iGEM 2023 — Team aboa-turku

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### Hey there and welcome to our project notebook!

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Here you can take a closer look on what we have been up to and when. Just hover over dates to see
date specific tasks and click on + sign for more information!

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{% block calendar %}

December

| Mon | Tue | Wed | Thu | Fri | Sat | Sun |
|  |  |  | 1 | 2 | 3 | 4 |
| 5 | 6 | 7 | 8 | 9 | 10 | 11 |
| 12 | 13 | 14 x14 / 12 / 2022  ---   Kick-off ABOA 2023 meeting  Talked about what iGEM is, what last year’s team did, what are the next steps in this project and then the team members got to know each other a little bit.   --- | 15 | 16 | 17 | 18 |
| 19 | 20 | 21 | 22 | 23 | 24 | 25 |
| 26 | 27 | 28 | 29 | 30 | 31 |  |

January

| Mon | Tue | Wed | Thu | Fri | Sat | Sun |
|  |  |  |  |  |  | 1 |
| 2 | 3 | 4 | 5 | 6 | 7 | 8 |
| 9 | 10 | 11 x11 / 01 / 2023  ---   1. team meeting  Held first official team meeting where we selected team leaders, went through some practicalities and started brainstorming.   --- | 12 | 13 | 14 | 15 |
| 16 | 17 | 18 | 19 | 20 | 21 | 22 |
| 23 | 24 x24 / 01 / 2023  ---   2. team meeting  We discussed office and work spaces, met a new team member and started brainstorming.   --- | 25 | 26 | 27 | 28 | 29 |
| 30 | 31 x31 / 01 / 2023  ---   3. team meeting  We decided sub team leaders for human practices, dry lab, wet lab and IT, talked about each group's responsibilities and met yet another new team member.   --- |  |  |  |  |  |

February

| Mon | Tue | Wed | Thu | Fri | Sat | Sun |
|  |  | 1 | 2 | 3 | 4 | 5 |
| 6 | 7 | 8 | 9 x09 / 02 / 2023  ---   Instagram post about the new team  First group photo of the team and a caption that says we are excited for this iGEM journey. ![Instagram post about the new team](https://static.igem.wiki/teams/4831/wiki/photos/notebook/ig-9-2.jpg)   --- | 10 | 11 | 12 |
| 13 | 14 x14 / 02 / 2023  ---   4. team meeting  We discussed this year’s team logo, making of one-pager and upcoming presentation for Suomen bioteollisuus ry.   ---   1. human practice meeting  Started to vision our team values, talked about Bioteollisuus Forum presentation, looked at the medal criteria and started to plan our education journey   --- | 15 | 16 | 17 | 18 | 19 |
| 20 | 21 | 22 | 23 | 24 | 25 | 26 x26 / 06 / 2023  ---   Instagram post about the new logo  Post about our logo with the new team colors that Jesse picked out (team voted which combo is the best). Quote from the caption: We wanted to carry on the tradition where the new ABOA team picks out their own new colors for the team logo! ![ Instagram post about the new logo](https://static.igem.wiki/teams/4831/wiki/photos/notebook/ig-26-2.jpg)   --- |
| 27 | 28 x28 / 02 / 2023  ---   5. team meeting  We agreed on future meeting time, what medals we would like to aim for and talked about labs.   --- |  |  |  |  |  |

March

| Mon | Tue | Wed | Thu | Fri | Sat | Sun |
|  |  | 1 | 2 | 3 | 4 | 5 |
| 6 x06 / 03 / 2023  ---   Presentation to ÅA students  Mariel and Ami went to present ABOA for 15 min to second-year biochemistry students to promote ABOA, iGEM and recruit new team members.   --- | 7 x07 / 03 / 2023  ---   6. team meeting  We agreed on time for a team photoshoot and brainstormed for professionals to contact about our project idea.   --- | 8 | 9 x09/ 03 / 2023  ---   Meeting with Pauli about the project idea  Meeting with our PI to talk about the project topics we have been thinking about. We narrowed down the possible topics to a few of them.   --- | 10 x10 / 03 / 2023  ---   2. fundraising meeting  Planned fundraising strategy, wrote an email base and set responsibilities to each subteam member.   --- | 11 x11 / 03 / 2023  ---   Team leaders meeting  Sara and Mariel went to have a cup of coffee and tea. We discussed what should be done and when. Basic team administration.   --- | 12 |
| 13 x13 / 03 / 2023  ---   Presentation to ÅA students  Sara and Saana went to present ABOA for 15 min to third-year students to promote ABOA, iGEM and recruit team members. We got two team members after this!!   --- | 14 x14 / 03 / 2023  ---   Photoshoot & hangout  We took our first professional group photos and individual photos at Quantum. After the photoshoot we went to a pub to hangout.   --- | 15 x15 / 03 / 2023  ---   7. team meeting  We met our new group members from Åbo Akademi, talked about starting a promotion video and discussed lab status.   --- | 16 | 17 | 18 | 19 |
| 20 | 21 x21 / 03 / 2023  ---   8. team meeting  Talked about fundraising status, future school visits and what researchers have been contacted.   --- | 22 | 23 x23 / 03 / 2023  ---   1. media meeting  Promo video planning, decided how to execute the individual introductions on Instagram and updated our social media accounts.   ---   Individual introduction begins on Instagram  We introduced every team member on Instagram: their name, what they study, what sub teams they are part of and favorite things.   --- | 24 | 25 | 26 |
| 27 | 28 x28 / 03 / 2023  ---   Human practice meeting with Aino (about the kindergarten visit)  We talked with Aino, who is a preschool teacher at Ilpoisten päiväkoti, to talk about how we should teach the children about synthetic biology.   --- | 29 x29 / 03 / 2023  ---   Meeting with Jesper  Satu and Joakim had a meeting with Jasper. They visited the lab, the warehouse and talked about the lab work in general.   ---   9. team meeting  Went through what lab teams are doing, what is the budget (750€) and agreed on team values.   --- | 30 | 31 x31 / 03 / 2023  ---   Collaboration with patras.med  Participated in a World Health Day collaboration that was organized by the Patras Medicine iGEM team. We made and decorated a poster about Joyce Meyer’s quote. We sent a photo of us with the poster to Patras Medicine and they made an Instagram compilation of iGEM teams who participated. ![Collaboration with patras.med](https://static.igem.wiki/teams/4831/wiki/photos/notebook/ig-31-3.jpg)   --- |  |  |

April

| Mon | Tue | Wed | Thu | Fri | Sat | Sun |
|  |  |  |  |  | 1 | 2 |
| 3 x03 / 04 / 2023  ---   Meeting with the vice faculty of UTU  Sara and Ami held a presentation about ABOA and our project. We got a grant from the University of Turku after this meeting.   --- | 4 x04 / 04 / 2023  ---   10. meeting  Talked about fundraising status, future school visits and team shirts.   ---   3. Fundraising meeting  We sent fundraising emails to companies together and applied for grants.   --- | 5 | 6 | 7 | 8 | 9 |
| 10 | 11 x11 / 04 / 2023  ---   Kindergarten visit  We visited Ilpoisten päiväkoti and taught 3- to 5-year-old children about lab safety and cell modification.   --- | 12 x12 / 04 / 2023  ---   11. team meeting  Talked about collaboration with the Aalto-Helsinki team and arranging a meeting with them. Discussed also about our research plan and team registration.   ---   Promo video meeting  We brainstormed promo video ideas and decided the plan and schedule for the execution.   --- | 13 x13 / 04 / 2023  ---   Meeting with Pauli about the team registration  We discussed how to do the team registration.   --- | 14 x14 / 04 / 2023  ---   Meeting with Juha Kurkela  Talked overall about Synechocystis, what things need to be considered when growing cyanobacteria, how to monitor cell functions and how to handle mercury in the lab.   --- | 15 | 16 |
| 17 | 18 x18 / 04 / 2023  ---   Clean up collab  In honor of Earth Day we participated in a Clean Up collaboration that was organized by the iGEM Bulgaria team. We cleaned up the area near our campuses. ![Clean up collab](https://static.igem.wiki/teams/4831/wiki/photos/notebook/ig-18-4.jpg)   ---   12. team meeting  Went through IT, media and lab statuses, talked about registration and made a plan for summer.   --- | 19 x19 / 04 / 2023  ---   Bioteollisuus Forum  We had an opportunity to talk about synthetic biology, our project and iGEM in general in the Bioteollisuus Forum that was organized by Finnish Bioindustries. Before our presentation we learn about the current situation of synthetic biology in Finland and the different Finnish companies in this field. After the presentations we had the chance to have eye-opening conversations with the representatives of various bioindustry companies and organizations. ![Bioteollisuus Forum](https://static.igem.wiki/teams/4831/wiki/photos/education/bioforum.jpg)   --- | 20 | 21 | 22 | 23 |
| 24 | 25 x25 / 04 / 2023  ---   Meeting with Pauli: team registration  Sara and Mariel had a meeting with our PI. They registered the team for the iGEM competition and discussed how to proceed. They also got some very good advice as team leaders from the PI.   --- | 26 | 27 | 28 | 29 | 30 |

