Difference between revisions of "Team:DTU-Denmark/Notebook"

Team Overview

Startup meeting - We, the team, met for the first time and we were split into subgroups.

Team Overview

After a few brainstorming sessions, we agreed to create a promoter library for eukaryotic cells as this year’s project.

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.

Team Overview

We made dinner together and played board games.

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.

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.

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!

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.

Team Overview

Kenneth Bruno and Grayson Wawrzyn from Zymergen kindly agreed to an interview about using filamentous fungi in production.

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.

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.

Wetlab

The first days in the lab were spent creating competent cells and the plasmids needed for the experiments.

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.

Team Overview

We had a fun barbeque evening to celebrate that exams were over and that we could finally begin work on the project.

Drylab

We analyzed RNA-Seq datasets to get a handle on the format and get used to the data structure.

Wetlab

Media and solutions for making protoplasts was prepared.

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.

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.

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!!!

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!

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.

Drylab

Start of coding part to make a general script so we don’t have to do the different parts manually.

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.

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.

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!

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.

Drylab

Gene candidates were picked to run in our program.

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.

Drylab

The promoters were ordered! - This calls for a celebration!

Wetlab

Fragments were prepared to be assembled into plasmids.

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!

Wetlab

We made another transformation of A. niger and got GFP producing transformants!
We additionally Gibson assembled fragments from last week.

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.

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.

Team Overview

We visited Novozymes for the second time to follow up on some questions.

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!

Team Overview

We had dinner together to take a small break from all of the lab work and celebrate how far we'd come.

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!

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!

Team Overview

The semester started again so we were busy following our classes. But there was still time to go to the lab.

Wetlab

We tried out our new Opentrons robot and got it to prepare a PCR. The official name of that robot is now Robert.

Wetlab

We inoculated shake flasks with 5 different promoters and sampled the flasks every 3 hours.

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.

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!

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!

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.

Team Overview

We spent the entire week writing the wiki and making it perfect :D

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.

Drylab

Final data analysis, everything is stressful but we are almost there.

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!