Team:Exeter/Poster
Poster: Exeter Team
Hello and welcome to our poster page! We are the 2020 University of Exeter iGEM team!
Please enjoy our interactive poster where you can learn about our project, our journey as a team and what we've achieved in the last months. Just click on the section you'd like to read and it should pop up, we suggest starting with our header!
We hope you find it informative and that it sparks your interest in our project. May the calcification operation begin!
CalcifEXE: A SynBio Method of Calcium Carbonate Precipitation
The Exeter iGem 2020 project aimed to design an ‘ink’ for a 3D bioprinter that produces calcium carbonate structures.
Introduction:
Conventional calcium carbonate production methods emit large amounts of carbon dioxide (McDonald et al., 2019) as well as many toxic compounds due to the reliance on fossil fuels in the manufacturing process. Our team is developing a novel precipitation method using bacteria engineered with enzymes capable of producing the carbonate ions required for precipitation of calcium carbonate. One enzyme we are focusing on is carbonic anhydrase (CA), an enzyme that facilitates the interconversion of carbon dioxide and bicarbonate in solution. CA should not only increase carbonate production, but also allow for atmospheric carbon dioxide to be used as the feedstock for the carbonate ions. Our method has the potential capability to be employed with a 3D-bioprinter that will print structures comprising a hydrogel and our engineered bacteria. On precipitation the calcium carbonate should take the shape of the hydrogel structure. One potential application, is the production of coral backbones used to facilitate coral regrowth.
Problem and Objective:
The Problem:
It was clear that the environment was a shared passion and concern amongst our team, which resulted in numerous ideas for an environmentally focused project. One of the leading concerns globally right now is climate change and the negative effects of global warming on our planet. A gas that contributes to this is CO2, one source of such emissions is in the production of precipitated calcium carbonate (PCC) that requires the thermal decomposition of calcium carbonate to be manufactured. This releases carbon dioxide and many other pollutants due to the fuel requirement. Therefore we set out to find a more carbon-friendly way of producing PCC that would also allow for production of a variety-specific products.
Objectives:
• Engineering Bacillus subtilis to enhance its ability of precipitating calcium carbonate (PCC).
• Modelling parts of the hydrogel to inform us of real-world implementation.
• Producing high quality educational content throughout.
• Experimenting on hydrogel for empirical data.
It was clear that the environment was a shared passion and concern amongst our team, which resulted in numerous ideas for an environmentally focused project. One of the leading concerns globally right now is climate change and the negative effects of global warming on our planet. A gas that contributes to this is CO2, one source of such emissions is in the production of precipitated calcium carbonate (PCC) that requires the thermal decomposition of calcium carbonate to be manufactured. This releases carbon dioxide and many other pollutants due to the fuel requirement. Therefore we set out to find a more carbon-friendly way of producing PCC that would also allow for production of a variety-specific products.
Objectives:
• Engineering Bacillus subtilis to enhance its ability of precipitating calcium carbonate (PCC).
• Modelling parts of the hydrogel to inform us of real-world implementation.
• Producing high quality educational content throughout.
• Experimenting on hydrogel for empirical data.
Education:
We've delivered a wide range of educational content; from educational videos designed for an online curriculum to our picture book, 'The Brilliant Biologists', which was written for young children to learn about synthetic biology whilst promoting diversity in science. We used social media to share engaging, accessible infographics on topics related to synthetic biology, to share the progress of our project and to collaborate with iGEM teams across the globe. Our online surveys helped to inform the direction of our project outreach efforts. At the end of September we attended the iGEM Coral Symposium, hosted by the University of New South Wales iGEM team. Here we shared our project to the public, experts, and other iGEM teams as well as what we have learned about syn bio approaches to solving problems facing coral reefs. We also taught university students about responsible innovation through our bioethics lecture!
Method
Two bacterial species were used for our project; Bacillus subtilis WB800N and Escherichia coli (strains BL21(DE3) and DH5α). E. coli DH5a was used during the cloning stages, where our chosen genes were inserted into a plasmid that would replicate in both E. coli and B. subtilis. B. subtilis was chosen to be our terminal chassis due to the species natural ability to tolerate changes in pH (Wilks et al., 2009).