May

| Mon | Tue | Wed | Thu | Fri | Sat | Sun |
| 1 | 2 x02 / 05 / 2023  ---   13. meeting  Went through our current budget, upcoming events and lab team got everyone up to date on what was happening.   --- | 3 | 4 x04 / 05 / 2023  ---   2. Media meeting  Discussed the promo video and its schedule, started to plan our education campaign and decided upon our project reveal.   ---   Meeting with Jesper  Discussed how to get started with lab, research and what things need to be considered in the different phases of the project.   --- | 5 | 6 | 7 |
| 8 | 9 x09 / 05 / 2023  ---   Beginning of the post series Synthetic biology with ABOA  We started our educational campaign on Instagram. We explained different synthetic biology terms and topics regarding our projects. ![Beginning of the post series Synthetic biology with ABOA](https://static.igem.wiki/teams/4831/wiki/photos/notebook/ig-9-5.jpg)   ---   Meeting with Aalto-Helsinki team  We had a Zoom meeting with the other Finnish team: Aalto-Helsinki. We presented our project ideas and talked about collaboration ideas. ![Meeting with Aalto-Helsinki team](https://static.igem.wiki/teams/4831/wiki/photos/notebook/aalto-9-5.jpg)   --- | 10 x10 / 05 / 2023  ---   14. meeting  Talked about upcoming lab orientation, collaboration status with the Aalto-Helsinki team and project status.   ---   4. Human practice meeting  Discussed our interest in Turku’s Comic Festival, decided the plan for Tiedekoulu visit, planned our future events and compiled a list of questions that you help start the conversations with our stakeholders.   --- | 11 | 12 x12 / 05 / 2023  ---   KSYK visit  Visited an upper secondary school in Helsinki called Kulosaaren yhteiskoulu. We taught them about our project, iGEM and different biology terms especially those regarding genetically modified organisms. To activate the audience and get the conversation following, the students got to brainstorm their own iGEM project and we went through those together in the classroom. ![KSYK visit](https://static.igem.wiki/teams/4831/wiki/photos/education/upper-secondary.jpg)   --- | 13 | 14 |
| 15 x15 / 05 / 2023  ---   Laboratory orientation  Everyone who wanted to work in the lab during the summer needed to participate in a laboratory orientation which Anniina Lepistö held in order to ensure the safety of us and others. We walked through the different lab facilities, learned protocols regarding autoclave and other devices, learned about the break room etiquette (if you drink coffee, you bring coffee) and said hi to various people working in the lab.   ---   Tiedekoulu visit  We visited Turku’s Tiedekoulu (Science school in English) and there we taught the children about cells, microscopes and together we extracted DNA from bananas. ![Tiedekoulu visit](https://static.igem.wiki/teams/4831/wiki/photos/notebook/tiedekoulu-15-5.jpg)   --- | 16 x16 / 05 / 2023  ---   Subteam leaders meeting  We had a meeting with the subteam leaders to check that everything is already for the upcoming summer months.   ---   15. meeting  Talked about the Biotop meeting, project name and future collaborations and upcoming events.   --- | 17 x17 / 05 / 2023  ---   Meeting with Pauli, Ville & Joakim on plasmids  Went over research to decide the genes we would be working with. Then started to design the genes and the construct.   --- | 18 | 19 | 20 | 21 |
| 22 x22 / 05 / 2023  ---   Lab meeting  Went over what progress has been made and how we should continue.   --- | 23 x23 / 05 / 2023  ---   Heureka meeting  Had a meeting with Aalto-Helsinki and Heureka. We discussed the possible workshops and dates for the education events.   --- | 24 x24 / 05 / 2023  ---   5. Human Practice meeting  We planned our future Human practice work and education journey: we planned how to organize   --- | 25 x25 / 05 / 2023  ---   Meeting with Celine  We discussed what are the sources and impacts of mercury in aquatic ecosystems.   ---   Meeting with Arja  We discussed forestry, its history and future, how forestry is done on marshlands. We discussed ditching of marshlands and what are its impacts on forests, ecosystems and mercury emissions.   --- | 26 | 27 | 28 |
| 29 x29 / 05 / 2023  ---   Meeting with Antti Kaseva  We discussed water purification technologies, ditching of marshlands, processed waters and the fertilization of marshland forests.   --- | 30 x30 / 05 / 2023  ---   16. team meeting  We went through the budget, talked about making a project reveal video and what was going on with human practises and collaborations.   --- | 31 x31 / 05 / 2023  ---   Meeting with Kimmo Kahilainen  We discussed mercury in ecosystems, its impact, Minamata convention and eutrophication of bodies of water. We also got some very good advice concerning scientific research.   --- |  |  |  |  |

June

| Mon | Tue | Wed | Thu | Fri | Sat | Sun |
|  |  |  | 1 x01 / 06 / 2023  ---   Meeting Linda Vuorijoki  Joakim met with Linda Vuorijoki and they discussed about codons and how to harmonize them.   ---   Team shirts arrived  Our team shirts arrived which were provided to us by Turku University of Applied Sciences.   --- | 2 | 3 | 4 |
| 5 x05 / 06 / 2023  ---   World Environment Day collab  To celebrate World Environment Day, we published the collaboration video we organized. We asked various iGEM teams to tell us how they are saving this environment. Huge thank you to all the amazing teams that participated: iGEM Thrace, iGEM MIT-MAHE, iGEM Aix-Marseille, iGEM TU Braunschweig, iGEM UniCRETE and iGEM TU Dresden. ![World Environment Day collab](https://static.igem.wiki/teams/4831/wiki/photos/notebook/tiktok-5-6.jpg)   ---   Lab day 1  We began our lab work! We started growing Escherichia coli (E. coli) culture to extract constructs. We prepared samples for P0, P1, P23S, P24S and P6.   --- | 6 x06 / 06 / 2023  ---   Lab day 2  We prepared glycerol samples of P0, P1, P23S, P24S and P6. We also performed plasmid minipreps on the above-mentioned E. coli cultures.   --- | 7 x07 / 06 / 2023  ---   Lab day 3  We prepared BG-11 medium for cyanobacteria.   --- | 8 x08 / 06 / 2023  ---   IT meeting  Discussed our ideas, the schedule and how are we preceding with the wiki building.   --- | 9 x09 / 06 / 2023  ---   Lab day 4  We made our first liquid culture of cyanobacteria.   --- | 10 | 11 |
| 12 x12 / 06 / 2023  ---   Lab day 5  We made Synechocystis sp. PCC6803 glycerol preps. We also grew more E. coli culture.   --- | 13 x13 / 06 / 2023  ---   Impact grant application sent  Sara and Mariel double checked our implication grant application and then sent it.   ---   17. meeting  Talked about the upcoming Turku comic festival, organizing group funding with Kulttuurilahja and contacting press and other media.   ---   Village decision done  We filled out the village decision preference form.   ---   Lab day 6  We made more E. coli minipreps. We also had a meeting with our primary assistant professor Pauli Kallio.   --- | 14 x14 / 06 / 2023  ---   Photoshoot with new team shirts  We took group photos and individual photos with our new team shirts. We also filmed a part of the promo video.   ---   Project reveal  We posted our project reveal on our social media platforms: Instagram, Facebook, LinkedIn and TikTok.   ---   Lab day 7  We had a research day when we planned our next experiments.   --- | 15 x15 / 06 / 2023  ---   Filming at a swamp  Promo video team took a trip to a nearby marshland called Pomponrahka. We filmed parts of the promo video.   ---   Lab day 8  We prepared competent E. coli cells for transformation.   --- | 16 | 17 | 18 x18 / 06 / 2023  ---   Project description ready (for the first deadline)  IT put up our project description on our wiki page.   --- |
| 19 x19 / 06 / 2023  ---   Preliminary Safety Form sent  Our PI and Satu looked over our safety form and sent the preliminary version.   ---   Lab day 9  We prepared antibiotic-free BG-11 growth plates with Synechocystis sp. PCC6803. We had our weekly lab meeting, too.   --- | 20 x20 / 06 / 2023  ---   Lab day 10  We prepared our genes (P3 sYFP2 and P4 RBS S4) and then performed heat-shock transformation to competent E. coli cells.   --- | 21 x21 / 06 / 2023  ---   Lab day 11  We made glycerol preps and minipreps of the transformed E. coli cultures (P3 sYFP2 and P4 RBS S4).   --- | 22 x22 / 06 / 2023  ---   Lab day 12  We measured the DNA concentrations in our E. coli (P3 sYFP2 and P4 RBS S4) miniprep samples using NanoDrop.   --- | 23 | 24 | 25 |
| 26 x26 / 06 / 2023  ---   Lab day 13  Weekly lab meeting.   --- | 27 x27 / 06 / 2023  ---   18. meeting  Talked about the upcoming Study in Turku event, Heureka collaboration and Aalto-Helsinki meet up. Lab team got everyone up to speed.   ---   Lab day 14  We prepared LB and LBA media and grew E. coli cultures for P1, RBS S5 and EFE. Additionally, we had a meeting with our primary assistant professor Pauli Kallio.   --- | 28 x28 / 06 / 2023  ---   Team leaders meeting  Discussed what to do, when and who is in charge. We also did team administration.   ---   Lab day 15  We made glycerol preps and minipreps of EFE, sYFP2, RBS S4, RBS S5 and P1 E. coli cultures.   --- | 29 x29 / 06 / 2023  ---   Lab day 16  We had a meeting with our primary assistant professor Pauli Kallio.   --- | 30 |  |  |

