Meetups to decide the project we are doing, which area in mining we were tackling and preparations to kick start the project.
We have officially started our internship/competition!! This was the week when the entire team came together! As a result, two other students from the Kwame Nkrumah University of Science and Technology (KNUST) with whom we collaborated, came to Ashesi University for a week to kick off the project.
We started the day excellently with two other team members in the AshesiGhana team outside our institution. They talked about what they do on campus, their interest and how excited they are about the project.
A brief introduction was followed by the Principal Instructor (PI), Dr Elena Rosca. We discussed what Synthetic biology is doing and how we can leverage it to solve world pressing problems, especially issues identified in the pre-exploratory stage of gold mining in Ghana.
We spent the afternoon brainstorming on the research and scoping the project topic. We were tasked with conducting additional research on biosensors, how gold can be explored, exploratory mining methods and principles, and so on. By the end of the day, we had clearly defined the project topic and identified some biological methods for detecting gold particles or ions.
We created a Slack page, Google Docs folder, and OneDrive folder to keep the team's files and important documents. In addition, different co-Leads/sub-teams were chosen for the various project responsibilities and components.
We also brainstormed and proposed human practices to ensure an integrated human practice.
We continued with a detailed suggestion of integrated human practices and education. The team investigated applying for the impact Grant; hence, we reviewed the questions. Our PI introduced us to Benching, a cloud-based platform for biotechnology and research. We explored some of the platform's features that would be useful to us in our project.
The team completed the Impact grant application and requested that the PI review it. Michael, the team's co-lead, ensured this and chaired the application process by gathering relevant information and conducting a literature review for the application.
Further discussion on our project resulted in us coming up with a problem statement and solid literature to back our research idea and proposed solution. At this stage, we hadn't looked at the biological composition of our solution; we just explored the potential solutions without considering the biological feasibility.
From there, we looked at the first level of detection of gold complexes or ions, bacteria culture for the specific detection and other pathfinder elements. We also asked the question; how do we turn off these bacteria after sensing?
We looked into iron, arsenic, and gold binding proteins. We also looked at genetic circuits and inducible promoters for the different pathfinders. A resource person took the team through benchling on how to do sequencing, take notes, perform digestion, ligation, and cloning, among others.
To turn the gene, we looked into inducible promoters for the selected pathfinders and catalytic enzymes. Also, our two team members from the other institution finally left us for school! 😔😢
The team brainstormed and researched solutions to the question, "How do we turn these bacteria off after sensing?" This session's ideas gave birth to our first safety and biosecurity stage, which includes using a UV kill switch to activate the T4 Endolysin. Given that we have a diverse team with little to no biology or SynBio background, two design team members designed their first sequence of the arsenic inducer in benchling (See figure below!). The rest of the team sat through benchling tutorials on bio-bricks, digestion and ligation, and golden gate assembly.
The team extensively discussed integrated human practices, education and collaborations with elaborate objectives and required actions. We also met with the Map the System (MTS) Ashesi Team, who also worked on "A systems analysis of the political, environmental, and socio-economic impacts of Illegal Mining in Ghana". Insights from this meeting helped us with a power dynamics 2x2 matrix, a stakeholder map and a clear list of other stakeholders we need to integrate into our project to create a user-centered solution.
Also, Gideon Bonsu and Vera Bordah migrated from benchling to snap gene to do the designs. The experience was a great one!
Sub teams were assigned a task to investigate the safety and biosecurity grant application, specific secretion of proteins outside the selected chassis that enables it to interact with the gold and other pathfinder ions or complexes.
Leeroy explored other ways of safety besides the autolytic kill switch. This resulted in the 2nd and 3rd phases of our biosecurity and safety level. Thus, developing a hydrogel encapsulation that prevents bacteria from escaping into the environment and, secondly, methanol dependency for our chassis (E-coli strain).
The team researched arsenic, iron and gold ions binding proteins concerning our chassis.
Also, Stony Brook iGEM 2022 team reached out for collaboration on their outreach project.
Further research on these were made:
The team explored to find the sequence for the arsenic promoter, thus Pars and arsR.
The team has a zoom call with our PI, Dr. Elena Rosca, via zoom to discuss our sequences, running them in benchling to enable us to submit our part to twist for sequencing. We also discussed a developing framework for documenting information from human practices: interviews, audio recordings, and write-ups, among others. Edith went ahead to contact Ghana Minerals Commission and Golden Star Mining Company. Lastly, Gideon helped the team with writing the project description on slack!
We researched why iron is an ideal element to be detected and the interesting relation between iron and our selected chassis (E-coli). From this research, we identified ferrous iron repressor FUR. We also explored some promoters controlled by FUR protein. These were:
Also, we found out that “although the FUR acts as a transcriptional repressor it can be turned into an activator by preventing or eliminating another transcriptional repressor’s ability to bind to DNA. These findings resulted in our first iron sensing module….
