Week 1: Introductions and Idea Pitches
Once our team had been determined, we began with the first steps of our project. Our first meeting consisted of introducing ourselves, our courses and our background in science. This was then followed by pitching of ideas for our potential project
Week 2: Team and Role Assignment
Now that we had gotten to know each other online, it was time to create teams and assign roles. We determined that seven subgroups were necessary to complete this project: Organization, External Communication, Programming, Scientific Communication, Journal, Funding, and the Drylab/Wetlab subgroups. Each team member chose 2-3 subgroups to be a part of. We also assigned a social media manager (Saskia) and team roles such as Team Managers (Anna and David), Team Leader (Tim/Kevin), and Secretary (Maaike).
Week 3: Goals Were Determined
To decide what type of project we wanted to make, different aspects were considered and certain project ideas were ruled out as either impractical, unnecessary or already attempted in past iGEM projects. This week our social media introductions also took place, where we were introduced ourselves to those that followed our Instagram account.
Week 4: Meeting Times and Topics Were Chosen
A presentation of topics occurred where 3 ideas were researched and weighed against each other by the dry lab team. From there they were presented to the rest of the team where we then voted to choose the topic of stopping ruminant methane emissions.
Week 5: Began Collaborations and
Pre-Requisites Were Distributed
The first teams were contacted by our external relations team and our first meeting was held (with Wageningen's graduate team). The iGEM deliverables were also divided up between the sub-teams in order to ensure that everything was covered.
Week 6: iGEM Registration and Medal Criteria Distribution
With our team officially registered for iGEM, each subgroup started doing research on what projects they wanted to complete. This includes looking into the individual medal and award criteria and determining which ones we would attempt to fulfill.
Week 7: Logo Design and First Monthly Report
The next big step in the identification of our team came about as our social media team created our logo. This was also when the first set of weekly reports were submitted and everyone got an overview of the whole project.
Week 8: First In-Person Meeting for iGEM Welcome Ceremony
As iGEM got set up, so did we. Finally, with the lifting of some Covid restrictions, we were able to meet some of our team mates face to face in order to watch the iGEM welcoming ceremony. The Journal team met with writing expert Joop Westra for the first time who helped develop the workshops.
Week 9: Exam Week
With most of the team busy preparing for exams, minimal work was done this week. Mostly consisting of report backs from collaboration meetings including the Uppsala, Tirupati and Pune teams.
Week 10: Project Period Planning
A meeting was held to create a plan for the weeks to come during project period. This is a 4 week interval schedule in the MSP program where students focus on completing projects, then writing a report and doing a presentation about them. Monthly feedback was also given.
Week 11: First In Person Meeting and Second Monthly Report
We had our first in person meeting and socializing event, where progress and ideas were discussed. The second monthly reports were also handed in to the organization team. Lab work also has begun:
Transformation with LB broth without Kanamycin. 100ul of culture were used on the plates.
Agar plates creation and plating. Competent cell creation.
Remaking of transformed cells
Week 12: Design Detailing
In person meetings allowed for a better flow of creative ideas and designs started to arise. Finally our project name was chosen to be MethaGone. In hopes of showing off our style at the Jamboree we also started designing our apparel and masks.
From the transformed cells, two colonies were chosen each for overnight cultures in LB + Kanamycin. Total of 8 vials - for miniprep
Band present in the gel. Overnight cultures were generated from the corresponding colony for PCR. Primers resuspension and working stock creation based specification sheets by IDT.
Resuspension of the gBlock constructs based on specification sheets given by IDT.
Week 13: New Calendar and More Lab Work
Moving to Taskade had a positive impact on our team and enabled us to be more organized in assigning tasks. The lab team continued working to linearize the plasmid (and we made some creative agar plates on the side). The programming and journal team created a website in order to help the communication with the journal participants. The construct designs were completed and the genes present in the lab.
Week 14: Waganingen Meetup and Project Period Report
An online event was organized by Wageninger and included all the teams from around the Netherlands. All teams gave a project pitch and then listened to guest speakers. With multiple members missing because of this and other circumstances, no meeting was held that week. The Journal team also wrote and compiled a report of our project up until this point as required by the university.
Successful Transformation of the genes, colony selection and overnight culture.
Duplex gene combos via Gibson Assembly done, transformation and plating carried out.
Single gene transformations were successful on plates however DNA wasn’t clearly present on the gel. Duplex gene transformations (Gene + Mbb2) had successful growth, 1 plate did not.