July

| Mon | Tue | Wed | Thu | Fri | Sat | Sun |
|  |  |  |  |  | 1 | 2 |
| 3 x03 / 07 / 2023  ---   Lab day 17  Weekly lab meeting.   --- | 4 x04 / 07 / 2023  ---   Lab day 18  We started growing P0, P1 and RBS S4 E. coli cultures.   --- | 5 x05 / 07 / 2023  ---   Lab day 19  We made glycerol preps and minipreps of P0, P1 and RBS S4 E. coli cultures and measured their DNA concentrations. We also made Synechocystis sp. PCC6803 preculture and E. coli cultures for P4 RBS S5, sYFP2, P2 RBS S3, P4 RBS S3 and P6 TT.   --- | 6 x06 / 07 / 2023  ---   Lab day 20  We performed minipreps of P4 RBS S5, sYFP2, P2 RBS S3, P4 RBS S3 and P6 TT E. coli cultures. We also performed midipreps of promoter and backbone.   --- | 7 x07 / 07 / 2023  ---   Lab day 21  We concentrated our DNA fragments by centrifugation.   --- | 8 | 9 |
| 10 x10 / 07 / 2023  ---   Team leaders meeting  Discussed the trip to Paris and came up with team rules to keep up the work. Also some project administration.   ---   Lab day 22  We performed an assembly reaction for sYFP2/EFE and merA/merB.   --- | 11 x11 / 07 / 2023  ---   Sub team leaders meeting  Went through what is going on in every subteam and how to proceed. We also agreed on the team rules.   ---   19. team meeting  Went through group funding status, talked about the upcoming HS Turku interview and how we would like to collaborate with the Edinburgh team.   ---   Lab day 23  We performed a heat-shock transformation (sYFP2/EFE and merA/merB) to E. coli.   --- | 12 x12 / 07 / 2023  ---   Lab day 24  We prepared an E. coli liquid culture and performed a colony PCR.   --- | 13 x13 / 07 / 2023  ---   HS Turku interview  Helsingin Sanomat Turku division came to our lab to interview us about our project and synthetic biology.   ---   Lab day 25  We performed an assembly reaction for sYFP2/EFE and merA/merB.   --- | 14 x14 / 07 / 2023  ---   Lab day 26  We performed a heat-shock transformation to E. coli and prepared agar plates.   --- | 15 x15 / 07 / 2023  ---   Lab day 27  We made secondary E. coli cultures.   --- | 16 |
| 17 x17 / 07 / 2023  ---   Lab day 28  We made new colonies and prepared colonies for a PCR reaction.   --- | 18 x18 / 07 / 2023  ---   New gene order  Joakim made new gene order with added cutsites for cloning in pUC-19   ---   Lab day 29  We ran electrophoresis to analyze the results of PCR.   --- | 19 x19 / 07 / 2023  ---   Lab day 30  We concentrated our DNA and ran a construct (P1, P2 RBS S3, P4 RBS S5 and P6 TT) assembly reaction.   --- | 20 x20 / 07 / 2023  ---   Implementation meeting  Discussed our implementation and who to contact to discuss our idea.   ---   Lab day 31  We performed a RecBCD reaction and transformation to competent E. coli cells.   --- | 21 x21 / 07 / 2023  ---   Lab day 32  We made secondary E. coli cultures for PCR.   --- | 22 x22 / 07 / 2023  ---   Our video was presented in JulyGEM  iGEM Calgary organized an event called JulyGEM in which we participated via recording.   --- | 23 |
| 24 x24 / 07 / 2023  ---   Aalto-Helsinki meet up  We invited Aalto-Helsinki team to Turku. We went sightseeing, gave them a tour of our lab and campus, ate in our student cafeteria and got to know each other. ![Aalto-Helsinki meet up](https://static.igem.wiki/teams/4831/wiki/photos/collaboration/2.png)   ---   Had a conversation with Ari Savikko from Suomen Kalakirjasto  We had a conversation with a fisherman called Ari Savikko. He helped us understand the current situation of fish and our waters by telling us about artificial lakes Finland has and the way they have affected the environments.   ---   Lab day 33  We performed a PCR reaction.   --- | 25 x25 / 07 / 2023  ---   Had a conversation with Jaakko Ikonen  We had a conversation with a fisherman called Jaakko Ikonen. We discussed the history of mercury, the current situation and how different things affect fish and fishers.   ---   20. team meeting  Went through our current budget, upcoming events and wiki text status.   ---   Lab day 34  We ran electrophoresis to analyze the results of PCR.   --- | 26 x26 / 07 / 2023  ---   Wet lab meeting  Discussed about gene reorder in vectors and decided on the ordering of primers. Also talked about the next upcoming weeks of the lab.   ---   Human Practice meeting  Discussed the Human Practice question upon the iGEM website and the vision about our work   ---   Lab day 35  We had a lab meeting where we discussed gene reorder in vectors and ordering of primers. We also planned the next upcoming weeks in the lab.   --- | 27 | 28 x28 / 07 / 2023  ---   Nordic iGEM Conference  Mariel and Joakim represented the whole team at the Nordic iGEM Conference, NiC for short, and we won the conference! Judges were impressed by our presentation and we got rewarded with the golden pipette. The whole weekend was so much fun: getting to know different Nordic and Baltic teams and their project and learning a lot by listening to lectures and attending Hackathon. Thank you iGEM SDU for organizing this great event and thank you for all the teams that made this weekend so fun! ![Nordic iGEM Conference](https://static.igem.wiki/teams/4831/wiki/photos/collaboration/4.png)   --- | 29 x29 / 07 / 2023  ---   Nordic iGEM Conference  Mariel and Joakim represented the whole team at the Nordic iGEM Conference, NiC for short, and we won the conference! Judges were impressed by our presentation and we got rewarded with the golden pipette. The whole weekend was so much fun: getting to know different Nordic and Baltic teams and their project and learning a lot by listening to lectures and attending Hackathon. Thank you iGEM SDU for organizing this great event and thank you for all the teams that made this weekend so fun! ![Nordic iGEM Conference](https://static.igem.wiki/teams/4831/wiki/photos/collaboration/4.png)   --- | 30 x30 / 07 / 2023  ---   Nordic iGEM Conference  Mariel and Joakim represented the whole team at the Nordic iGEM Conference, NiC for short, and we won the conference! Judges were impressed by our presentation and we got rewarded with the golden pipette. The whole weekend was so much fun: getting to know different Nordic and Baltic teams and their project and learning a lot by listening to lectures and attending Hackathon. Thank you iGEM SDU for organizing this great event and thank you for all the teams that made this weekend so fun! ![Nordic iGEM Conference](https://static.igem.wiki/teams/4831/wiki/photos/collaboration/4.png)   --- |
| 31 |  |  |  |  |  |  |

