Team:Imperial College/Demonstrate
Results
Building and Testing Pixcell Constructs
Assembling the Pixcell Constructs
The PixCell Construct 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 molecule or by oxidative stress, SoxR binds and activates transcription of GFP downstream of pSoxS.
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.
Pixcell construct was assembled together using the Golden Gate assembly method. With the two inserts; the SOXRS regulon and GFP, being originally amplified out from the E. coli genome and a storage vector respectively. Gel electrophoresis showed that a construct of approximately the right length was produced and the construct was then sequence verified.
Figure 1. PCR results of genomic extraction of SoxR from E. coli (including bi-directional promoter and SoxR protein gene)
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Characterizing Pixcell Constructs
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.
These results demonstrate that the assembled PixCell construct work as expected.
In order to demonstrate if the PixCell construct response is responsive to oxidation signals, we started by inducing the circuit with oxidised pyocyanin, as it relatively easier than using the electrode set-up. Once we proved that oxidised pyocyanin activates the circuit by oxidising SoxR, we performed the experiment by oxidising pyocyaninusing oxidising voltages.
Figure 1.Pyocyanin dose response
Figure 2.Ferricyanide dose response
Figure 3.Ferrocyanide dose response
Explain results here.
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Electrochemistry of Redox Modulators
We investigated the redox status of the our redox modulators with cyclic voltammeter (CV) and by measuring the absorbance at 390 nm with the plate reader. Pyocyanin in its oxidised form has an absorption peak at 390 nm, therefore we were able to use this as a signal for the concentration of oxidised pyocyanin.
These results that with our set-up we are able to selectively and reversibly oxidise and reduce our redox modulator. 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.
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Figure 1.Pyocyanin reduction by the reducing agent Sodium Sulfite
Sodium sulfite is a reducing agent. We measured the concentration of oxidised pyocyanin in solutions with increasing sulfite concentration. As we expected, the more sulphite added, the less absorption at 390 nm occurs (peak for oxidised pyocyanin). However this linear relationhiop resulted to be true only up to ca. 5% of sulphite. After that point it seems that an oxidised species is being produced.
Figure 2.Production of Pyocyanin Sulfonate at 2.5% Sulfite
To dig deeper into the reasons of the unexpected non-linear relationship described above, we played with lower suite concentration. Reding a thesis, we hypothesised that sulphite could react with pyocyanin, producing pyocyanin sultanate. because pyocyanin sulfanate has an absorption peak at 400 nm, we performed the same experiment as above but this time measuring the OD at both 390 and 400 nm. We found that at a concentration of 2.5 % of sulfite, there is appear of absorption at 400nm, suggesting that pyocyanin sultanate is being produced. Becausepyocyanin sultanate is toxic to cells, , we carried the next experiments with lower sulfite concentration
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These results describe the first ever aerobic electro genetic control system, that works both in liquid and solid E. coli cultures.
Spatial Electronic Control of Gene Induction
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Figure 1. PCR results of genomic extraction of SoxR from E. coli (including bi-directional promoter and SoxR protein gene)
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Building and Characterizing the Pixcell Library
Creating the Sox Library
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Figure 1. PCR results of genomic extraction of SoxR from E. coli (including bi-directional promoter and SoxR protein gene)
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Characterising the Sox Library
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Figure 1. PCR results of genomic extraction of SoxR from E. coli (including bi-directional promoter and SoxR protein gene)
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Testing Alternative Redox Modulators
Testing Phenazine Methosulfate
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Figure 1. Phenazine as as Cheaper, Non-toxic Redox Modulator
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Applications
Biocontainment- Gp2 Growth Inhibition
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Figure 1.pSoxS-Gp2 is an electronic-responsive growth inhibition
Figure 2.Negative controls
Explain results here.
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Fabric Bioprinting - Melanin
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Figure 1. PCR results of genomic extraction of SoxR from E. coli (including bi-directional promoter and SoxR protein gene)
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