Week 15: G-Block Arrive and Summer Planning
Finally, the long awaited G-Blocks arrived from IDT and the lab team was ready to start modifying. Within the week they make competent cells of the bacteria, suspended and inserted DNA as well as inserted multiple genes into the same bacteria, both those that coded for bromoform and peroxide production. Monthly reports were due again and progress was shared between the subgroups, the goals for each subgroup over the summer (month of July) were also established during this meeting.
Miniprep of Mbb4 and combinations of mbb2+mbb1/mbb4/ccMbb3. Restriction digestion and gel electrophoresis
More agar plates containing Kanaymcin. Linearization of pET39b+ ( further stock). Neb Hifi Assembly:
Competent cell transformation and plating on agar.
Minipreps and Restriction Digest using XbaI and NdeI. Visualization on Gel.
New working primer stock solution for other primers. Lb broth and agar plates creation.
Week 16: Brightlands Funding Pitch and the OJS
The journal website was officially up and running with a fully functioning Open Journaling System (OJS). This was an exciting step for the MSP Vector as it allowed a platform to organize submissions and distribution of articles for the journal. From the Science Communication team, filming, editing and feedback was underway for the promotional video that was due in the upcoming week. A pitch was also done to the Brightlands Venture Capital which was a great experience for the funding team and earned us €250.
Week 17: Moving
This week was intensely disruptive as our university labs were moving from the Brightlands campus to a Maastricht Campus. This caused our lab to be split and ment that we had to organize to keep all the supplies we needed from being moved to the new labs. Our project remained at the same campus due to regulation restrictions and for the sake of consistency.
Week 18: Promotional Video
The promotional video was worked on and finalized this week thanks to the hard work of the science communication team. Filming at the lab and at a cattle farm in the area produced some amazing shots that made this possible. This video can be seen at the bottom of the home page.
Week 19: Managerial Involvement and Safety meeting
Due to holidays, members from the organizational team became more directly involved in the subgroups in order to get an overview of their progress and upcoming goals. A meeting with RIVM which is the dutch National Institute for Public Health and the Environment (Rijksinstituut voor Volksgezondheid en Milieu) was organized to talk about GMO safety.
Lb broth creation, overnight culture for competent cells. A HiFi assembly was created.
Agar plates. Gene transformations and plating.
No colonies grew overnight. A new hifi assembly was conducted with a different linearized plasmid.
Single colonies were re-plated. The genes were PCR with specific gene primers.
Week 20: GMO and Farmer Survey Launched
Simultaneously, a survey directed at farmers in collaboration with iGEM Uppsala, and a survey directed at the general public about GMOs in collaboration with iGEM Tirupati, Pune and Aachen was launched. The workshops for the journal were uploaded to youtube.
Purification and miniprep of genes and gel. Followed by gel extraction. Gel was not showing good results. PCR of the genes.
Ran a gel from PCR, and got better results. Gel extraction of mbb2, not strong concentration.
Gel extractions concentrations were not high enough. Another gel with the previous PCR was done and gel extractions were conducted. Liquid cultures were made with single cultures.
PCR of Mbb1 & Mbb4 were redone since clear bands were not present. Modification of the PCR annealing temperature. Miniprepp of other single gene constructs.
Week 21: Started Business Plan
A subset of the funding team began to create a business plan in order to fulfil the criteria for the Entrepreneurship medal. This involved brainstorming to create schematics in canva that can be seen on the page. These schematics were then written up into the pdf business plan.
Restriction digestion of the genes, single genes in pET39b and followed by a gel electrophoresis.
Gel electrophoresis was carried out and the single PCRed genes were checked following PCR clean up.
Massive gel, with Restriction Digestion genes, PCRed genes and the original genes to compare.
ccMbb1 and ccMbb3 appeared to be correct in the gel, sent off for sequencing
Week 22: Crowdfunding Went Online
After getting input from some advisors within the crowdfunding field, it was time to launch our crowdfunding campaign. The original soft launch was to persuade our warm contact (people we are personally connected to) to donate and later it was more widely distributed to the general public.
Re-did the single genes constructs. They were transformed and plated on kanamycin agar plates.
Growth was not detected on the plates therefore they were left to go further.
Week 23: Swiss School Workshop
This workshop was run by two of our members over Zoom to a class of teenagers in Switzerland. This workshop created an environment to debate about GMOs and other ethical issues while also teaching a higher level of English to the German-speaking students.