August

| Mon | Tue | Wed | Thu | Fri | Sat | Sun |
|  | 1 | 2 | 4 x04 / 08 / 2023  ---   Feedback conversation  We discussed and went over feedback we had about team members.   --- | 4 | 5 | 6 |
| 7 x07 / 08 / 2023  ---   Lab day 36.  We ran PCR to amplify our old gene fragments and our new merB gene (Pseudomonas aeruginosa).   --- | 8 x08 / 08 / 2023  ---   21. team meeting  We talked about who is going to Paris and booked our tickets!   ---   7. Human practice meeting  Discussed the contribution, values and a possible field trip to a local lake and swamp area with an expert   ---   Lab day 37.  We made construct assemblies using new PCR amplified gene fragments: P0 pdF-C, P1 promoter, P2 RBS S3, P3 merA, P4 RBS S3, P5 merB, P6 terminator.   --- | 9 x09 / 08 / 2023  ---   Lab day 38.  We performed exonuclease reactions on the assemblies that we made the day before and transformed the assemblies into competent E. coli cells.   --- | 10 | 11 x11 / 08 / 2023  ---   Lab day 39.  We ran PCR with the construct assemblies that were made latest.   --- | 12 x12 / 08 / 2023  ---   Lab day 40.  We ran electrophoresis with the constructs that were amplified the day before.   --- | 13 x13 / 08 / 2023  ---   Lab day 41.  We made cultures of the E. coli cells that we transformed with the latest plasmid constructs.   --- |
| 14 x14 / 08 / 2023  ---   Lab day 42.  We performed colony PCR on transformed E. coli cells. We also made glycerol preps and midipreps of the cell cultures that were made the day before. Additionally, we started a Synechocystis sp. PCC6803 preculture.   --- | 15 x15 / 08 / 2023  ---   22. team meeting  We planned a workshop and wiki home page   ---   1. presentation video meeting  Talked about what the presentation video is, the schedule and the division of the workload   ---   Lab day 43.  We run agarose gel electrophorensis with the PCR samples from the day before. We also started liquid cultures on transformed E. coli plate cultures.   --- | 16 x16 / 08 / 2023  ---   Meeting with Elisa Männistö about swamps  We had a conversation with Elisa Männistö about swamps, ecology and the role they have in our ecosystems. We gain a lot from the conversation.   ---   Lab day 44.  We run PCR on four different plasmid constructs. We also made minipreps and glycerol preps of merA and merB E. coli. Additionally, we had a lab meeting.   --- | 17 x17 / 08 / 2023  ---   Lab day 45.  We run electrophoresis with construct samples that were amplified with PCR. We also started the assembly of new constructs.   --- | 18 x18 / 08 / 2023  ---   Meeting with Laura Höijer about Baltic Sea Action Group and the Baltic Sea in genera  We met with Laura to discuss the protection of the Baltic Sea, what things to consider in these types of projects and how our project can have a continuous impact on our waters. We received a lot of great points and questions we need to find answers to.   ---   Lab day 46.  We performed a RecBCD reaction to the new constructs that were started the day before. Then we transformed competent E. coli cells with the constructs.   --- | 19 | 20 |
| 21 x21 / 08 / 2023  ---   Lab day 47.  We started a new Synechocystis main culture.   --- | 22 x22 / 08 / 2023  ---   Meeting with Aalto-Helsinki team about Heureka & Science Basement  We planned the Heureka workshops and Science Basement talk we are doing as a collaboration with Aalto-Helsinki. Unfortunately Aalto-Helsinki could not make it to the meeting but we got a lot of work done and sent our ideas to them.   ---   23. group meeting  Went through our project budget and collaboration plans.   ---   Lab day 48.  We performed colony PCR on transformed E. coli cells. Then we run electrophoresis on the PCR samples.   --- | 23 x23 / 08 / 2023  ---   Lab day 49.  We measured concentrations of our gene fragments and prepared LBA for our cell cultures.   --- | 24 | 25 x25 / 08 / 2023  ---   Lab day 50.  We started the assembly of new constructs by preparing a fragment mastermix.   --- | 26 | 27 |
| 28 x28 / 08 / 2023  ---   Lab meeting about mercury measurement  The lab team researched, planned and contacted experts about mercury and how to handle it responsibly.   ---   Lab day 51.  We performed a RecBCD reaction to the new constructs that were started the day before. Then we transformed competent E. coli cells with the constructs. We also made cultures for P2 RBS S4 and P2 RBS S3.   --- | 29 x29 / 08 / 2023  ---   Visit to Kakolanmäki Wastewater Treatment Plant  We visited a local underground wastewater treatment plant. We took a tour of their facilities, learned about their work and had a meeting with an expert to discuss biological purification and what to consider in wastewater treatment.   ---   24. group meeting  Talked about future events and went through every subteams status.   ---   Lab day 52.  We prepared new LBA and started cultures for P2 RBS S4 and P2 RBS S3 again.   --- | 30 x30 / 08 / 2023  ---   Study In Turku fair  We had a stand about our team, project and synthetic biology at the fair for new students of Turku. We met numerous new students, guided them with pipetting and got to answer and ask interesting questions.   ---   Lab day 53.  We made transformation with our constructs to competent E. coli cells.   --- | 31 x31 / 08 / 2023  ---   2. meeting with iGEM Edinburgh about our cyanobacteria collaboration  We had a Zoom meeting about our collaboration and decided to do a report about cyanobacteria and biosafety. Also we discussed our projects and shared feelings about the slow growth rate of Synechocystis.   ---   Lab day 54.  We plated E. coli colonies to LBA plates with spectinomycin to choose cells with transformed plasmids.   --- |  |  |  |

September

| Mon | Tue | Wed | Thu | Fri | Sat | Sun |
|  |  |  |  | 1 x01 / 09 / 2023  ---   Lab day 55.  We performed colony PCR on transformed E. coli cells. Then we run electrophoresis on the PCR samples.   --- | 2 x02 / 09 / 2023  ---   Heureka collaboration with Aalto-Helsinki  We organized a DNA, RNA & Protein workshop in Heureka, a science center in Vantaa, with the Aalto-Helsinki team. In the workshop the visitors learned about the power trio of life and got to build DNA & RNA themselves.   --- | 3 |
| 4 x04 / 09 / 2023  ---   Lab day 56.  We made plasmid preparations of P2 RBS S3 and PDF-C backbone, and measured the concentrations.   --- | 5 x05 / 09 / 2023  ---   Meeting with Katja Rytkönen from Turun pursiseura (Turku’s yacht club)  We talked about what Katja and the Turun pursiseura does, the insights Turun pursiseura has regarding sailing and the Baltic Sea and then how we should consider them in our project.   ---   25. team meeting  Went through collaborations and lab stuff. ![25. team meeting](https://static.igem.wiki/teams/4831/wiki/photos/notebook/meeting-5-9.png)   ---   Lab day 57.  We started the assembly of six new constructs.   --- | 6 x06 / 09 / 2023  ---   Meeting with PI about implementation  We met our PI Pauli Kallio and discussed our implementation text and how to present our ideas the best way with visual aids.   ---   Lab day 58.  We performed a RecBCD reaction to the new constructs that were started the day before.   --- | 7 x07 / 09 / 2023  ---   Team Leader meeting  The Team Leaders discussed what has to be done and with what urgency. We did a delegation and planned the next steps of our project.   ---   Lab day 59.  We transformed our latest assemblies into competent E. coli cells.   --- | 8 x08 / 09 / 2023  ---   Meeting with Risto Sulkava from Hiilipörssi (Carbon Market)  We talked about the restoration of peatlands, what Hiilipörssi does and what things we should take into consideration regarding restoration in our project.   ---   Lab day 60.  We plated colonies that were transformed with our latest plasmid constructs the day before.   --- | 9 | 10 |
| 11 x11 / 09 / 2023  ---   Advisor meeting about lab wiki texts  We went over the first versions of our lab wiki texts with our advisors. They gave us really good comments and insights. We will take these points under consideration.   ---   Advisor meeting about Human Practice and other texts  We went over the first versions of our Human Practice and other wiki texts with our advisors. They gave us really good comments and insights. We will take these points under consideration.   ---   Lab day 61.  We performed colony PCR reaction with cells that were transformed with our latest plasmid constructs. We also started a new Synechocystis sp. PCC803 main culture.   --- | 12 x12 / 09 / 2023  ---   26. team meeting  We went through our current budget and talked about the lab team's future plans.   --- | 13 x13 / 09 / 2023  ---   2. presentation video meeting  We discussed the overall vision for the presentation video and after informed the rest of the presentation video group about it.   --- | 14 | 15 | 16 x16 / 09 / 2023  ---   Heureka collaboration with Aalto-Helsinki  We organized a DNA, RNA & Protein workshop in Heureka, a science center in Vantaa, with the Aalto-Helsinki team. In the workshop the visitors learned about the power trio of life and got to build DNA & RNA themselves. The second time we added pipetting to the workshop in order to be more interactive and exciting.   --- | 17 |
| 18 | 19 x19 / 09 / 2023  ---   Meeting with our PI Pauli Kallio  We talked about our achievements, how they should be reported and how we should continue in the lab. In addition we ate apple pie that our team leader Sara had made and talked about this journey we have faced. ![Meeting with our PI Pauli Kallio](https://static.igem.wiki/teams/4831/wiki/photos/notebook/meeting-19-9.png)   ---   27. team meeting  Short meeting, went through our plans regarding upcoming Science Basement talk   --- | 20 x20 / 09 / 2023  ---   1. filming day of presentation video  We filmed the Design, Education, Ending and Introduction scenes.   --- | 21 x21 / 09 / 2023  ---   2. filming day of presentation video  We filmed the Human Practice, Methodology, Safety and Next steps scenes.   ---   9. Human practice meeting  Went over the Human Practice and other wiki texts. Discussed animal rights and our project goals   ---   Lab day 62.  We made another colony PCR with cells that were transformed with our latest plasmid constructs.   --- | 22 x22 / 09 / 2023  ---   Lab day 63.  We made elektrophoresis analysis on the PCR samples from the day before to see if the merA-merB plasmid assembly worked.   --- | 23 | 24 |
| 25 x25 / 09 / 2023  ---   Ruusukortteli hyvinvointikeskus (in English Ruusukortteli’s wellness center) visit  We visited a local wellness center to teach their customers about the basics of synthetic biology. We held a presentation with interactive discussion sessions and at the end showed our everyday laboratory equipment.   --- | 26 x26 / 09 / 2023  ---   28. team meeting  Went through wiki and each pages status.   --- | 27 x27 / 09 / 2023  ---   Meeting with the GU-Frankfurt team  We met with the GU-Frankfurt team via Zoom. We discussed our projects and wastewater treatment in our countries. It was wonderful to share our thoughts and hear how other iGEM projects are going.   --- | 28 | 29 | 30 x30 / 09 / 2023  ---   The Science Basement talk with Aalto-Helsinki team  We gave an afternoon talk about bioremediation with our collaborating team at Helsinki main library Oodi. The talk can be found here ![The Science Basement talk with Aalto-Helsinki team](https://static.igem.wiki/teams/4831/wiki/photos/communication/the-science-basement.jpg)   --- |  |

