Difference between revisions of "Team:Imperial College/Demonstrate"
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<p> 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.</p> | <p> 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.</p> | ||
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Revision as of 00:00, 16 October 2018
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.
Figure 1. Illustration of the PixCell Construct
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 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.
Figure 1. PCR results from the amplification of the entire construct
In order to lower the basal level of GFP expression a degradation tag was added. Therefore, any increase in GFP expression induced by the oxidation of SoxR would be more predominant.
Figure 1. Illustration of the PixCell Construct DegTag
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.
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 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.
The above setup was used to perform Square-wave voltammetry to investigate bulk oxidisation and reduction of our system.
Figure 1. Square-wave voltammetry of redox modulator
The graphs above summarise the results showing that our system was consistently bulk reduced at -0.3V and bulk oxidised at +0.5V. The addition of Sulfite and cell containing the pixCell construct did not significantly alter the reduction and oxidation profile. These results led us to chose +0.5V as our oxidising potential in the following experiments testing the spatial electronic control of gene induction.
Figure 2. 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 3.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)
Lorem ipsum dolor sit, amet consectetur adipisicing elit. Excepturi, nisi illum. Consequuntur cum, sequi quo, esse facere corrupti voluptatum ipsum vel consequatur error quasi tenetur, repellat voluptatibus aspernatur optio mollitia! Lorem ipsum dolor sit, amet consectetur adipisicing elit. Excepturi, nisi illum. Consequuntur cum, sequi quo, esse facere corrupti voluptatum ipsum vel consequatur error quasi tenetur, repellat voluptatibus aspernatur optio mollitia!
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Building and Characterizing the Pixcell Library
Creating the Sox Library
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Lorem ipsum dolor, sit amet consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat.
Lorem ipsum dolor, sit amet consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat. Lorem ipsum dolor, sit amet consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat.
Figure 1. PCR results of genomic extraction of SoxR from E. coli (including bi-directional promoter and SoxR protein gene)
Lorem ipsum dolor sit, amet consectetur adipisicing elit. Excepturi, nisi illum. Consequuntur cum, sequi quo, esse facere corrupti voluptatum ipsum vel consequatur error quasi tenetur, repellat voluptatibus aspernatur optio mollitia! Lorem ipsum dolor sit, amet consectetur adipisicing elit. Excepturi, nisi illum. Consequuntur cum, sequi quo, esse facere corrupti voluptatum ipsum vel consequatur error quasi tenetur, repellat voluptatibus aspernatur optio mollitia!
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Characterising the Sox Library
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Lorem ipsum dolor, sit amet consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat.
Lorem ipsum dolor, sit amet consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat. Lorem ipsum dolor, sit amet consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat.
Figure 1. PCR results of genomic extraction of SoxR from E. coli (including bi-directional promoter and SoxR protein gene)
Lorem ipsum dolor sit, amet consectetur adipisicing elit. Excepturi, nisi illum. Consequuntur cum, sequi quo, esse facere corrupti voluptatum ipsum vel consequatur error quasi tenetur, repellat voluptatibus aspernatur optio mollitia! Lorem ipsum dolor sit, amet consectetur adipisicing elit. Excepturi, nisi illum. Consequuntur cum, sequi quo, esse facere corrupti voluptatum ipsum vel consequatur error quasi tenetur, repellat voluptatibus aspernatur optio mollitia!
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Testing Alternative Redox Modulators
Testing Phenazine Methosulfate
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The above setup was used to perform Sqaure-wave voltammetry confirming that ferrocyanide was bulk reduced at -0.3V and bulk oxidised at +0.5V.
Figure 1. Phenazine as as Cheaper, Non-toxic Redox Modulator
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Applications
Biocontainment- Gp2 Growth Inhibition
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Lorem ipsum dolor, sit amet consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat. Lorem ipsum dolor, sit amet consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat.
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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Lorem ipsum dolor, sit amet consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat.
Lorem ipsum dolor, sit amet consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat. Lorem ipsum dolor, sit amet consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat.
Figure 1. PCR results of genomic extraction of SoxR from E. coli (including bi-directional promoter and SoxR protein gene)
Lorem ipsum dolor sit, amet consectetur adipisicing elit. Excepturi, nisi illum. Consequuntur cum, sequi quo, esse facere corrupti voluptatum ipsum vel consequatur error quasi tenetur, repellat voluptatibus aspernatur optio mollitia! Lorem ipsum dolor sit, amet consectetur adipisicing elit. Excepturi, nisi illum. Consequuntur cum, sequi quo, esse facere corrupti voluptatum ipsum vel consequatur error quasi tenetur, repellat voluptatibus aspernatur optio mollitia!
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