Team:UCL/Partnership
Overview
Part 1: University of Exeter iGEM 2020
Exeter project summary
Damaged coral reef environments do not have physical structures to house new coral and these can take thousands of years to regenerate. Currently, most reef restoration projects use concrete (which comes at a huge carbon cost) as an artificial coral backbone. Their project ‘CalcifEXE’ aims to produce a new carbon negative 3D bio-printer that would enable calcium carbonate (limestone) to printed into custom shapes using metabolic pathways in B. subtilis. These shapes could be used to replicate pieces of reef which could act in place of the natural limestone backbones produced by corals. This could be used to massively speed up coral regeneration in damaged reefs.
To learn more about their project, please check out Exeter's page
Partnership summary
Early in this year’s competition, we attended a virtual meetup hosted by the University of St. Andrews, allowing us to introduce ourselves to the 8 other teams from the UK. After taking a keen interest in Exeter’s project, we began communicating with their team very closely. After initial virtual meetings, we were able to arrive at two shared objectives that were central to the success of our respective projects:
- Attempt to characterise the expression and function of our PETase-MHETase fusion protein construct
- Perform Flux Balance Analysis modelling on their bacterium’s metabolism to gain critical insight into synthetic biology approaches to achieve desired outcomes
Section 1: How did your collaborative work inform and shape your project at different stages?
June 2020
At this stage, our team were still finalising the direction of our project as we had just finished our university summer examinations. We thought we were going to try to combine enzymatic PET degradation with Microbial Desalination Cell technology, but we weren’t 100% sure due to COVID-19 restrictions. Through initial virtual meetings with Exeter, we were able to learn about their previous iGEM teams’ experience with PET degrading enzymes, which we thought would be very useful if we were to go down this route. This encouraged us to look further into the topic, as we had more confidence that enzymatic PET degradation could be achieved.
July 2020
As the season shifted into a higher gear and the COVID-19 pandemic maintained the restrictions in the UK, we realised our chances of getting laboratory access were decreasing. Considering this, we decided to aim for the gold-level partnership medal with another team who did have laboratory access, so that they could help us with experiments. Fortunately, Exeter were as keen as we were. The second step of our engineering cycle (building on our in silico modelling) would be a crucial one: to express and characterise the PET degrading enzymes in our expression system of choice. Therefore, we confirmed this as a shared objective.
This shaped the two types of models we were going to build, as they would constitute the next two steps of our proposed device. The first would simulate the PET degradation pathway and the second would simulate the electricity production of our device.
August 2020
Through collaboration with academics from Portsmouth University, we were able to gain insight into a PETase-MHETase fusion protein that had enhanced PET degradation activity compared to the individual enzymes. This informed the design of four separate DNA constructs we began building in Benchling, which were ordered from GenScript at the end of August.
After this, supervisors and team members from both teams met virtually to discuss the construct designs and their implications on expression, purification and characterisation. This meeting was essential for checking that Exeter had access to the required technical equipment, bacterial hosts and laboratory time to attempt the expression and characterisation of our constructs. Thankfully they did, meaning we were not forced to re-think our expression system or synthetic biology approaches.
September 2020
With the DNA constructs being synthesised by GenScript, we moved onto communicating the experimental protocol workflow to Exeter. We explained how we would like the first experiments to attempt the expression of our DNA constructs in E. coli., then measure lactate production from the products of PET degradation in P. putida and finally to measure electricity produced by S. oneidensis. We also supplied Exeter with protocols for the individual experiments within each step. These arrangements were ready for our DNA constructs to arrive on the 18th October.
October 2020
As the season ended, we were conscious that we were very short on time for Exeter to perform these experiments for us. Therefore, we agreed to minimise the number of prospective experiments early in the month to relieve Exeter of some pressure in the last week before the wiki and presentation deadlines. Unfortunately, the DNA constructs we ordered took longer than expected to arrive meaning Exeter were unable to conduct the desired experiments for us. This made us adapt our plans for next year’s team as part of our 2-year project.
June 2020
At this stage, our team was still finalising the direction of our project as we had just finished our university summer examinations. Due to COVID-19 restrictions in the UK, we became more and more certain that we would be attempting a fully virtual project with no laboratory access ourselves. After deciding to pursue the integration of enzymatic PET degradation with Microbial Desalination Cell technology, we began brainstorming which computational modelling techniques would suit our aims best. Through regular meetings with Exeter, we learned that the Flux Balance Analysis (FBA) model we were planning to create could also be applied to Exeter’s bacterium.