October

| Mon | Tue | Wed | Thu | Fri | Sat | Sun |
|  |  |  |  |  |  | 1 |
| 2 | 3 x03 / 10 / 2023  ---   29. team meeting  Went through wiki status and budget. Talked about our plans for Paris.   --- | 4 | 5 x05 / 10 / 2023  ---   3. presentation video filming day  We planned to film our Results scenes.   ---   Lab day 64.  We performed colony PCR using three colonies from a plate containing E. coli cells with merA/merB constructs. After running the PCR, we made gel electrophoresis with the samples to analyze their content.   --- | 6 x06 / 10 / 2023  ---   Lab day 65  We made minipreps from the three merA/merB colonies from the day before.   --- | 7 | 8 |
| 9 | 10 x10 / 10 / 2023  ---   30. team meeting  Went through wiki and talked about our plans for the Jamboree.   --- | 11 | 12 | 13 | 14 x14 / 10 / 2023  ---   Turku’s Comic Festival  We will attend Turku’s Comic Festival and the idea is to have an interactive workshop that mixes science and art.   --- | 15 |
| 16 | 17 | 18 | 19 | 20 | 21 | 22 |
| 23 | 24 x24 / 10 / 2023  ---   31. team meeting  Planned team meeting   --- | 25 | 26 | 27 | 28 | 29 |
| 30 | 31 x31 / 10 / 2023  ---   32. team meeting  Planned team meeting   --- |  |  |  |  |  |

November

| Mon | Tue | Wed | Thu | Fri | Sat | Sun |
|  |  | 1 | 2 x02 / 11 / 2023  ---   Grand Jamboree in Paris  First day of the Jamboree   --- | 3 x03 / 11 / 2023  ---   Grand Jamboree in Paris  Jambree day 2   --- | 4 x04 / 11 / 2023  ---   Grand Jamboree in Paris  Jamboree day 3   --- | 5 x05 / 11 / 2023  ---   Grand Jamboree in Paris  Final day of the Jamboree   --- |
| 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| 13 | 14 | 15 | 16 | 17 | 18 | 19 |
| 20 | 21 | 22 | 23 | 24 | 25 | 26 |
| 27 | 28 | 29 | 30 |  |  |  |

{% endblock %}
{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the
engineering design cycle.{% endblock %}
{% block title\_class %}engineering-title{% endblock %}
{% block additional\_css %}
{% endblock %}
{% block page\_content %}

In iGEM, the engineering mindset is in a spotlight to turn scientific research into innovations that promote
bioindustry, sustainable solutions and creating something unforeseen. To create optimized and the most
efficient
solutions the engineering cycle – Design, Build, Test and Learn (DBTL) – is often used as a guideline. To
create
the most optimized strain of Synechocystis, our host organism, there were nested
engineering cycles (Figure 1).

---

![](https://static.igem.wiki/teams/4831/wiki/photos/engineering/d-3.png)

Figure 1. A visualization of the Engineering Process our team went through in the laboratory work process.
Within our primary engineering cycle to genetically modify our host organism, the Synechocystis, we achieved multiple engineering cycles whilst aiming to create
optimized constructs in the host.

---

## Table of contents

- [Intro](#intro)
- [Design](#design)
- [Build](#build)

- [Test](#test)
- [Learn](#learn)
- [DBTL within building our constructs](#DBTL)

---

# Design

MercuLess in about remediating waters from the toxic methylmercury with the means of synthetic biology. To
achieve this we had to find effective genes merA and merB
of the proteins MerA and MerB, respectively, which would work in our host organism Synechocystis sp. PCC 6803. The MerB protein is an alkylmercury lyase that separates the
mercury from the methyl group creating ionic mercury. The MerA is a mercuric reductase that reduces the ionic
mercury into its elemental form. We also had to choose the right ribosomal binding sites (RBS) for our genes
of interest but this was not simple. Although Synechocystis is a model organism, it
is much less known about than for example Escherichia coli and required more effort
and creativity from our team to find the best choice. Because there are no standard in
silico models for choosing the right RBSs in Synechocystis we had to go
with the old trial and error way of engineering. We were also not certain how well the genes would work. Here
we saw the possibility for testing two different merA and merB homologs to find the most suitable one.

While deciding which homologs to use we had to make sure the genes of interest were compatible to be
translated efficiently in Synechocystis . This issue was especially relevant since
half our genes are from Pseudomonas Aeruginosa , a Gram-negative aerobic bacteria,
which has a really high guanine-cytosine concentration unlike Synechocystis and a
whole different codon usage. To tackle this problem, we decided to try codon harmonizing software
“CodonWizard”
[(Rehbein et al., 2019)](https://www.sciencedirect.com/science/article/pii/S1046592819301214),
which matches the codons by their corresponding rarity.

To measure the performance of our constructs and to help us select the best candidate for the future, we had
to design a suitable measuring protocol. Because not much work is done with methylmercury and its measurement,
there are no standard protocols for measuring it, especially for Synechocystis. Due
to this we had to also engineer our measurement system to suit our purposes. We decided to take inspiration
from team Minnesota's 2014 project with merA and merB and
their experimental setup to study the activity of our genes of interest, and modified it for our needs. More
information on the modification process can be found from our
[Design page](https://2023.igem.wiki/aboa-turku/design).

---

# Build

Because of the need for modularity in our construct design we were stirred into the way of Golden Gate
assembly. Fortunately for our team the Department of Molecular Plant Biology at the University of Turku had
just finished their new Golden Gate Cloning based assembly system for RBS optimization. This means they
already had a library of parts for the assembly, so we only had to design our genes of interest to fit the
assembly. We got the opportunity to use their cloning system, which was great because it allowed us to
conveniently make multiple different constructs with different RBSs and genes of interest. This assembly
system also allowed us to speed up the engineering cycle by studying multiple variables of the same construct
simultaneously.

To find out more about our part design and our final construct with optimized RBS go check our
[Parts page](https://2023.igem.wiki/aboa-turku/parts).

---

# Test

To test the performance of our genetically modified Synechocystis the object is to
test their efficiency in catalyzing methylmercury into elemental mercury. This is to be done by treating our
cells with methylmercury and then measuring the concentration afterwards.
[(Click here for a more detailed protocol)](https://2023.igem.wiki/aboa-turku/protocols).
To get better certainty of our constructs’ toxicity to the cells, we also measure the oxygen usage
of the cells. To get the most optimal result,, the aim is to test different variations of the same construct.

To get certainty of the proper function of our measurement system the objective is to use a
negative control
A controlling method that gives us the information of the success when it does not work in given conditions
.
To save time we decided to go for a setup that was fast, affordable and did not require a collaboration with a
third party. This was an essential decision, since shipping samples requires a lot of extra time and effort
especially because our samples contain methylmercury and are extremely dangerous.

Our experimental setup needed to prove to us that both of our genes of interest, merA and merB , had activity. Because our genes are operating
under the same promoter, we had to somehow separately measure which changes are caused by merA and which by merB. To achieve this, we made single gene
constructs, where the other gene of interest was replaced with a reporter gene, either sYFP2 or EFE . This way even though we know that merA catalyzes the reduction of the ionic mercury and merB the
cleavage of the methyl group, we can with certainty know what causes what. We will also be treating the cells
with methylmercury and mercury chloride separately, to be sure.

