Team:DTU-Denmark/Notebook
Notebook
Choose a week
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Team Overview
Startup meeting - We, the team, met for the first time and we were split into subgroups.
Week 7 (Feb 11 - Feb 17)
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Team Overview
After a few brainstorming sessions, we agreed to create a promoter library for eukaryotic cells as this year’s project.
Week 8 (Feb 18 - Feb 24)
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Team Overview
We started the preparations for the annual BioBrick Tutorial and attend the annual DTU Open House where high school students can come and hear about the different fields of study and opportunities at DTU.
Week 10 (Mar 4 - Mar 10)
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Team Overview
We made dinner together and played board games.
Week 13 (Mar 25 - Mar 31)
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Team Overview
We attended a talk about bioethics in genetically modified insects to be better prepared for working with GMO and sharing our work with the general public.
Week 14 (Apr 1 - Apr 7)
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Team Overview
The annual BioBrick Tutorial took place with 69 guests from Sweden, Denmark, Norway, and Finland. The meetup offered both an introduction to lab work and to iGEM in general.
Week 15 (Apr 8 - Apr 14)
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Team Overview
The things needed for the Science EXPO were prepared.
We attempted an escape room with a laboratory theme and excelled in solving the different puzzles and finished with 29 minutes and 16 seconds to spare - one of the fastest teams ever!
Week 17 (Apr 22 - Apr 28)
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Team Overview
We attended the annual Science EXPO competition for primary school and high school students to spread the word about iGEM and talk about our project.
Students were given the opportunity to come and try pipetting droplets of water with food coloring onto paper.
Week 18 (Apr 29 - May 5)
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Team Overview
Kenneth Bruno and Grayson Wawrzyn from Zymergen kindly agreed to an interview about using filamentous fungi in production.
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Drylab
First meeting of the drylab group with advisor Chris. We discussed gene expression and the true meaning of “constitutive” (regulation is always present in some way). Moreover, the first goals were decided: looking for relevant datasets and finding literature.
Week 19 (May 6 - May 12)
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Team Overview
We attended the Nordic iGEM conference (NiC) hosted by the UCopenhagen team. Here, we presented the project and a poster to the other Nordic teams.
Week 20 (May 13 - May 19)
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Wetlab
The first days in the lab were spent creating competent cells and the plasmids needed for the experiments.
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Drylab
The search for relevant literature continues. The group members started searching for information on the inducibility and leakiness of the promoters. First datasets to start our tests were found.
Week 21 (May 20 - May 26)
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Team Overview
We had a fun barbeque evening to celebrate that exams were over and that we could finally begin work on the project.
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Drylab
We analyzed RNA-Seq datasets to get a handle on the format and get used to the data structure.
Week 22 (May 27 - Jun 2)
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Wetlab
Media and solutions for making protoplasts was prepared.
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Drylab
We discussed the interpretation of the found data and how to handle data noise in datasets. Our supervisor Chris suggested many bioinformatics tools that would be useful in the journey forward. The group members started learning how to make these tools work.
Week 23 (Jun 3 - Jun 9)
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Team Overview
We went to The Hague to attend the iGEM meetup for teams and supervisors hosted by Netherlands National Institute for Public Health and the Environment. Here, we participated in workshops regarding i.a. safety by design and learned about storytelling and human practices. We also made a lot of contacts and collaboration agreements with other teams.
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Drylab
At this point, we made a more specific plan of action. Looking at how to identify the best candidate genes based on the expression level in RNA-Seq data. Several Aspergillus genomes were obtained and we tried to generate the first promoter. The results were analyzed. Stuff makes sense!!!
Week 24 (Jun 10 - Jun 16)
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Team Overview
We visited the Novozymes site in Lyngby, got a tour at the site, and learned a lot about what the industry is looking for in a promoter library!
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Wetlab
The first attempt at making protoplasts of A. niger. This sadly failed. We talked to some professors who gave us advice on what to improve on the protocol. Furthermore, the first four fragments that will make up the initial integration plasmid were PCR amplified.
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Drylab
Start of coding part to make a general script so we don’t have to do the different parts manually.
Week 25 (Jun 17 - Jun 23)
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Wetlab
The PCR fragments were assembled into the first whole plasmid of our project! Following this, work began on inserting a GFP marker into the basic plasmid.
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Drylab
At this point, we made a more specific plan of action. The first goal was to identify the best candidate genes based on the expression level.
Week 26 (Jun 24 - Jun 30)
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Team Overview
We talked to Peter Richard from the VTT Technical Research Centre of Finland. He gave us excellent feedback on our project - we will definitely take this into consideration!
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Wetlab
The second attempt at making protoplasts of A. niger, this time with an improved protocol. This time, the attempt succeeded and we successfully made protoplasts and transformants, even following two different protocols for transformation!
Furthermore, in preparation for a Gibson assembly, we amplified several fragments by PCR and on Friday they were assembled into one.
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Drylab
Gene candidates were picked to run in our program.
