Team:DTU-Denmark/Notebook

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Notebook

February March April May June July August September October

February

February 15th 2021: 1st General meeting

On this day we had our fisrt online team meeting! We met our supervisors and introduced ourselves to eachother. Our supervisors introduced us to the world of iGEM and we split into 3 teams to do our homework for next time: brainstorm about 2 project ideas per group.

February 22nd 2021: 2nd General meeting

On this day we formed our subgroups and aligned expectations within the groups. Following this we pitched our developed project ideas to eachother. Our homework after this meeting was to further discuss and develop our ideas. Team building also started to plan activities for us to get to know eachother better.

March

March 1st 2021: 3rd General meeting

On this day we presented our further developed project ideas to eachother and brainstormed together, how they could be even better. We established expectations from drylab and wetlab teams. Human practices and PR subgroups gave an overview of interviews and stakeholders contacted.

March 8th 2021: 4th General meeting

On this day, we worked on further developing our projects ideas and voted on the different ones, to decide which one we will work with. We started creating a budget for our project with the help of the economy subgroup. The PR/SoME subgroup got access to all social media platforms and started working on potential collaboration ideas.

March 15th 2021: 5th General meeting

Based on the project assessment template the scoring from each group was validated. Working on methane assimilation got the most votes! We discussed the different time slots to participate in the Ambassador call organized by iGEM.

March 22nd 2021: 6th General meeting

We met in person for the first time to identify potential areas of interest in the project. Decided to assimilate methane in 2 organisms via co-culturing with pMMO subunit being able to metabolize methane. Admin updates to create iGEM account and get the team code. Decided to use Benchling as a platform to document details of our project. We joined the European Ambassador meeting with other iGEM teams from Tubingen, Boku Vienna, Moscow to discuss status of project and covid crisis affecting project workflow.

Souvenir from the Ambassador meeting:

April

April 8th 2021: 7th General meeting

On this day a new supervisor joined our team: Benjamin Schwetz. We discussed the compartmentalisation of our project into two parts: i) methane to methanol and ii) methanol utilization. Furthermore we made a brief project description. Our economy team applied for our first of many grants! This was the day when we have found out that we can start working in the lab on June 1st, which we were really excited about and started planning right away! We also discussed the on-going hearings in the Danish parliament about gene editing and agreed that we should be ready for upcoming political discussions.

April 12th 2021: 8th General meeting

On this day we had a discussion about the possibilities of co-culturing two microorganisms - one converting methane to methanol (mutant methanotrophic bacteria), another to uptake methanol and produce protein of interest (mutant yeast). The idea of joining a political festival in Bornholm was brought up for the first time. We said good bye to Annette, who decided to leave our team.

April 26th 2021: 9th General meeting

On the 9th meeting we decided to split into 3 groups to design the required parts simultaneously. Each group has established separate meetings on top of the general meeting. We decided to use Golden Gate Assembly as one of our cloning methods and Emil gave a plasmid crash course for the whole team to create the best possible designes. We also discussed digital content for Folkemødet.

This month we could also meet in person for the first time!

May

May 1st 2021: Saturday meeting

We decided to meet up on the weekend to work on our constructs: Looked into plasmid based USER cloning and discussed the strategies for using available strains and plasmids codon optimised for K. phaffii , humans, S. cerevisiae .

May 5th 2021: 11th General meeting

On this day, wetlab and drylab made the last preparations to enter the lab: we ordered NEB kits, did the codon optimisation of our constructs, brainstormed more technical questions and had a meeting with Tomas Strucko. The human practices subgroup was preparing for Folkemødet and our economy group sent more grant applications. We made a logo competition within the team, this is how we decided on our final logo. The PR subgroup had a meeting about editing our website and reached out to Mikkel Schwarz for the team photoshoot.

May 10th 2021: 12th General meeting

On our 12th general meeting we discussed strategies on education and reaching out to elementary and high schools. Wetlab and drylab were looking for methanotroph promoters and did some research on glucose repression. We reached out to Unibio and scheduled a meeting with Solange. In the beginning of the project we picked Mambalgin-1 as our protein of interest. The human practices subgroup further planned the GMO discussions at Folkemødet and started planning discussions with high school students.

May 17th 2021: 13th General meeting

This week wetlab and drylab went through the USER protocol and started determining integration sites in K. phaffii genome. Before going to the lab we had to fill out a lab safety form and we all also signed a team contract stating the expectations in the duration of the project. Karen and Karl were working hard on preparing for Folkemødet, furthermore we also hired Mikkel Schwarz for our photoshoot and promotion video.