---

# Learn

To get the most optimal construct to transform methylmercury into elemental mercury in Synechocystis, we would iterate the process and learn from all results. In the next
iterations the aim is to try other RBSs to see if more efficient dissociation of methylmercury and reduction
of the ionic mercury would be achieved. The objective is also to multiply the number of merA genes to find out if that would make the reaction more efficient. Also, since there
are multiple varieties of merA homologs we would test them out and see if any of
them would be better than the one we are currently using. On top of these iteration cycles the aim is to test
the effects of adding
IPTG
Isopropyl β-D-1-thiogalactopyranoside, a molecular biology reagent
in our system and studying the effects of different promoters.

---

# DBTL within building our constructs

Our laboratory work faced some trouble when trying to assemble our constructs. Although this slowed down our
process of measuring the process of turning methylmercury into elemental mercury we got valuable experience of
iterating the engineering cycle (Figure 2 a-e).

Designing the construct:
we designed as good a construct as we were able to with our current knowhow by using the
pDF backbone
The base of the plasmid, where our genes of interest are added
.

Building the construct:
we built the construct carefully with the new Golden Gate inspired assembly method.

Testing the construct:
we tested if the constructs are transformed into our host organism with antibiotic resistance using an
antibiotic, Spectinomycin. We also see that the constructs are correct by amplifying the constructs with PCR
and by running an electrophoresis.

Learning of the results:
when getting the results of the antibiotic selection and the electrophoresis we see if the resulting bands are
correct. If the bands are not what we were looking for, we get back to the design phase and make another
iteration of the engineering cycle to get a properly functioning assembly.

---

1. iteration

2. iteration

3. iteration

4. iteration

5. iteration

---

![](https://static.igem.wiki/teams/4831/wiki/photos/engineering/iteration-1.png)

![](https://static.igem.wiki/teams/4831/wiki/photos/engineering/iteration-2.png)

![](https://static.igem.wiki/teams/4831/wiki/photos/engineering/iteration-3.png)

![](https://static.igem.wiki/teams/4831/wiki/photos/engineering/iteration-4.png)

![](https://static.igem.wiki/teams/4831/wiki/photos/engineering/iteration-5.png)

Figure 2. Our engineering cycles on creating the optimized construct visualized. A) The first iteration of
our engineering cycle. The goal was to create a seven-part construct including our genes of interest with
linear fragments with the new assembly method based on the Golden Gate. B) The second iteration of our
engineering cycle. The first cycle was modified by having higher concentrations of the linear fragment to
enhance the reaction. C) The third iteration of our engineering cycle. The objective was to achieve creating
optimal constructs by using circular fragments subcloned from the linear fragments used in the previous
cycle. D) The fourth iteration of our engineering cycle. The goal was to build the constructs by using the
modified Golden Gate assembly method but using commercial fragments. E) The fifth iteration of our
engineering cycle. Repeating and modifying the previous cycle with new knowledge.

---

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block title\_class %}experiments-title{% endblock %}
{% block page\_content %}

No scientific progress can be achieved without experimenting. We have begun the experiments on the first days
of June 2023 by using protocols we have received from different sources e.g. professors and the internet. Well
planned is half done, so we seek to plan and execute our plans with care every step of the way. All
experiments are carried out taking different safety aspects into account.

In our lab experiments, our goal was to assemble plasmid constructs with merA and
merB genes using a Golden Gate inspired method. The plasmid construct that we
designed included the following fragments: P0 pDF backbone, P1 promoter, P2 RBS, P3 merA , P4 RBS, P5 merB and P6 terminator. The construct parts,
except from merA and merB genes, were extracted from
existing Eschericia coli cultures. After successful assembly we will transform the
constructs firstly to E. coli and then to Synechocystis
sp. PCC 6803 cells.

---

## Table of contents

- [Construct part extraction from E. coli cultures](#construct part)
- [Preparing the construct assembly](#preparing)
- [Construct assemblies and
  screening](#construct assemblies)
- [Growing Synechocystis](#growing)
- [Synechocystis
  transformation and screening (future experiments)](#transformation)
- [Mercury experiments (future experiments)](#mercury)
- [References](#references)

---

# Construct part extraction from E. coli cultures

### Growing E. coli cultures for extracting construct parts

Cultures for all gene fragments from the library were made: P0 backbone, P1 promoter, P2 RBS S3, P2 RBS S4, P3
sYFP2, P4 RBS S3, P4 RBS S4, P4 RBS S5, P5 EFE and P6 terminator. Antibiotic selection was conducted with
spectinomycin and ampicillin.

### Preparing glycerol samples of construct part E. coli strains

Glycerol preps were made from the E. coli cultures. The samples were placed in the
freezer (-80°C) for later use.

### Making plasmid preparations of E. coli cultures

Plasmid preparations were made from E. coli cultures using either the Monarch
Plasmid Miniprep Kit (NEB) or Qiagen Midiprep kit (pDF and promoter) following the protocols provided by the
manufacturers.

---

# Preparing the construct assembly

### Preparing competent E. coli cells for transformation

Competent cells were prepared for transformation by making a culture with inoculated DH5α E. coli cells. The culture was centrifuged, the pellet resuspended with CaCl₂ and stored
in the freezer (-80°C) for later use.

### Preparing commercial genes

We ordered the merA (from Synechocystis and Pseudomonas aeruginosa) and merB genes (E.
coli and Pseudomonas aeruginosa) from IDT and Twist. The genes were
prepared following the instructions provided by the manufacturers.

---

# Construct assemblies and screening

### Modified Golden Gate assembly

In our experiments we used a modified Golden Gate assembly to create plasmid constructs with different
combinations of our merA and merB genes and different
ribosome binding sites (RBS). We made several assemblies with this one-pot Golden Gate reaction with all the
fragments in a single tube in order to construct several complete plasmids simultaneously. The general
structure of our plasmids was as follows: starting with a plasmid backbone, then a promoter at position 1, RBS
at position 2, one of our merA homologs at position 3, another RBS at position 4, one of our merB homologs at
position 5 and a transcription terminator at position 6.

For each assembly, we also prepared positive controls by replacing our genes of interest with sYFP2 and EFE. Negative controls were also prepared by using
only the plasmid backbone. Later we also tried creating assemblies with only one gene of interest, while the
other was replaced with either sYFP2 or EFE depending on
the position of the replaced gene.

For the assembly reaction, two mastermixes were prepared: a reagent mastermix with all the necessary PCR
reagents and a fragment mastermix. The fragment mastermix was prepared with our genes of interest, RBSs,
promoter, transcription terminator and plasmid backbone. The genes and ribosome binding sites were different
in each assembly reaction and the amount of each varied between assemblies due to different concentrations. A
digestion ligation cycle (RecBCD) was run on the samples.

### Heat-shock transformation (E. coli )

PCR samples were transformed into the competent DH5α E. coli cells using heat-shock.
DNA-cell samples are briefly exposed to a high-temperature and then immediately placed back on ice. This
method is based on the principle of thermal stress which makes the bacteria more likely to accept foreign DNA.
The control included a plate with only the backbone as well as a plate with everything else except backbone
verifying the success of transformation.

### Colony screening by PCR (E. coli )

After a successful transformation the possibility of positive results were high. To verify that our assembly
had worked and that we had successfully constructed modified plasmids, we did colony screening by PCR. The
forward primer in the PCR binds towards the end of the promoter and the reverse primer to the other end of the
pDF vector. This means the PCR covers the entire length of all inserts as well as a small portion of the
promoter and pDF backbone. E. coli samples were randomly selected colonies from our
LBA plates. The amount of samples varied between transformations, but the PCR master mix was always prepared
15% more than our sample size.

### Agarose gel electrophoresis (E. coli )

The PCR results were studied with 1% agarose gel electrophoresis with positive and negative controls.

---

# Growing Synechocystis

### Making a liquid culture of cyanobacteria

Liquid culture was made from Synechocystis sp. PCC 6803. The cyanobacteria were
flushed from a growth plate with BG-11 medium. The culture was put in a growth chamber where the main liquid
cultures were made from the previous one with a starting OD750 of 0,1A

### Making Synechocystis sp. PCC803 storage cells

Once the absorbance of the liquid culture had reached 0,597 A at 750 nm. Glycerol preps were made from the
Synechocystis culture. The samples were frozen with liquid nitrogen and put in the freezer at -70 °C for later
use.

### Making Synechocystis sp. PCC6803 BG-11 growth plates without antibiotics

BG-11 growth plates were prepared with bacto-agar. Samples were then plated with no antibiotics and put to
grow in a growth chamber.

---

# Synechocystis transformation and screening (future experiments)

### Synechocystis sp. PCC6803 transformation

We have a modified transformation protocol based on the findings of Zang et al., and Lauri Kakko’s, a doctoral
researcher at the Molecular Plant Biology unit of University of Turku, modifications. In this protocol EDTA
was
added to the liquid main Synechocystis sp.
PCC6803 before the culture reached a mid logarithmic growth phase. The cells were plated on BG-11 plates first
with no antibiotics. After a green film forms the antibiotics are added underneath the agar. Then the plates
are covered with a paper sheet to reduce light intensity. The goal of all of these modifications are to add
efficiency and reduce the growth and selection period of Synechocystis.