Pc -> -> (RBS)->Fur -> (T) -> PAceB ->-> (RBS)-> lacI-> (T) -> PlacI -> eforRed->(T)
Also, we officially met with Stony Brook to discuss the terms of a possible partnership. They proposed that we participate in their journal initiative, where we describe the societal impact of our project. And we also proposed that they could help us with modelling.
The team decided to investigate alternative designs due to the issue of leakages in our first designs. Our initial design had limited control and operated in a direct "input-output” paradigm. To prevent false positives would lead to an ineffective biosensor that will further worsen the problem we seek to address by giving false indications of areas that may not have gold. Thus, strong terminators insulate against read-through of genes from neighboring promoters.
Early Monday morning this week, Gideon and Vera worked on updating the iron sensing module so we could submit it to IDT and twist. The updated iron sensing module was: Pc -> (RBS)->Fur -> (T) -> PAceB ->-> (RBS)-> TetR-> (T) -> pTetR ->RBS -> eforRed->(T) Thus, we replaced the lacI repressor and its regulatory promoter with TetR and pTetR respectively. In preparation for our meeting with the minerals commission of Ghana, as part of human practices, the team used this time to prepare for the meeting adequately. The co-lead of the team, Michael, helped with the slides.
We designed the kill switch gene sequence in snap gene, and all other designs that were submitted to IDT and Twist. pUV ->RBS->T4 Endolysin->Terminator
Some team members, Gideon, Edith, Michael, and Trish, met with the Minerals Commission of Ghana to understand the current pre-exploratory gold mining activities, the consequences or the impact of the current approaches and any chance of using synthetic biology in this pre-exploratory stage. The team pitched our solution to them and got relevant feedback to improve it.
We got our safety training! We also had a brainstorming session following our meeting with Ghana's minerals commission to discuss their feedback and how to incorporate it into our design. This birthed the staking idea. To develop the stake design, we first demonstrated with a tube filled with a slime where the slime represented the hydrogel we planned to design as a safety measure.
A day after the safety training, the team set up the lab space to kickstart lab work for the project.
We looked at the different lab equipment and what they do and performed simple lab activities such as pipetting, using the water bath, centrifuge etc. We also prepared LB Media and made plates with and without ampicillin before the transformation we were going to do.
Some team members were doing wet lab work, and others were developing content for the wiki page. The two co-leads for the team, Michael and Trish, and Gideon met with team Exeter to discuss a collaboration regarding examining their CAD model. We agreed to the collaboration and ask of them in return to troubleshoot our initial models after we complete them.
It was an exciting day for the team! Approximately 80% of us had no previous experience with synthetic biology. As a result, today's experiments in engineering bacteria strain were an exciting part of our journey. We formed the first competent cells! It was a fun moment, a learning experience, and the starting point for everything we needed to do in the lab to complete our project.
As part of education under human practices, the team had a session with some students in the United Student Achievers Program in Zimbabwe. These high schoolers were working on degrading plastic utilizing an engineered microorganism and wanted help with some information to aid them in achieving their goal.
The team compiled all the parts we used for our design and provided the links to them on the iGEM registry to aid in efficient parts identification and their functions (the role they play in the sequence).
We checked the protocol for transformation since it was the next thing for us to do. Also, we delegated tasks to populate the wiki content document. Edith was tasked to write on the project description and landing page, Vera with design, Gideon with modeling, Trish with collaborations, Leeroy with Safety, Rosemond with Teams page and Michael with human practices.
Individuals continued to work on populating the wiki page content document. We also had a photoshoot for the wiki page, promo video and to thank Inquaba BioTech for providing us with some reagents, lab coats and other lab items. We finalized the designs of our genetic circuits and developed graphic representations of them in PowerPoint.
We met with Stony Brook via zoom to further discuss the project's direction and the entities to model in the system. They suggested possible modeling approaches.
The deadline for the promo video was approaching fast. Led by Michael, Elijah, and Gideon, we brainstormed ideas for the promo video. Gideon also developed models for reactions in the genetic circuits using the Simbiology library in MATLAB. He sent the models to the University of Exeter for their review and feedback, given our collaboration with them.
July was very busy, demanding and a step closer to the final jamboree. We started the Month with a high spirit. We were one of the teams to win the Impact Grant! How exciting is that?
In the first week of August, we made a transformation with amilGFP, amilCP an meffRED from the iGEM kits we got. Unfortunately, our ice marker got spoilt hence we had to make ice from frozen sachet water by crashing it with a mallet. That was a great way to resolve the problem with the shortest time. We also had a meeting with Exeter to work on our collaboration.