Week 27 (Jul 1 - Jul 7)
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Wetlab
This week, we confirmed that the plasmids had been assembled correctly and we linearised them to be able to transform them into our fungi.
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Drylab
The promoters were ordered! - This calls for a celebration!
Week 28 (Jul 8 - Jul 14)
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Wetlab
Fragments were prepared to be assembled into plasmids.
Week 29 (Jul 15 - Jul 21)
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Team Overview
This weekend, we participated in the Danish iGEM meetup hosted by the SDU team on the south of Funen. We had so much fun and got to discuss how to make our wikis, how to be better at presenting, and we discussed starting a national Danish iGEM community. Thank you so much to SDU iGEM for hosting this great event!
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Wetlab
We made another transformation of A. niger and got GFP producing transformants!
We additionally Gibson assembled fragments from last week.
Week 30 (Jul 22 - Jul 28)
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Wetlab
We Gibson assembled the plasmid pGIA2P2e which is the foundation for our testing calibration vector. The plasmid was sent for sequencing and PCR verified. From this, we saw that we had successfully assembled the plasmid. Concurrently, assembly of the plasmid pGIA2P2o began but this turned out to be problematic.
We additionally began to prepare the fragments required for Golden Gate assembly of the next layer of plasmids.
Week 31 (Jul 29 - Aug 4)
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Wetlab
The pGIA2P2e plasmid was linearized and prepared for fungal transformation.
We also received a kanamycin backbone from the SDU-Denmark team. This means that we can now continue the assembly process for the pGIA2P2o plasmid. However, the assembled plasmid turned out not to work so we retraced our steps and tried again. While this was going on, we Golden Gate assembled the plasmid pBB006 which we will use in the future for fungal transformation.
Week 32 (Aug 5 - Aug 11)
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Team Overview
We visited Novozymes for the second time to follow up on some questions.
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Wetlab
We realized that the second attempt at Gibson assembling the pGIA2P2o plasmid had failed and tried a third time - which also failed :'(. On a more positive note, pBB006 was linearised. A fungal promoter was inserted and the plasmid was then linearised successfully!
Our synthetic promoters arrived and we were all very happy!
Week 33 (Aug 12 - Aug 18)
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Team Overview
We had dinner together to take a small break from all of the lab work and celebrate how far we'd come.
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Wetlab
The promoters arrived so we worked like Trojans to get ready to run the Biolector next week. During this, we inserted the promoters into our plasmid backbones using Golden Gate, inoculated liquid cultures, and miniprepped the 40 promoters in triplicate, totaling 120 plasmid samples!
Week 34 (Aug 19 - Aug 25)
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Wetlab
Despite setbacks by the fungi's lack of will to grow, we were able to run the first batch in the biolector and got some nice results!
Week 35 (Aug 26 - Sep 1)
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Team Overview
The semester started again so we were busy following our classes. But there was still time to go to the lab.
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Wetlab
We tried out our new Opentrons robot and got it to prepare a PCR. The official name of that robot is now Robert.
Week 36 (Sep 2 - Sep 8)
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Team Overview
On Tuesday, we visited Bagsværd Kostskole og Gymnasium to talk about synthetic biology and our iGEM project.
On Sunday, we gathered and worked on the wiki. We got a lot of stuff done, including, but not limited to; checking the safety page, documenting our results until now, and describing our collaborations with other teams.
Week 39 (Sep 23 - Sep 29)
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Wetlab
We inoculated shake flasks with 5 different promoters and sampled the flasks every 3 hours.
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Drylab
The drylab team started to clean up the software so other people could use it and began the analysis of data from the Biolector.
Week 40 (Sep 30 - Oct 6)
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Team Overview
On Friday, we visited Birkerød High School to talk about the project and synthetic biology. We had a blast and the students were incredibly interested in our presentation!
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Wetlab
While still running the shake flasks and Biolector, we started the bioreactors in order to test the scalability of our synthetic promoters. Additionally, we examined the transformed fungi with our promoters under a confocal microscope, seeing that both the conidia and hyphae were red, proving that our promoters had worked as intended!
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Drylab
A guide for the software and for how to find data for the software was developed. This was sent to the Brown-Stanford-Princeton team for them to test and evaluate the software.
Week 41 (Oct 7 - Oct 13)
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Team Overview
We spent the entire week writing the wiki and making it perfect :D
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Wetlab
The samples taken from the shake flasks and the bioreactor were analyzed in multiple ways. The biomass was separated and its proteins purified so we could determine the amount of mCherry produced by the different promoters. Additionally, the samples were also tested for the amount of remaining glucose to indicate how far they were in their growth phases at the different sampling times.
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Drylab
Final data analysis, everything is stressful but we are almost there.
Week 42 (Oct 14 - Oct 20)
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Team Overview
We're almost there and we've worked so very hard on our wiki. Wiki freeze is here and Boston is on the horizon!
Week 43 (Oct 21 - Oct 27)