### Colony screening by PCR and gel electrophoresis (Synechocystis)

The colony screening with Synechocystis includes freeze-thaw cycles.

The protocol for gel electrophoresis is the same as above with E. coli DNA samples.

---

# Mercury experiments (future experiments)

To test the function of the Synechocystis sp. PCC 6803 cell strains that have been
transformed with merA and merB genes, our idea was to
perform mercuric resistance experiments on the modified cyanobacteria. The ability of the modified cells to
grow in the presence of mercury compounds can be used as an indicator of the function of the merA and merB genes. Our team did not have time to perform
these experiments with offers a unique opportunity for further research. The protocols for mercury resistance
experiments would have been based on the protocols by iGEM team Minnesota 2014 (Mercury(II) Chloride Plate
test protocol - parts.igem.org, n.d.). The protocols would have been slightly modified and adapted to our
cyanobacteria-based system.

Our plan was to make two different assays to test the function of merA and merB genes separately. In the experiments, we would have prepared agar plates with a
filter disk spotted with methylmercury or mercury(II) chloride in the middle of the plates. In one assay, we
would make agar plates with 10µL of 0.1M methylmercury on a filter disk to screen the function of the merB gene. The diameter of the area where the cells can not grow would have been
measured and compared between different cell strains. The plates would have contained cells with constructs
that either include the merB gene alone or both merB and
overexpressed merA. The mercury resistance of modified cyanobacteria would have been
compared to unmodified cyanobacteria. Because the assembly was performed in E. coli
, we would also have tested and compared the function of the constructs in E. coli
versus Synechocystis sp. PCC 6803.

In the other assay, we would have made agar plates containing 10µL of 0.1M mercury(II) chloride on a filter
disk to test the function of the merA gene. The mercury(II) chloride resistance of
Synechocystis with overexpressed merA would have been
compared to unmodified Synechocystis that has a natural merA gene. The results would also have been compared with E.
coli cells with the merA containing plasmid construct.

Later, had the constructs been qualitatively shown to function in the resistance assays, we would also have
wanted to test the constructs quantitatively using ICP-MS to measure the changes in methylmercury and mercury
ion concentrations. This would have required preprocessing of the samples since ICP-MS only can detect the
total concentration of mercury.

---

[Link to Protocols](https://static.igem.wiki/teams/4831/wiki/documents/protocols-of-experiments.pdf)

[Link to Lab notebook](https://static.igem.wiki/teams/4831/wiki/documents/lab-notebook.pdf)

---

## References and sources

Mercury(II) Chloride Plate test protocol—Parts.igem.org. (n.d.). Retrieved from
[https://parts.igem.org/Mercury(II)\_Chloride\_Plate\_test\_protocol](https://parts.igem.org/Mercury%28II%29_Chloride_Plate_test_protocol)

Rippka, R., Deruelles, J., Waterbury, J. B., Herdman, M., & Stanier, R. Y. (1979). Generic
Assignments, Strain Histories and Properties of Pure Cultures of Cyanobacteria. Microbiology, 111(1),
1–61. <https://doi.org/10.1099/00221287-111-1-1>

Zang, X., Liu, S., Arunakumara, K. K. I. U., & Zhang, X. (2007). Optimum conditions for transformation
of Synechocystis sp PCC 6803. Journal of microbiology (Seoul, Korea), 45, 241–245.

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## Table of contents

- [Results](#results)
- [Planning future experiments](#future)
- [References](#references)

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Our project aimed to create a bioremediation tool to purify mercury-contaminated waters using cyanobacteria.
The design of our detoxification system is based on two genes, merA and merB, which code for mercury degrading enzymes. First we aimed to assemble plasmid
constructs with these genes of interest that we could then transform to Escherichia coli
(E. coli). After confirming the success of the assembly, we would transform them to a naturally
competent genus of cyanobacteria, Synechocystis sp. PCC 6803. The modified Synechocystis sp. PCC 6803 strains would then be tested for their ability to resist and
detoxify organic mercury compounds. This text will discuss our lab results and achievements.

### Submitting the Project safety form

After careful evaluation of the safety protocols needed for our project and that our activities were
compatible with iGEM regulations, we filled the Project safety form which was submitted by our Principal
Investigator.

### Acquiring a lab space

Our team acquired a lab space from the Department of Life Technologies in the University of Turku. We went
through an orientation and safety training arranged by the department before beginning lab work.

### Selecting the target genes for the project

As candidate expression targets for the project, we chose to use two merA genes: one
native gene from Synechocystis and the other from Pseudomonas
aeruginosa. As merB genes we chose to use gene homologs from E. coli and P. aeruginosa.

### Choosing the expression host

We selected the photoautotrophic model organism, a genus of cyanobacteria, Synechocystis sp. PCC 6803 as the expression host for our project since we wanted to
create a sustainable bioremediation tool that can produce its own organic compounds using light and carbon
dioxide readily available for large-scale systems.

### Selecting the construct assembly platform

An experimental Golden Gate based assembly system developed specifically for translation-level optimization of
heterologous genes and operons in Synechocystis sp. PCC 6803 was selected for
building the expression constructs.

### Designing the expression system

An autonomous replicative expression vector pDF with an IPTG-inducible lac-promoter variant was selected as
the expression construct. The specific translational control elements, ribosomal binding sites (RBS), to be
fused with merA and merB were selected based on the
article by Thiel et al. (2018). We used three different RBS’s (RBS S3, RBS S4 and RBS S5) for P2 and P4
positions in our plasmids. We designed a plasmid construct containing seven fragments: P0 pDF backbone, P1
promoter, P2 RBS, P3 merA, P4 RBS, P5 merB and P6
transcription terminator (Fig. 1). In the control assemblies, we used reporter genes EFE and sYFP2 instead of the merA
and merB genes and we also made some assemblies with merA
or merB with EFE or sYFP2.

![](https://static.igem.wiki/teams/4831/wiki/photos/results/figure1-a.jpg)

A

![](https://static.igem.wiki/teams/4831/wiki/photos/results/figure1-b.jpg)

B

![](https://static.igem.wiki/teams/4831/wiki/photos/results/figure1-c.jpg)

C

![](https://static.igem.wiki/teams/4831/wiki/photos/results/figure1-d.jpg)

D

Figure 1. Plasmid maps for our constructs using P2 RBS S3 and P4 RBS S5 for our RBS’s: A) sYFP2/EFE B)
merA/EFE C) sYFP2/merB D) merA/merB

### Modifying the target genes for the assembly

The target gene sequences were codon harmonized for Synechocystis sp. PCC 6803. We
also modified the gene sequences to be compatible with the modified Golden Gate assembly system by removing
internal BbsI restriction sites and introducing site-specific flanks.The sequences were also checked for no
BsaI or SapI restriction sites to be compatible with iGEM systems.

### Confirming the design by in silico assembly

The intercompatibility of the designed genetic fragments was verified in silico by
assembling the fragments into expression plasmids in different combinations (Fig. 2). The assembly was tested
with the different RBS elements in one-gene and two-gene constructs using merA,
merB, and a control reporter gene sYFP2.

![](https://static.igem.wiki/teams/4831/wiki/photos/results/figure2.jpg)

Figure 2. The expression plasmid was assembled in silico using SnapGene. The
backbone (pDF) is cut out of the figure. Our target genes were designed to fit into positions 3 and 5,
with the promoter in position 1, RBS’s in positions 2 and 4, and the transcription terminator in position
6.

### Preparing the genetic element library

We completed a part library consisting of all the genetic elements (apart from merA
and merB) needed for the assembly by amplifying the respective pre-existing
constructs in E. coli. The plasmid minipreps and the respective E. coli glycerol preps were indexed in an electronic library designated for our team,
and stored at -80 °C for later use.

### Ordering merA and merB as synthetic fragments

The designed merA genes (Synechocystis and P. aeruginosa) and merB genes (E.
coli and P. aeruginosa) were first ordered as synthetic linear gene
fragments.

### Testing the assembly system using the ordered linear gene fragments

We first evaluated the construct assembly for merA-merB operons using ordered linear
merA and merB fragments. Control reactions with
plasmid-based reporter genes sYFP2 and EFE were
successful, but
the merA-merB reactions did not yield positive clones. (Fig. 3).