We decided to use a multi-enzyme approach to increase microbial induced calcium carbonate precipitation in our chassis.
We identified three enzymes that precipitated calcium carbonate. These were ureases, carbonic anhydrases and coral acid rich proteins (CARPs).
Ureases hydrolyse urea and the products equilibritate in water to produce bicarbonate ions and hydroxide ions. Carbonic anhydrases catalyze the conversion of atmospheric CO2 and water into carbonic acid and bicarbonate ions. These bicarbonate ions could join with calcium ions to make calcium carbonate – this reaction may be mediated by CARPs.
We used Type IIs cloning to build constructs with the coding sequences for our chosen enzymes and a variety of promoters, RBS and terminators The team built two sets of constructs. One set was designed for protein expression in E.coli BL21(DE3) while the second set was designed for protein expression in our terminal chassis, B. Subtilis WB800N.
We decided to use a multi-enzyme approach to increase microbial induced calcium carbonate precipitation in our chassis.
We identified three enzymes that precipitated calcium carbonate. These were ureases, carbonic anhydrases and coral acid rich proteins (CARPs).
Ureases hydrolyse urea and the products equilibritate in water to produce bicarbonate ions and hydroxide ions. Carbonic anhydrases catalyze the conversion of atmospheric CO2 and water into carbonic acid and bicarbonate ions. These bicarbonate ions could join with calcium ions to make calcium carbonate – this reaction may be mediated by CARPs.
We used Type IIs cloning to build constructs with the coding sequences for our chosen enzymes and a variety of promoters, RBS and terminators The team built two sets of constructs. One set was designed for protein expression in E.coli BL21(DE3) while the second set was designed for protein expression in our terminal chassis, B. Subtilis WB800N.
Modelling:
The modelling team had a particular focus on modelling the properties of Hydrogel. Our model for the viscoelastic properties of hydrogel containing 90% water showed that when highly stretched, the stress forces cease to increase therefore suitable for use in a bioprinter. After this validation of our printer concept, we made a simulation of how carbon dioxide diffuses into the hydrogel, drawing inferences for the optimal thickness of the hydrogel.
Our model for the viscoelastic property of hydrogel containing 90% water showed that when it is highly stretched, the stress forces cease to increase. This makes it a suitable choice for use in a bioprinter.
Our model for the viscoelastic property of hydrogel containing 90% water showed that when it is highly stretched, the stress forces cease to increase. This makes it a suitable choice for use in a bioprinter.
Results:
Due to the COVID-19 pandemic, we relied heavily on the modelling aspects of our project to help supplement the 'wet-lab' results we managed to achieve with limited lab access and restrictions.
Diffusion of CO2 into agarose-based hydrogel (experiments):
Agarose hydrogel containing Universal indicator was poured into beakers (3 replicates) and put into an 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:
The graph above shows that CO2 diffusion through the hydrogel plateaus after 20 hours/1200 minutes.
Diffusion of CO2 into agarose-based hydrogel (modelling):
We developed a code in MatLab that shows a 2D representation of carbon dioxide gas diffusing through a hydrogel medium.
The non-dark-blue pixels above represent the diffusion of CO2 through the hydrogel matrix. The model predicted that maximum diffusion of CO2 through the hydrogel (shown above) would occur after 1440 minutes.
We also modelled the mechanical properties of hydrogel, to achieve this we compared the Maxwell and Kelvin-Voigt models of stress/strain on a viscoelastic material to predict how the hydrogel would respond to stress:
Left: Maxwell material held at a constant stress of σ0=100kPa. Right: Strain deformation response showing a linear increase over time.
Left: Kelvin-Voigt material held at a constant stress of σ0=200kPa. Right: The strain response over time showing an exponential rise asymptotically approaching the value σ0/E.
From this we determined the Kelvin-Voigt model was a more accurate model for predicting strain on the hydrogel.
Flux Balance Analysis (FBA):
While collaborating with University College London, we used FBA to measure the flux through varying product pathways of our B. subtilis system.
