Part:BBa_K2862021

PixCell Construct with deg tag

This part consists of a repurposed version of the soxRS regulon from E. coli, consisting of SoxR and GFP with a novel degradation tag 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, thereby 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 could be used for programmable cell patterning.

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

Biology

The soxRS regulon in E. coli has the same architecture as this device, although transcriptional activation of pSoxS allows for expression of ~15 genes, instead of just GFP, 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, soxR 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. The electrical stimulus was transferred to the cells via oxidation and reduction of redox-mediators at an electrode, and their subsequent diffusion in the medium. These systems provide programmable spatiotemporal control of gene expression within 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 electrical stimulus. The device functions by oxidising pyocyanin and ferrocyanide with an electrode. Oxidised pyocyanin in turn oxidises SoxR to activate the device. The 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 as 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. A novel degradation tag was ligated before the STOP codon of GFP by overlap PCR in order to reduce leakiness of the device.

Figure 2: Circuit of PixCell construct Deg Tag.

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 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 sixfold induction in fluorescence was seen in the device when comparing values at 0μM and 2.5μM of pyocyanin, as shown in Fig. 4. Also shown are model curves that illustrate the predicted maximal response of the system were high levels of pyocyanin not toxic; fitting details are provided under the modelling tab on the wiki.

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.

Oxidisation of pyocyanin, without intentional induction, was observed in the aerobic environment. To prevent this behaviour, sodium sulfite (an oxygen scavenger) was added to the medium to maintain an OFF state in the presence 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.

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. 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 an tenfold 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.

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.

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. A reference electrode (Ag/AgCl) was placed in proximity to the working electrode to ensure a standard in voltage measurements. 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.

The system was also tested with an alternative redox-cycling drug: PMS (phenzine methosulfate). This was tested as part of our integrated human practices in order to reduce the toxicity of our system. A series of PMS concentrations (0-1 nM) were tested. As significant cell stress was observed above 0.3 nm it is recommended as the working concentration for the system.

Figure 8: Growth of cells containing the construct in a range of PMS 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.

This working concentration provided an tenfold increase in GFP expression. Previous research suggests this response could be improved ~10-fold further by supplementing the growth media with branched-chain amino acids, the synthesis of which is inhibited by PMS leading to cell death. PMS is also incredibly cheap, as detailed in the integrated human practices section of our wiki. This demonstrates the SoxR/pSoxS system can be used a cheap and effective tool for chemical, as well as electronic, gene induction.

Figure 9: GFP fluorescence normalised with respect to OD600 at a range of PMS 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.

Characterization/Improvement - 2019 NYU Shanghai

NYU Shanghai 2019 contributed to this part by developing a much simpler method. Instead of using potentiostat, we attached the alligator clips from the power supply and adjust the value of the voltmeter to 0.5V (Fig 10).

Figure 10: Making Sure that the Electric Setup has 0.5 V

We had the following settings for redox modulators: +/- 2.5 uM Pyocyanin; 5mM Ferricyanide; 5mM Ferrocyanide; and 0.02% Sodium sulfite. We made 4 plates for the analysis (Fig 11). The plates had the electric potential for 1 hour at RT and then cultured in the 37˚C incubator overnight. On the next day, the GFP expression level is quantified by pixel intensity analysis. The quantitative result is shown in Fig 12.

Figure 11: Agar Plate Setup

Figure 12: Results of PixCell Construct

As the result is shown in Fig 12, the electric stimulation properly worked on the existing parts in a much simpler method with a normal power supply than the previous method with an expensive potentiostat. This result characterizes this part and improves it since we introduced a much simpler method of electric activation and created the reporter gene of more complicated pathways regulated by electric stimulation, which serves as one of our Bronze and Gold medal criteria.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 790
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 49
  Illegal BsaI.rc site found at 1436