July 2020
As our project matured and we decided on the second computational modelling method for our project – Cellular Automata (CA) – we maintained our weekly meetings with Exeter to stay on top of changes they were making in their system due to their engineering cycle process, meaning we could make the necessary changes in our approach for applying FBA modelling to their bacterium’s metabolism. This required our FBA team to ask for new guidance from academic FBA experts.
August 2020
As our own FBA model began to materialise and produce some initial results, our team began to build the FBA model for Exeter’s bacterium. In order to do this, we received information from Exeter regarding their bacterium’s food source (Lactose) and critical details about the bacterium’s ureolytic pathway for fixing CO2 into calcium carbonate, as well as the aerobic conditions required.
We also organised a virtual meetup between our two partners (Exeter and Ashesi) to foster further collaboration between them, as our three projects shared some overlap. We were elated to see how this influenced each of their projects, as Exeter were able to receive an improved design for the nozzle of their device thanks to Ashesi’s mechanical engineering expertise.
Email from Ashesi to Exeter detailing their improved nozzle solution:
September 2020
At this stage, our own FBA model was almost complete and had informed our approach for attempting to co-culture our three-species bacteria co-culture. This allowed our FBA team to focus almost completely on Exeter’s model, leading us to complete the model and refine the research questions that Exeter were hoping to answer with our model:
- Simulate effect of lactate uptake rate on B. subtilis aerobic biomass growth
- Simulate effect of urea and CO2 uptake rates on carbonate ion production rate
- Simulate the effect of a single gene knockout to maximize carbonate ion production rate (on the basis of urea and CO2 uptake rates)
October 2020
The final stage of this shared objective was to obtain the results for Exeter’s research questions. The FBA model we built gave them answers to their research questions, allowing them to plan for their next steps: to experimentally measure the effect of knocking out.
Section 2: How did your teams work together throughout the season?
Throughout most of the season, our teams worked together using the online meeting software Zoom. This allowed us to initially present our respective project ideas and future directions, and to propose the idea of partnering. Through Zoom we were able to conduct the following types of meetings:
- Weekly updates to discuss progress in our respective tasks for the shared objectives
- One-off meetings to brainstorm new tasks that would advance the progress of our shared objectives
- Tutorial meetings where one team could teach the other how to use certain software such as Benchling or how to code content into the wiki
The only exception was when some of us were able to meet in person in late August, as coincidentally 2 of our team members and one of Exeter’s team members had organised separate visits to Bulgaria. In this meetup, our teams were able to work in-person and make progress with respect to our shared objectives.
Section 3: How did each team in the partnership benefit from the collaboration?
At each step of the process from construct design, to experimental design and overall methodology, our partnership with Exeter allowed us to go through the necessary steps to develop our Benchling DNA constructs despite our lack of laboratory access (see below). Despite the delays in DNA construct delivery from GenScript, this allowed us to develop them from mere theory into proteins that can one day be expressed and assayed. Without this, our project would have been strictly limited to in silico modelling, meaning we would not have been able to access further steps in the engineering cycle.
With their help, we were able to develop our methodology overview for our PETase-MHETase constructs to include assembly of DNA, cloning and ligation, purification and more (see below). These extra steps have greatly advanced the development of our proposed device, and to give the second year of our project a head-start. Exeter were also able to benefit from this shared objective, as it gave them additional experience in protocol, experimental and methodology design.
For the details of the engineering cycle and wet lab methodology, please visit Engineering
Exeter benefited greatly from the second shared objective, as we were able to obtain data from in silico modelling on the three research questions noted above. This was central to the success of their project, as it allowed them to A) estimate the optimal lactate uptake rate for B. subtilis growth, B) estimate their carbonate ion production rate from a range of urea and CO2 uptake rates, and C) a single-gene knockout they could attempt to maximise carbonate ion production rate (ugtP - Ensembl database code BSU21920). From this data, they have adapted their next steps and with more time they would have tested the single gene knockout’s effect on carbonate ion production experimentally.
Exeter also discussed the issues they had faced with their B. subtilis transformation protocol. We then provided Exeter with contact details for Dr Rebekka Biedendieck from the Technical University of Braunschweig, a Team Leader and Bacillus specialist. On the 18th September, Dr Biedendieck sent Exeter an improved B. subtilis protocol that allowed Exeter to successfully transform their bacteria.
