Team:Exeter/Results
Results
Due to the COVID-19 pandemic we were unable to access the laboratory for much of our project. Because of this we have relied heavily on the modelling aspects of our project to help supplement what lab results we managed to achieve.
Hydrogel Diffusion Experiments
The diffusion rate of carbon dioxide into various concentrations of agarose-based hydrogels was measured to obtain data which we could then use alongside our models.
Results
These images show the diffusion of carbon dioxide into our agarose-based hydrogels. The image on the left is at the start of the experiment and the image on the right is at the end. We ran images of the hydrogels, taken at ten minute intervals over a 24 hour period, through our pixel-intensity code. This data was then used to inform the parameters our diffusion model.
Agarose hydrogel containing Universal indicator was poured into beakers (3 replicates) and put into a incubator with CO2 supply (10%). As carbon dioxide diffused into the hydrogel the universal indicator changed colour to red. By analysing the change in red pixel intensity in time lapse photographs from a Raspberry Pi, we gathered gas diffusion data useful in parameterising our model.
Hydrogel Diffusion Modelling
We have developed a code in MatLab that shows the diffusion of carbon dioxide through hydrogels. This model creates a 2D representation of how the carbon dioxide gas will dissipate throughout a hydrogel medium.
See the model and detailed results here!
Results
The increase and then stabilisation of the number of red pixels supports the model’s stagnation in CO2 diffusion. The conclusion we can draw from this is that there is a limitation on the depth CO2 can penetrate into the hydrogel. Therefore when we lay the ink inside the 3D printer, we need to make sure that the layer is thin enough for CO2 to diffuse well. If we need to print something thicker, we need to allow for CO2 diffusion to happen thoroughly in the layer below before building on top.
Figure (11) gives us a great deal of information about when peak CO2 diffusion has occurred. Around 20 hours after the number of red pixels has reached a plateau. Since the camera was programmed to take a picture every 10 minutes, this means that peak CO2 diffusion takes about 1200 minutes to occur in 1% agarose hydrogel.
The height of the hydrogel in the beaker is 2.6 cm and the depth penetrated by the CO2 in the 1% agarose hydrogel after roughly 300 minutes is 0.6 cm from the Dirichlet boundary. Therefore, if we were to use this hydrogel in a printer with omnidirectional Dirichlet boundaries, the layer can be up to 1.2 cm thick.
Hydrogel Mechanical Properties Modelling
The aim of this model was to analyse the viscoeleastic effects of the fluid, as these would impact how our printer mechanism would work.
See the model here!
Results
When hydrogel is stretched to a significant fraction of its original length the stress forces cease to increase, unlike with a regular elastic material. We also found that the shear modulus G no longer becomes a constant and will become a function of the strain itself. This was found to be true for hydrogels even when they are in a swollen state.
Flux Balance Analysis
Flux Balance Analysis (FBA) was used to measure the flux through varying product pathways of our B.subtilis system. This was performed to predict the production rate of carbonate ions in our bacteria, as well as to inform us of any potential knockout genes that could help increase carbonate production in the future.
We would like to thank the University College London iGEM team for performing this analysis for us. The analysis can be found here!
Results
The results showed that:
1. Lactose uptake rate of around 4 mmol/gDCW/hr gave us the greatest growth rate for the least amount of lactose. At higher uptake rates there was no further increase in growth.
2. The minimum carbonate production rate was 42.85 mmol/gDCW/h (at an uptake rate of -1mmol/gDCW/hr for both carbon dioxde and urea).
3. The realistic carbonate production rate was 80.85 mmol/gDCW/hr (at an uptake rate of -1mmol/gDCW/hr for both carbon dioxide and urea).
4. Carbonate production could be increased by knocking out the non-essential BSU21920 gene which encodes UDP-glucose diacylglyceroltransferase.
Promoter Experiments
We investigated 30 putative consitutive promoters from the genome of B. subtilis 168 to see whether any of them were active and if so whether they were suitable for our project.
We found two promoters that were active in E. coli - yqgW and rho
To see a detailed description and discussion of our results please visit our contribution page!