The above graphs show the minimum rate of carbonate ion production in our bacteria (42.85 mmol/gDCW/h) and the realistic rate (80.85 mmol/gDCW/hr) (both relying on an uptake rate of -1mmol/gDCW/hr for carbon dioxide and urea).
Characterising two new B. subtilis promoters for use in E. coli
Previously our supervisory team had identified 30 putative constitutive promoters from the genome of B. subtilis 168. We inserted the promoters upstream of the coding sequence for either AmilCP (blue chromoprotein) or MeffCP (pink chromoprotein). From these transformations two promoters were found to be active in E. coli DH5 alpha; yqgW (K3371000) and rho (K3371001).
Above is a graph showing that use of the yqgW promoter (P_30) resulted in a 3 hour lag in expression of AmilCP compared to rho (P_15). We contributed this data to the registry page for AmilCP (K592009).
Diffusion of CO2 into agarose-based hydrogel (experiments):
Agarose hydrogel containing Universal indicator was poured into beakers (3 replicates) and put into an 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:
The graph above shows that CO2 diffusion through the hydrogel plateaus after 20 hours/1200 minutes.
Diffusion of CO2 into agarose-based hydrogel (modelling):
We developed a code in MatLab that shows a 2D representation of carbon dioxide gas diffusing through a hydrogel medium.
The non-dark-blue pixels above represent the diffusion of CO2 through the hydrogel matrix. The model predicted that maximum diffusion of CO2 through the hydrogel (shown above) would occur after 1440 minutes.
We also modelled the mechanical properties of hydrogel, to achieve this we compared the Maxwell and Kelvin-Voigt models of stress/strain on a viscoelastic material to predict how the hydrogel would respond to stress:
Left: Maxwell material held at a constant stress of σ0=100kPa. Right: Strain deformation response showing a linear increase over time.
Left: Kelvin-Voigt material held at a constant stress of σ0=200kPa. Right: The strain response over time showing an exponential rise asymptotically approaching the value σ0/E.
From this we determined the Kelvin-Voigt model was a more accurate model for predicting strain on the hydrogel.
Flux Balance Analysis (FBA):
While collaborating with University College London, we used FBA to measure the flux through varying product pathways of our B. subtilis system.
The above graphs show the minimum rate of carbonate ion production in our bacteria (42.85 mmol/gDCW/h) and the realistic rate (80.85 mmol/gDCW/hr) (both relying on an uptake rate of -1mmol/gDCW/hr for carbon dioxide and urea).
Characterising two new B. subtilis promoters for use in E. coli
Previously our supervisory team had identified 30 putative constitutive promoters from the genome of B. subtilis 168. We inserted the promoters upstream of the coding sequence for either AmilCP (blue chromoprotein) or MeffCP (pink chromoprotein). From these transformations two promoters were found to be active in E. coli DH5 alpha; yqgW (K3371000) and rho (K3371001).
Above is a graph showing that use of the yqgW promoter (P_30) resulted in a 3 hour lag in expression of AmilCP compared to rho (P_15). We contributed this data to the registry page for AmilCP (K592009).
Conclusion
3D Bioprinter (with ink composed of hydrogel and calcium-carbonate precipitating B. subtilis):
• Determined the rate of diffusion of CO2 through agarose-based hydrogel using a combination of experiments and modelling, which together showed that CO2 takes 20-24 hours to diffuse through 1% agarose hydrogel and can reach a maximum depth of 1.2cm.
• Discovered, using Kelvin-Voigt modelling, that when the hydrogel is stretched to a certain point, stress forces cease to increase unlike with a regular elastic material which makes it an ideal material for printing into custom shapes because it won't deform.
• Designed a nozzle with help from Team Ashesi Ghana which mixes bacteria and hydrogel using a vortex (eliminating the need for a separate mixing chamber which would require the nozzle to be periodically replaced) and uses filleting/an extra layer around the joints to prevent cracks caused by pressure within the nozzle.
• Through collaboration with coral expert Professor Steve Simpson, discovered that coral would more readily colonise rough, porous 3D-printed calcium carbonate shapes and that these shapes should have open spaces which could house other marine life such as herbivorous fish.
