Difference between revisions of "Part:BBa K2862022"

Revision as of 01:50, 18 October 2018

PixCell Construct

This part consists of a repurposed version of the soxRS regulon from E. coli, consisting of SoxR and GFP being expressed from either side of the pSoxR/pSoxS bidirectional promoter. pSoxR provides constitutive expression of SoxR. When oxidised, either directly by redox-cycling molecules or by oxidative stress, SoxR binds and activates transcription of GFP downstream of pSoxS. This circuit acts as a reporter for various redox-cycling drugs, toxins, antibiotics, heavy metals, hydrogen peroxide and nitric oxide: providing various applications in the development of environmental and therapeutic devices. By coupling oxidation of redox-cycling species to an electrode, the 2018 Imperial College London iGEM team (PixCell) were able to activate this device electronically. They proved electronic pulses could induce spatially controlled expression of GFP about an electrode, demonstrating how electrogenetic control can be used for programmable cell patterning.

This part is optimised for use in a plasmid with a pMB1 origin and is compatible for BioBrick, BASIC and Golden Gate assembly.

Biology

The soxRS regulon in E. coli consists of the same architecture as this device, although transcriptional activation of pSoxS allows for expression of ~15 genes providing resistance to oxidative stress. SoxR acts as the sensor of the system. It is constitutively expressed from pSoxR providing a steady state of ~75 molecules per cell. Upon oxidation of SoxR in conditions of oxidative stress it activates transcription from pSoxS causing downstream activation of the soxRS regulon.

Usage

This device acts as a functional sensor of redox-cycling drugs and oxidative stress, making it a useful part for the creation of biosensors or devices activated by redox-cycling drugs, toxins, antibiotics, certain organic molecules, heavy metals, nitric oxide and hydrogen peroxide: all of which can exert oxidative stress on cells.

The 2018 Imperial College London iGEM project (PixCell) utilised SoxR in electrogenetic devices capable of activating gene expression in response to an electrical stimulus. This was achieved via oxidation and reduction of redox-mediators at an electrode. These systems provide programmable spatiotemporal control of gene expression with an inexpensive experimental set up.

The induction of this system by redox-cycling drugs makes it a particularly cheap system to use for chemical induction of gene expression, with the molecule PMS (phenazine methosulfate) being cheaper per reaction than several other common chemical inducers.

Parts within this device were redesigned as part of the PixCell library: a series of SoxR and pSoxS parts which allow for modulation of the response of this device.

Characterisation

PixCell, the 2018 Imperial College iGEM team, characterised this device in a series of steps in order to make it respond to an electronic input. The device functions by oxidising pyocyanin and ferrocyanide with an electrode. Pyocyanin oxidises SoxR to activate the device, allowing for activation of the device with the application of an oxidising potential at the electrode. This potential also oxidises ferrocyanide to ferricyanide, allowing electrons to be drawn away from pyocyanin via the quinone pool to amplify this response. The full details of the mechanism of the electrogenetic device alongside more in depth characterisation results are available on the PixCell Wiki.

Figure 1: Schematic illustration of electrochemical and biological modules.

This device was constructed by Golden Gate assembly using a colony PCR product of SoxR and pSoxR/pSoxS from the E. coli MG1655 genome as well a GFP part. Both of these parts included terminators downstream of their respective coding sequences. The device was integrated into a pMB1 plasmid so that copy-number was sufficiently high to provide detectable expression.

Figure 2: Circuit of PixCell construct.

The construct was firstly 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 health.

Figure 3: Growth of cells containing the construct in a range of pyocyanin concentrations, with untransformed DJ901 as a negative control and DJ901 transformed with a GFP expression cassette as a positive control. Data obtained from three biological replicas.

A significant X-fold induction in fluorescence was seen in the device when comparing values at 0μM and 2.5μM of pyocyanin. This response curve was also normalised with the fluorescence of the positive control in order to fit a sigmoidal response curve using our biological model. This shows the theoretical maximum fold-induction that can be achieved by the device.

Figure 4: GFP fluorescence normalised with respect to OD600 at a range of pyocyanin concentrations, with untransformed DJ901 as a negative control and DJ901 transformed with a GFP expression cassette as a positive control. Data obtained from three biological replicas. Model curves predict the response of the system in a hypothetical environment in which high levels of pyocyanin are not toxic.

Due to pyocyanin being oxidised in the presence of oxygen, sodium sulfite (an oxygen scavenger) was used to prevent this oxidation from occuring so the device could be maintained in an OFF state in the presence of pyocyanin. Without this, 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 the presence of 2.5μM pyocyanin and 2.5mM ferricyanide. Solution results show the fluorescence decrease is a result of changing the systems redox conditions rather than a lack of oxygen causing a decrease in GFP fluorescence because fluorescence of the positive control does not vary with sodium sulfite.

Figure 5: Agar plates of construct-bearing cells with varying sodium sulfite conditions, imaged under UV transluminescence, showing that 0.02% is sufficient to suppress unintentional induction.

Next the concentration of ferrocyanide was optimised. 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. A final condition of 10mM was selected because higher concentration of ferricyanide significantly impacted cell growth.

Figure 6: GFP flourescence as a function of ferricyanide and ferrocyanide concentration, taken at steady state (700 mins), normalised with respect to OD600, and averaged over three biological replicas, for circuits with and without deg tag and the same controls as Fig. 4. Experiments performed at a sodium sulfite concentration of 0.02% and with 2.5μM pyocyanin.

A 10mM concentration of ferricyanide provided an X-fold 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.

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Square-wave voltammetry was used to confirm that ferrocyanide was bulk reduced at -0.3V and bulk oxidised at +0.5V. An electrode rig was set up to apply these potentials 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 electrode. 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 7: Fluorescent images of agar plates with 2.5μM pyocyanin, 0.02% sodium sulfite and 10mM ferrocyanide and subjected to sustained voltages via electrodes. Top left: construct without decay tag. Top right: construct with decay tag. Bottom left: positive control (DJ901 transformed with a GFP expression cassette as a positive control). Bottom right: negative control (untransformed DJ901). Successful functioning of the circuit is shown by the induction of additional fluorescence in the vicinity of the working electrode for the construct-containing cells at positive applied voltages, but not in the vicinity of working electrodes for negative applied voltages.

Sequence and Features