---

A

![](https://static.igem.wiki/teams/4831/wiki/photos/results/figure3-a.jpg)

B

![](https://static.igem.wiki/teams/4831/wiki/photos/results/figure3-b.jpg)

Figure 3. 1% standard agarose-gel analysis of the sYFP2/EFE, backbone and
merA-merB constructs using linear gene fragments. Quick-Load 1 kb Extend DNA
Ladder was used as a marker (sample M). For the RBS’s we used P2 RBS S3 and P4 RBS S5. A) Sample 1
contains a positive control with YFP/EFE, sample 2 contains only the
backbone
and sample 3 contains the negative control with water. Samples 4-14 are the sYFP2/EFE constructs and samples 15-22 are backbone samples. B) Sample 1
contains
only backbone and sample 2 the negative control. Samples 3-19 are the merA-merB constructs and samples 20-23 the backbone.

### Testing the assembly system using PCR-amplified linear gene fragments

To increase the concentration of the merA and merB
fragments in the assembly, we amplified the genes with PCR using primers designed for this specific purpose.
Both the control reactions with plasmid-based reporter genes and the merA-merB
plasmid constructs were unsuccessful (Fig. 4).

![](https://static.igem.wiki/teams/4831/wiki/photos/results/figure4.jpg)

Figure 4. 1% standard agarose-gel analysis of the merA-merB constructs with
PCR-amplified linear genes. NEB Quick-Load 1 kb Extend DNA Ladder was used as a marker (sample M) in the A
and B gels and NEB Quick-Load 1 kb Plus DNA Ladder in the C and D gels (sample M). A) Sample 1 was the
positive control with YFP/EFE, sample 2 only the backbone and sample 3 a
negative control with water. C) Sample 1 was a negative control with water, sample 2 only the backbone and
sample 3 the positive control with YFP/EFE. Samples in the A and B gels are
PCR-amplified linear genes and in the C and D gels samples were more concentrated before PCR. All of the
samples except the controls contain merA-merB constructs with P2 RBS S3 and
P4 RBS S5.

### Ordering merA and merB as plasmid-based gene fragments

Since we could not produce the designed merA-merB plasmid constructs using linear
gene fragments, we ordered the genes in plasmid vectors.

### Testing the assembly with plasmid-based gene fragments

We performed the assembly reaction with different combinations of the ordered merA
and merB genes that were already in plasmids. We did not get positive PCR results
for the merA and merB containing plasmid clones although
the transformation results were promising (Fig. 6, 7).

![](https://static.igem.wiki/teams/4831/wiki/photos/results/figure6.jpg)

Figure 6. Transformation results of construct assemblies. The constructs contained: A) the pDF backgone B)
the part of expression plasmids that do not contain the pDF backbone but an insert consisting of 9
fragments C) merA from Synechocystis and merB from E. coli D) merA from Synechocystis and EFE E) merB from E.
coli and sYFP2 F) sYFP2 and EFE.

---

![](https://static.igem.wiki/teams/4831/wiki/photos/results/figure7-a.jpg)

A

![](https://static.igem.wiki/teams/4831/wiki/photos/results/figure7-b.jpg)

B

![](https://static.igem.wiki/teams/4831/wiki/photos/results/figure7-c.jpg)

C

![](https://static.igem.wiki/teams/4831/wiki/photos/results/figure7-d.jpg)

D

![](https://static.igem.wiki/teams/4831/wiki/photos/results/figure7-e.jpg)

E

Figure 7. 1% standard agarose gel with plasmid-based gene fragments. NEB Quick-Load 1 kb Plus DNA Ladder
was used as a marker (sample M). A) sample 1 was the positive control (YFP/EFE),
sample 2 only the backbone and sample 3 the negative control with water. Samples 4-7 were merA(P)/EFE constructs with P2 RBS S3 and P4 RBS S4, samples 8-15 merA(S)/merB(P) constructs with P2 RBS S3 and P4 RBS S4 and samples 16-24 merA(S)/merB(P) constructs with P2 RBS S3 and P4 RBS S4. B) Samples 1-8 were
merA(P)/merB(P) constructs with P2 RBS S3 and P4 RBS S4, samples 9-16 were
merA(P)/merB(P) constructs with P2 RBS S4 and P4 RBS S3 and samples 17-24
were sYFP2/merB constructs with P2 RBS S3 and P4 RBS S3. C) Samples 1-8 were
sYFP2/merB(P) constructs with P2 RBS S3 and P4 RBS S4, samples 9-16 were
merA(S)/EFE constructs with P2 RBS S3 and P4 RBS S5 and samples 17-24 sYFP2/merB(P) constructs with P2 RBS S3 and P4 RBS S3. D) Sample 1 was only
backbone, sample 2 a negative control with water and sample 3 the positive control with a 9-fragment
construct. For the RBS’s we used P2 RBS S3 and P4 RBS S5. Samples 4-15 were merA(S)/merB(E) constructs and samples were 16-24 merA(S)/EFE. E) For the RBS’s we used P2 RBS S3 and P4 RBS S5. Samples 1-3 were
merA(S)/merB(E) constructs, samples 4-14 were sYFP2/merB(E) and samples 15-24 were sYFP2/EFE
constructs.

---

# Planning future experiments

### Confirming successful assembly of plasmid constructs

Although we did not have time to assemble working merA-merB plasmid constructs, we
planned out our next experiments which we would have performed after successful assembly. First, we would have
confirmed the obtained E. coli clones using colony PCR and SDS-PAGE analysis.
Successful assembly of sYFP2-merA reporter constructs would have been verified by
fluorescence analysis. After that, we would have deposited the verified E. coli
clones in our library.

### Transforming the verified expression constructs in Synechocystis sp. PCC 6803

After extracting the assembled plasmids from the E. coli clones, we would have
transformed Synechocystis sp. PCC 6803 with the assembled plasmids containing the
merA and/or merB genes. Using antibiotic selection, we
would have chosen antibiotic-resistant colonies and later also confirmed that they contain the desired plasmid
constructs. After the confirmation, we would have deposited the verified cyanobacteria expression strains in
our library.

### Analysis of the cyanobacterial expression strains

To test the function of the Synechocystis sp. PCC 6803 cell strains that had been
transformed with merA and merB genes, our goal was to
perform qualitative and quantitative analysis on the expression strains. We would have compared merA-merB constructs and constructs with only merA or merB. Different gene homologs and RBS would also have been compared. First, we would
have performed methylmercury and mercury(II) chloride resistance experiments on the modified cyanobacteria to
indirectly get information about the function of MerA and MerB proteins. We could also have used proteomics to
show that the MerA and MerB proteins are present in the expression strains.

We also contacted SYKE (FInnish Environment Institute) and Ruokavirasto (Finnish Food Authority) to determine
if quantitative mercury analysis using ICP-MS would be possible. With ICP-MS, we would have measured the
changes in methylmercury and mercury ion concentrations in different cell cultures. This would have required
preprocessing of the samples since ICP-MS only can detect the total concentration of mercury.

Later, the constructs could additionally be tested with real contaminated water to see how the expression
strains function in a biologically relevant environment. After the analytical steps and comparison, the
expression strains could have been optimized and then analyzed again. Our final implementation plan is to
build a MercuLess-truck but before it is possible, many additional steps are needed after creating a
functioning cyanobacteria expression strain.

### Discussion

Despite careful construct design, the construct assembly turned out to be difficult to implement. Our
transformation and antibiotic selection results of the last assembly indicated that the transformed colonies
had plasmid constructs inside them but our colony-PCR results were inconclusive. Therefore, it is impossible
to draw reliable conclusions of the last, and also most promising, assembly results. More time would be needed
to perform the assembly reactions again and proceed to analytical steps in making a merA-merB expression strain of Synechocystis sp PCC 6803.

Since our transformation and antibiotic selection, as seen in the plates (Fig.6), were successful, we can
safely presume that leading up to the colony-pcr all the reagents and conditions were accurate. We can see
from the lack of colonies in the control plate B that the antibiotic selection worked and a couple colonies in
the backbone plate A indicates that there is only little background. All of our agarose gel-electrophoresis
results had a successful positive control which shows that our reaction setup and the reagents used were
correct. Therefore the issue may be specific to e.g primers used in the colony-PCR or a part of the construct
we were trying to assemble. More experimentation is needed before we can pinpoint the exact issue.

Since we did not have enough time to assemble and test the function of the desired merA-merB constructs, there are good opportunities for future research and iGEM teams to
continue studying the opportunities of the use of mercury modifying genes in cyanobacteria. We believe that
our cyanobacteria-based system is promising and since cyanobacteria are photoautotrophic, the system has
potential to become a sustainable bioremediation tool needed for environmental protection.

---

---

## References and sources

Thiel, K., Mulaku, E., Dandapani, H., Nagy, C., Aro, E.-M., Kallio, P. Translation efficiency of
heterologous proteins is significantly affected by the genetic context of RBS sequences in engineered
cyanobacterium Synechocystis sp. PCC 6803. Microb Cell Fact 17, 34 (2018).
<https://doi.org/10.1186/s12934-018-0882-2>

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