• With help from University College London, predicted the production rate of carbonate ions in our bacteria, worked out the ideal lactose uptake and identified a gene which we could knock out to increase the production of carbonate (gene BSU21920).
Parts:
• Characterised two new B. subtilis promoters (yqgW and rho) for use in E. coli.
• Contributed to registry page for existing part K592009 (AmilCP) after discovering that use of the yqgW promoter leads to a 3 hour lag in expression of AmilCP compared to the rho promoter.
• Determined the rate of diffusion of CO2 through agarose-based hydrogel using a combination of experiments and modelling, which together showed that CO2 takes 20-24 hours to diffuse through 1% agarose hydrogel and can reach a maximum depth of 1.2cm.
• Discovered, using Kelvin-Voigt modelling, that when the hydrogel is stretched to a certain point, stress forces cease to increase unlike with a regular elastic material which makes it an ideal material for printing into custom shapes because it won't deform.
• Designed a nozzle with help from Team Ashesi Ghana which mixes bacteria and hydrogel using a vortex (eliminating the need for a separate mixing chamber which would require the nozzle to be periodically replaced) and uses filleting/an extra layer around the joints to prevent cracks caused by pressure within the nozzle.
• Through collaboration with coral expert Professor Steve Simpson, discovered that coral would more readily colonise rough, porous 3D-printed calcium carbonate shapes and that these shapes should have open spaces which could house other marine life such as herbivorous fish.
• With help from University College London, predicted the production rate of carbonate ions in our bacteria, worked out the ideal lactose uptake and identified a gene which we could knock out to increase the production of carbonate (gene BSU21920).
Parts:
• Characterised two new B. subtilis promoters (yqgW and rho) for use in E. coli.
• Contributed to registry page for existing part K592009 (AmilCP) after discovering that use of the yqgW promoter leads to a 3 hour lag in expression of AmilCP compared to the rho promoter.
Next Steps:
The COVID-19 pandemic has meant that we didn't manage to complete all we had hoped for. To take our project further, we would like to run some tests to see if our co-culturing plan would have successfully increased the yield of calcium carbonate production like we had hoped, as well as successfully transform our plasmids into Bacillus subtilis- lab restrictions meant we didn't have time to troubleshoot the problem. We would like to combine our hydrogel with our bacteria and record the rate and yield of calcium carbonate precipitation, in addition to recording the carbon input and output to quantify the extent of carbon mitigation.
Our ultimate aim is to have CalcifEXE corals growing around the world! Their implementation on the seabed would therefore require coral attachment tests and investigations into the removal of the GM bacteria before being put into the environment.
Team and Partnerships:
Student Team:
Adam Bainbridge, BSc Biochemistry.
Anna Donnan, BSc Biological Sciences.
Ariane Goudie, MPhys Physics.
Bethan Rimmer, MSc Medical Sciences.
Eloise Martin, MPhys Physics.
Isabelle Browne, MPhys Physics.
Matthew Turk, BSc Biological Sciences.
Nina Senna, BSc Biochemistry.
Pazzy Boardman, MSc Physics.
Velizar Kirkow, MMath Mathematics.
Supervisors:
Professor John Love, Chair of Synthetic Biology and Director of Exeter Microbial Biofuels Group & BioEconomy Centre.
Dr. Chloe Singleton, Research Fellow and Lecturer, Exeter Microbial Biofuels Group.
Dr. Mark Hewlett, Research Fellow, Exeter Microbial Biofuels Group.
Dr. Paul James, Industrial Research Fellow, Exeter Microbial Biofuels Group.
Partnerships:
We'd like to take a moment to acknowledge our fruitful partnership with the University College London iGEM team . We are incredibly thankful for all of the work they did to assist us with our FBA modelling which provided us with critical insight into our bacteria’s metabolism, and we hope that the expression and characterisation of their PETase-MHETase fusion protein construct proves equally helpful for the future of their project.
We also recognise our partnership with the Ashesi University of Ghana iGEM team where we shared outsourced knowledge to amplify both projects reach.
Adam Bainbridge, BSc Biochemistry.
Anna Donnan, BSc Biological Sciences.
Ariane Goudie, MPhys Physics.