May 24th 2021: 14th General meeting

August joined our team as a supervisor and after discussions with him, we decided to change the protein of interest to hemoglobin, we had discussion on using soy or human hemoglobin, as human hemoglobin could also possibly be used therapeutically, but ethically it is not fit for food production, thus we picked soy leghemoglobin as final protein for the proof-of-concept. When it comes to our human practices subgroup, Karl and Karen were working on the video advertising for Folkemødet and we also had a meeting with Kenneth from Novozymes.

June

June 2nd 2021: 15th General meeting

During the first week of June we all finished the lab safety test and training, so we were ready to go to the lab. We received assistance from Niels, from the 2020 DTU iGEM team for our drylab modeling work. The human practices team had 2 great interview with Kenneth and with Irini, and we officially got accepted to Folkemødet. On Friday morning we had our first common breakfast, which became a habit on Fridays during the summer months.

June 9th 2021: 16th General meeting

This week we had a meeting with Ampliqon to finalize the details of our sponsorship and talked about a possible facility visit. We ordered the gRNAs for our 4 identified integration sites and made cryostocks for K. phaffi GS115 Δku70 strain. Our drylab team had a meeting with Nikolaus Sonnenschein from DTU Bioengineering for Genomic Scale Metabolic modeling to analyze potential things to do with the GS115 model. Possible risks associated with using pMMO were validated and it was agreed to look for yeast which have native methane activity or to keep sMMO as a backup.

June 16th 2021: 17th General meeting

Wetlab started working on pDIV151 purification and PCR amplification for all of our fragments, the general workflow of all the experiments were made and documentation on Benchling started. Drylab discussed the aspects of the GSM model to further optimize the existing model. This week we had our portraits taken by Mikkel, furthermore Karen and Karl gave an amazing presentation at Folkemødet.

June 23rd 2021: 18th General meeting

This week the wetlab group realized that our gRNA primers did not have the right sequence, thus we ordered new primers, which finally resulted in successful experiments. Drylab was working on the GSM model based on Niko's advices. Our economy team booked the summer house for our wiki freeze week and the PR team was working on writing the children's book.

June 29th 2021: 19th General meeting

Wetlab had great news this week, the gRNA primers finally worked, and started doing the USER cloning experiments, while drylab worked on further perfecting the GSM model. We filmed our presentation video this week, which required a lot of preparation during the days leading up to the filming. Our PR team made a nice post card for a collaboration with iGEM Düsseldorf and started planning the Ampliqon facility visit with iGEM SDU.

July

July 6th 2021: 20th General meeting

Our wetlab team had to troubleshoot this week for the failed USER transformations with the help of August and Louise, and started plannig the methanol kill curve experiment. Drylab started working on the gene knock-out using cobrapy and trying to optimize protein production and they ran the first attempts using GSM model which show that including sorbitol and/or glycerol in the medium gives a better protein production. Furthermore our human practices team applied to participate in Naturvidenskabelig festival.

July 13th 2021: 21st General meeting

This week, we have found the cause of USER cloning failing - wrong combination of RE & nicking enzymes and with this information in mind we corrected our protocols and continued with our experiments. Drylab this week was working on starting training on Kivy for app development and on growth curves with R script. We successfully submitted the promotion video and signed up for the iGEM Nordic conference.

July 20th 2021: 22nd General meeting

Our wetlab team successfully carried out USER cloning for IS1C1 and IS2C1. One out of four available crRNA candidates per IS were tested. We got great colony PCR results for crRNA candidates targeting IS4 and IS5, also confirmed by sequencing. Drylab continued working on documenting everything for the GSM model. Human practices had a busy week, with interviews with Unibio and Novozymes. This week we posted our last team member introduction on all our social media platforms and translated our promotion video into all of our teammember's native and spoken languages!

July 27th 2021: 23rd General meeting

This week we designed primers and homology arms and ordered them for further experiments, we also started planning yeast transformation for the following week. Our team started preparing for the Nordic meetup. Drylab and wetlab started collaborating more closely, in order to improve our models. Human practices was working on future meetings and signed up for the iGEM CRISPR conference.

August

August 3rd 2021: 24th General meeting

This week during the general meeting we decided to split up the wetlab team into 3 subgroups: pMMO, Hemoglobin and AOX knockout groups to be more efficient. Drylab had a final discussion about the GSM model with Niko and Benjamin, finalized the kinetic model and prepared it for presentation. Human practices hosted a GMO discussion with other iGEM teams and started discussing collaboration possibilities with the Gröningen iGEM team. Our initial thought was to collaborate by model verification and/or app development. We started planning the high school visit as part of our education outreach. Unfortunately we had to say goodbye to Cristina, because she decided to go on exchange in the US.