Part 2: Ashesi University iGEM 2020
Ashesi Project Summary
They aim to tackle the issue of coastal erosion and plastic pollution which severely affects Ghana. To address this problem, they are developing self-healing bio-concrete tetrapods containing bacteria able to seal gaps and microfractures in the structures to increase their lifetime and structural strength. In order to achieve this with synthetic biology, they are employing the same ureolytic pathway for CaCO3 generation and concrete regeneration as Exeter. Additionally, they proposed the integration of I. sakaiensis populations (with native PET-degrading metabolism) with these tetrapods to tackle plastic pollution in coastal regions.
To learn more about their project, please check out Ashesi's page
Partnership summary
Without a target location, our proposed Microbial Desalination Cell device was not much of a solution to plastic pollution and freshwater scarcity. To this end, we contacted Ashesi University’s iGEM team to enquire about a potential partnership. Fortunately, they were keen to collaborate with us since they were also attempting to tackle plastic pollution (as well as coastal erosion) in coastal regions of Ghana. This led us to decide on one shared objective:
- Conducting a research survey targeting potential stakeholders to improve the human practices & implementation approaches of our respective projects
Section 1: How did your collaborative work inform and shape your project at different stages?
June 2020
Aside from purely experimental efforts, we were also aiming to partner with a team from a target location for our Microbial Desalination Cell device. After an analysis of locations that suffered from plastic pollution and freshwater scarcity, we identified Ghana as a leading target. Therefore, we contacted Ashesi University’s iGEM team and discussed the general directions of our projects and areas of overlap.
July 2020
A vital part of our project this year would be to receive the potential stakeholders’ opinions about our device as a potential solution to Ghana’s plastic pollution and freshwater scarcity issues. Therefore, Ashesi agreed to help us design and distribute a research survey, asking stakeholders about different aspects of both our projects. As we learned of their aims to combine self-healing bio concrete with PET degradation, we conducted zoom tutorials to show them our construct designs and added.
August 2020
In early August, we began designing a survey to release to citizens living in Ghana. We consulted with Ashesi’s team and their academic contacts to design the questions to maximise the benefits of the research. This helped us to refine our research survey so that both our teams could gain more valuable insight. Without these refined questions, we would have both come away with considerably less information on how to tackle human practices & implementation.
We also organised a virtual meetup between our two partners (Exeter and Ashesi) to foster further collaboration between them, as our three projects shared some overlap. We were elated to see how this influenced each of their projects, as Ashesi were able to gain insight into bacterial growth protocols that Exeter had successfully employed to test the ureolytic pathway both teams were using in their projects.
September 2020
Thanks to Ashesi’s contacts throughout their region, we were able to spread our survey to potential stakeholders of our device and Ashesi’s project, whilst we exchanged updates about our respective projects throughout the month.
October 2020
We received answers to our survey which managed to get 99 responses. In the answers, we learned of the most critical human practices issues that our device and Ashesi’s project may cause, such as concern about the use of GMOs. More specifically, we gained insight into the main areas of knowledge that participants would like to learn more about to become more comfortable with the device (see below). This allowed us to shape ours and Ashesi’s human practices next steps to target educating stakeholders about GMO safety concerns.
Section 2: How did your teams work together throughout the season?
Throughout the season, our teams worked together using the online meeting software Zoom. This allowed us to initially present our respective project ideas and future directions, and to propose the idea of partnering. Through Zoom we were able to conduct the following types of meetings:
- Weekly updates to discuss progress in our respective tasks for the shared objective
- One-off meetings to brainstorm new tasks that would advance the progress of our shared objectives
- Tutorial meetings where one team could teach the other about relevant research on S. oneidensis
Section 3: How did each team in the partnership benefit from the collaboration?
An example of the critical concerns of stakeholders that ours and Ashesi’s subsequent human practices strategies can target to maximise success in our respective projects’ next steps. The three most critical concerns were summaries of the organisms our respective devices would be using, summaries of the modifications we would be making to these organisms. Specific to our device, we found that the most critical concern was that stakeholders wished to see a list of potential effects these GMOs could have on the freshwater produced by our Microbial Desalination Cell.
Furthermore, we found that 85.9% of respondents would feel more comfortable consuming crops grown using the freshwater produced by our GMO-containing device by learning more about the safety considerations of both of our GMOs.
Specific to Ashesi’s project, we found that their potential stakeholders would mostly be in support or strongly in support of their self-healing bio-concrete block solution.