Bethan Rimmer, MSc Medical Sciences.
Eloise Martin, MPhys Physics.
Isabelle Browne, MPhys Physics.
Matthew Turk, BSc Biological Sciences.
Nina Senna, BSc Biochemistry.
Pazzy Boardman, MSc Physics.
Velizar Kirkow, MMath Mathematics.
Supervisors:
Professor John Love, Chair of Synthetic Biology and Director of Exeter Microbial Biofuels Group & BioEconomy Centre.
Dr. Chloe Singleton, Research Fellow and Lecturer, Exeter Microbial Biofuels Group.
Dr. Mark Hewlett, Research Fellow, Exeter Microbial Biofuels Group.
Dr. Paul James, Industrial Research Fellow, Exeter Microbial Biofuels Group.
Partnerships:
We'd like to take a moment to acknowledge our fruitful partnership with the University College London iGEM team . We are incredibly thankful for all of the work they did to assist us with our FBA modelling which provided us with critical insight into our bacteria’s metabolism, and we hope that the expression and characterisation of their PETase-MHETase fusion protein construct proves equally helpful for the future of their project.
We also recognise our partnership with the Ashesi University of Ghana iGEM team where we shared outsourced knowledge to amplify both projects reach.
Acknoeledgements and Sponsors:
We'd like to thank our sponsors and supporters:
Outsourced Knowledge:
We were amazed by the response, encouragement and level of support we received from experts. We gained a comprehensive understanding of the current challenges industries and scientists face when tackling our issue. We'd like to thank:
Professor Mike Allen, Associate Professor of Single Cell Genomics.
Mr Tim Gordan, PhD student.
Mr Edward Hollis, MSc Engineering.
Dr Rebecca Hooper, British Lime Association Director and Senior Energy and Environment Advisor at MPA.
Ms Jenny Rusk, Science Communication expert.
Professor Sarah Hartley, Interdisciplinary Social Scientist.
Professor Steve Simpson, Professor of Marine Biology & Global Change.
Mr Jonathan Teague, PhD student.
Dr Sam Stevens, Biophysics Lecturer.
Our Sponsors:
University of Exeter
Biotechnology and Biological Sciences Research Council (BBSRC)
SnapGene
Geneious
Outsourced Knowledge:
We were amazed by the response, encouragement and level of support we received from experts. We gained a comprehensive understanding of the current challenges industries and scientists face when tackling our issue. We'd like to thank:
Professor Mike Allen, Associate Professor of Single Cell Genomics.
Mr Tim Gordan, PhD student.
Mr Edward Hollis, MSc Engineering.
Dr Rebecca Hooper, British Lime Association Director and Senior Energy and Environment Advisor at MPA.
Ms Jenny Rusk, Science Communication expert.
Professor Sarah Hartley, Interdisciplinary Social Scientist.
Professor Steve Simpson, Professor of Marine Biology & Global Change.
Mr Jonathan Teague, PhD student.
Dr Sam Stevens, Biophysics Lecturer.
Our Sponsors:
University of Exeter
Biotechnology and Biological Sciences Research Council (BBSRC)
SnapGene
Geneious
References:
1) McDonald L., Glasser F. P., Mohammed S. I. (2019) A New Carbon-Negative Precipitated Calcium Carbonate Admixture (PCC-A) for Low Carbon Portland Cements. Materials, 12(4):554. DOI:10.3390/ma12040554.
2) Wilks, J. C., Kitko, R. D., Cleeton, S. H., Lee, G. E., Ugwu, C. S., Jones, B. D., Bondurant, S. S., & Slonczewski, J. L. (2009). Acid and base stress and transcriptomic responses in Bacillus subtilis. Applied and Environmental Microbiology, 75(4), 981–990. DOI: 10.1128/AEM.01652-08.
2) Wilks, J. C., Kitko, R. D., Cleeton, S. H., Lee, G. E., Ugwu, C. S., Jones, B. D., Bondurant, S. S., & Slonczewski, J. L. (2009). Acid and base stress and transcriptomic responses in Bacillus subtilis. Applied and Environmental Microbiology, 75(4), 981–990. DOI: 10.1128/AEM.01652-08.