August 10th 2021: 25th General meeting

The pMMO subgorup started working on gRNA vector construction, two candidates for each chromosome based on advice from our supervisors. The hemoglobin subgroup created an overview of required inserts, figuring out measurement methods for leghemoglobin content. Discussed different promoters to determine expression levels, while the AOX groups started working on methanol shake flask experiments. Drylab was working hard on the Kivy app development and finished the GSM model! Our team finally registered for the Giant Jamboree and had a meeting with Jens Preben Morth about protein expression and pMMO integration.

August 17th 2021: 26th General meeting

This week the hemoglobin subgroup performed their first Golden Gate Assembly with the new kits, the pMMO subgroup was working on PCR amplification of new primers for homology arms and plasmid extraction, while the AOX group, after many failed USER clonings, retried with new generic PCR fragments and newly linearized plasmids. They also started experiments to investigate if K. phaffii can be frozen down immediately before electroporation and used at a later time. Drylab was working on uploading parts to the registry and on the app development. Our team also started working on ideas for the presentation video.

August 24th 2021: 27th General meeting

This week the pMMO subgroup had some troubles with the Golden Gate Assembly and started trouble shooting with the help of our supervisors. AOX subgroup started working on yeast colony PCRs and they confirmed their first successful knockout: aox2. The hemoglobin subgroup was working hard on Golden Gate Assembly and they sent their first constructs for sequencing. This time around drylab started working on the structure of the wiki and the struggles with the navbar began. Later this week we had an amazing facility visit to Ampliqon in Odense with the SDU iGEM team.

August 31st 2021: 28th General meeting

This week the AOX group conducted their last knockout, while the hemoglobin group started experiencing some problems with the sequencing results. The pMMO group obtained colonies for construct 6 and designed new primers for better sequencing. Drylab was working in close collaboration with the Gröningen iGEM team and continued working on the wiki. Our team started having meetings about the presentation video with Mikkel.

September

September 7th 2021: 29th General meeting

Our wetlab subgroups were experiencing difficulties with colony PCR and sequencing this week and were working hard brainstorming and troubleshooting with the help of our supervisors. Drylab further developed the GSM model with the option to include different organisms, protein sequence of choice and different activators and repressors and started planning new methanol kill curve experiments. Our whole team was working hard on the manuscript for the presentation video and we decided on the final track of choice.

September 14th 2021: 30th General meeting

Wetlab was working hard this week to produce nice results, but they experienced some further problems with K. phaffii transformation and false positives. Drylab continued working on the methanol kill curve experiment and started putting more time into the collaboration with iGEM Gröningen. Signe, Karen and Emil went to Roskilde to hold a workshop to high school students with great success! Furthermore our human practices team had a meeting with Alexandre Huchelmann, member of the European Commision, DG Health and Food Safety.

September 21st 2021: 31st General meeting

The pMMO group was successful with assembling construct 3 and 6, which later got confirmed by sequencing as well, however they were unsuccessful with the transformation of K. phaffii . Hemoglobin subgroup has success with construct 6, while the AOX group experienced problems with false positives. Human practices group finished working on the children's book this week and started looking into publishing it. Drylab was running further Biolector experiments to test different carbon sources for the GSM model.

September 28th 2021: 32nd General meeting

AOX subgroup successfully created a double knockout, verified by sequencing and started setting up the experiments for the last knock out. The hemoglobin subgroup was testing many colonies hoping to find one with the right plasmid, while pMMO subgroup had to order new primers because of an incorrect restriction site in the gblock sequences. Drylab made the last modifications on the app and worked hard to prepare the wiki for the wiki freeze week.

October

October 5th 2021: 33rd General meeting

For the pMMO subgroup E. coli colony PCR of Pmo_A and Pmo_C resulted in no bands on the gel. Venus was consistently missing from constructs and yeast colony PCR of construct 3 and 6 sequencing results did not confirm genomic integration. AOX group conducted their last transformation, but unfortunately the triple knockout was unsuccessful. For the hemoglobin subgroup results showed that the inserts were not present in the constructs sent for sequencing, additionally K. phaffii transformation with construct 6 was unsuccessful. This week during the weekens we filmed our presentation video, participated in an SBE event and started planning the wiki freeze week!

October 12th 2021: 34th General meeting

This week we stopped all lab work and started focusing on collecting every information for the wiki. Started working on the design and animations and on Saturday we left for the wiki freeze week. In the last week before wiki freeze our team took a trip to a summer house to design and perfect our wiki page while enjoying each other’s company!

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