2 illustrate the setup of the electrode rig. The working, counter and reference electrodes are penetrating the plate through holes. Cells were plated onto an agar plate containing the final working concentrations: 10mM FCN(R), 2.5 uM pyocyanin, 0.03% sulfite, ampicillin and kanamycin. Agar is fully covering the electrodes which are controlled by a potentiostat. An oxidising potential of +0.5% was applied.

Figure 3. Expected outcome for the electronic control of gene expression on an agar plate

Figure 3 illustrates the expected increased fluorescence around the working electrode. The oxidising potential results in bulk oxidation and activation of our system.

Figure 4. GFP expression after electric stimulation

We demonstrated elevated GFP expression around the working electrode in figure 4.

Figure 5. Comparison of PixCell Construct, PixCell Construct Deg Tag, positive control and negative control after application of an oxidising potential.

We next investigated GFP expression of PixCell Construct and PixCell Construct Deg Tag relative to the negative control (DJ901) and the positive control which constitutively expresses GFP. Fluorescence image analysis shows increased fluorescence around the working electrodes demonstrating electronic control of spatial gene expression. The addition of a degradation tag to GFP substantially reduces background fluorescence.

Figure 6. 3D surface plot showing relative fluorescence of PixCell Construct, PixCell Construct Deg Tag, positive control and negative control after electric stimulation. Clear peaks are evident at the working electrode sites for cells containing the PixCell Constructs.

To better visualise the results 3D surface plots were made showing GFP expression in the z axis. GFP expression level from the positive and negative control was used to set a relative scale of GFP expression. Peaks around the working electrodes clear indicate increased GFP expression. PixCell construct Deg Tag showed lower background fluorescence compared to PixCell construct.

Figure 7. Comparison of PixCell Construct, PixCell Construct Deg Tag, positive control and negative control after application of a reducing potential

Next the 4 cell types were exposed to a reducing potential of -0.3V (figure 7). Working electrodes do not show increased fluorescence. Higher background fluorescence in the plate with cells containing PixCell Construct compared to the the previous fluorescence images exposed to an oxidising potential is due to variation in spreading.

Figure 8. GFP expression on a plate with final conditions in the absence of pyocyanin

After successfully inducing spatially controlled activation of gene expression the system was tested in the absence of the redox modulator pyocyanin. This suggests that there is an electrode-induced mechanism for triggering the pSoxS pathway in the absence of pyocyanin. The plate contains 10mM FCN(R), 0.02% sulfite, Ampicillin, Kanamycin. Results show that increased expression levels of GFP can be achieved around the working electrode in the absence of pyocyanin. Further fine tuning of the system will be required to achieve the level of precision obtained on a plate containing our final working conditions.

These results demonstrate precise electronic control of spatial gene expression, in solid cultures grown in aerobic condition.

These results expand the electrogenetic synthetic biology toolkit.

These results allowed us to evaluate whether we had created parts that could be used in a modular fashion

Taking the steady state fluorescence values we can show GFP expression (and therefore construct activity) as a function of pyocyanin activity. This shows a standard sigmoidal behaviour as predicted by our model and exhibited by PixCell Construct.