Our transformation worked! Yaaay. We were excited to see our transformation work given that it was the first time for about 80% of us. With the help of our PI, we sent sponsorship letters to different organizations such as Tullow Ghana, AngloGold Ashanti etc.; we also went through the content we had written for the wiki page to edit and finalize some parts of it. Later in the day, some team members met with an engineer from the exploratory division of Golden Star Mining Company. We presented our project to her and found out more about the exploration process and exactly where our solution would fit.
Each member helped to perform a mini prep using the zymoPURE miniprep kit. Also, videos of the team working in the lab were taken by the communications department from Ashesi university.
We prepared another batch of competent cells. As part of human practices, we brainstormed ideas for synthetic biology info session at a high school called African Science Academy. Edith went on to arrange this session with the school.
We had an info session with the Ashesi University community where we talked about synthetic biology and its application and encouraged students to enrol in the newly introduced synthetic biology course at the university.
Four of the team members went on a week-long break to take care of other essential engagements they had. However, Leeroy and Gideon helped to design and finalize a project on a biosensor for Stony Brook because we collaborated with them.
Also, the modeling team got feedback from Mat, university of Exeter.
This was a week before the start of the academic year; hence, team members were allowed to prepare for the academic year. We drew a detailed schedule showing each person busy and available to aid in planning to continue the project while doing academic work. On Wednesday, August 17th, Gideon met with Mat to further discuss the feedback from his initial assessment and send over the designs of our genetic circuits for him to help us develop the elementary differential equations. On Friday, August 19th, Mat from Stony Brook sent over the finished differential equations for gold and iron sensing models and walked us through how to represent them in Simbiology. Also, we received a google doc containing a modelling guide, differential equations and parameter values for our gold sensing module. This is very helpful!
Later in the day, we got some exciting news! We won the Safety and Biosecurity Grant.
Our IDT and Twist parts arrived. We started by digesting and ligating the parts. This was a tedious experience. We transformed the DNA after digestion and ligation, but there was no growth. That was devastating, but it was only the tip of the iceberg. Elijah does his best to populate the wiki page with as much information as possible before it freezes.
The wiki guy lost his code…Shocking right? This week involved a lot of work in the wet lab and weekend work in the lab. Rosemond and Vera were busy troubleshooting the cause of the plasmid not ligating through serious digestion and ligation approaches. This included digesting pUC19 with two restriction enzymes, XbaI and SpeI. Michael was busy working on infographics and the content of our human practice, whiles Elijah was on the wiki.
From the results we obtained from our gel electrophoresis, we noticed that the pUC 19 wasn't showing. Hence, to rectify that, we made different concentrations of pUC 19 that we used to do different transformations. This was all we worked on for the entire week while other teammates were working on the wiki.
Finally, we had some light at the end of the tunnel, a different miniprep, and the gel result showed a plasmid. Also, on the 13th of September, some team members met with engineers and planners (E & P). E & P are a leading indigenous Mining and Construction company in the West Africa sub-region. Their primary services include Mining, Construction & Civil Works, and Consulting Services. Following our presentation to their representatives, they provided inquiries, recommendations, and contributions
We started this week with a lot of excitement; with the new pUC 19 plasmids, we were able to get growth on the E Coli, and there was a bacteria glow; thus, green fluoresce since we had GFP reporter gene.
On 21st of September, which was a government holiday, as part of education and collaboration, the team had a fun day with synthetic biology at African Science Academy (A-level school in Ghana), where:
1. We introduced the students to synthetic biology and the amazing things being done in the field
2. Extracted some DNA from bananas
3. Created DNA strand with Haribo and gummy bears
4. Finally, we demonstrated and worked through how to design a plasmid using coloured beads and a locker to represent the different parts of the plasmid.
Also, to know the concentration of the pUC 19 used to obtain the successful results, Rosemond and Vera, with a supporting Lab assistant, performed gel Electrophoresis, which whored that the pUC 19 with a ratio of 1:10 was the best choice.
We conducted digestion, ligation and transformation, including control setups with the 1:10 Puc 19 part to troubleshoot if our ligation and digestion worked. We also wanted to determine which digestion approach was the best for our experiment, whether the digestion approach using a mastermix or without a mastermix. Troubleshooting was successful, which we confirmed from the growth on our amp plates and gel electrophoresis. We confirmed that the digestion without the master mix gave the desired results; hence, we proceeded with our experiment with that information.
Officially the month of wiki freeze! Wrapping Up! We conducted our final digestion, ligation and transformation experiment with our genetic parts. We then continued with a miniprep and Polymerase Chain Reaction (PCR) to amplify our gene of interest in order to conduct a gel electrophoresis.