Colonies were present on the plates and an overnight culture was created.
The overnight culture of the single genes was miniprepped and the pieces were sent for sequencing
Week 24: Card Game and Peer Review for Journal
The card game for our Science Communication was completed and a test round was then played by our team members. It was then left in the facility common room for other people to play and give feedback on. The journal peer-review pairs were also assigned and sent out this week. Each team was given 2 other teams to peer review and then received 2 back that could then be used to make improvements on their article before submission.
We redid the triplex gene PCR and a Gibson assembly and transformation into DH5a competent cells. They were then plated on Kanamycin plates and left to grow in the incubator.
The plates were left to grow for 3 days to have a great amount of colonies for testing. From this we extracted the cells and created an overnight culture.
We miniprepped the overnight culture and then we ran a restriction digestion to visualize the bands. Due to excess noise we were going to repeat it the following lab day.
We redid the gel however the bands were not in the proper location, which meant two things: either our restriction digest was not successful or our genes do not contain the triplex genes. We selected 3 colonies of each gene combination and grew them in an overnight culture.
Week 25: Invited to Complete Blog Posts and Crowdfunding Video Was Made
Biotechnologie invited our team to create a series of 6 blog posts documenting our experience in iGEM as well as explaining our project. We completed and uploaded our promotional video with a funding task to the crowdfunding platform as we opened it up to the wider public.
We received the triplex gene sequences back and they contained the sequence of the original vector. Therefore we opted to switch with duplex genes only, they would all contain Mbb2 and the other respective gene.
We ran a PCR on our genes so that we could create the duplex combinations. Following this PCR we ran a Gibson Assembly and a transformation and plated the cells.
We were able to see growth on the plates and we created an overnight culture from 3 different colonies.
We mini-prepped the colonies and we sent them off to sequencing to gather the data and check if our duplex combination transformations were correct.
We continued with our single gene constructs; Mbb1 and ccMbb1, we replated these from the successful colonies so that we could generate more. We left these growing overnight on plates in the incubator.
Week 26: Surveys Closed and Analyzed
The survey collaborations were close with a total of 275 responses for the GMO and 10 responses for the Farmers survey. The data was then taken by each team and analyzed separately. For the GMO survey, empirical data were generated and analyzed in graphs. For the farmer survey, each response was individually processed to address individual concerns.
Editing of the documentary has officially started requiring footage licenses to be requested. The final questions for the interviews with various farmers were finished.
A cost structure for future field testing was established in the financing team. Stakeholders were contacted that were mentioned in the business plan. A radio interview was pre-recorded.
We then selected a few and grew them in an overnight culture.
We miniprepped them and we prepared the solutions and buffers required to create an SDS-PAGE.
From here we treated our samples and placed them in a gel and ran it for 40 mins. Following this the gel was removed and we placed it in a staining solution overnight.
Week 27: Video Presentation Preperation
The first blogpost for Biotechnologie was posted, which introduces our team and our project. Peer-reviewing was assigned within the group for different parts of our wiki texts. This week and A person was assigned to create a team that is in charge of a storyboard for the presentation video. With the final weeks of the project ddawing near, the journal team struggled to receive the final articles from the participants.
We then checked if any bands were present on the gel, however, the staining solution was too deep to visualize anything. We placed the gel in a destaining solution overnight.
Our duplex gene sequence results returned and unfortunately they did not represent the actual genes, only our original vector.
Week 28: Disabled Children's workshop and Politician interview took place
Two interviews with politicians took place, one in person in the Hague, and one online in order to acquire footage and information for the documentary. A timeline was simultaneously created for the filming and editing of the presentation video. With all the equipment in place, the ruminal simulation was able to take place. A few of our members traced to a nearby slaughterhouse to acquire a cow's ruminal fluid and solids, and then brought that to the lab for testing in the rumen simulation.
Week 29: Wiki freeze
This week marks the final stages of the iGEM competition with the wiki freeze approaching on the 21st of October. There is a rush to finalize the wiki pages as well as make the final edits on the documentary and the journal.
Week 30: Attending the BeNaLux Mini Jamboree
We will be attending the mini jamboree hosted in Eindhoven. We plan to travel by train to the Eindhoven to meet other iGEM teams from the Netherlands and present our project.
Week 31: iGEM judging will take place on the 5/11/2021
Our project is scheduled for judging on the 5th of November