Team:DTU-Denmark/Notebook
Week 5 (Jan 29 - Feb 4)
-
Team Overview
The DTU BioBuilders had the first official meeting. Fun team building activities were planned and the members got the first real taste of what iGEM is really all about.
Week 15 (Apr 9 - Apr 15)
-
Team Overview
The annual BioBrick Tutorial was held and 89 exited and talented iGEM participants were gathered at DTU for an exiting weekend.
Week 17 (Apr 23 - Apr 29)
-
Team Overview
The team participated in Science in Forum, where they had a stand and told interested high school students about the wonders that is iGEM. The team based their explanations on the previous team's project and on the many project ideas that they had discussed during their time in iGEM.
Week 18 (Apr 30 - May 6)
-
Team Overview
Lo and behold! After many hours of discussing various projects and weighing the pros and cons of each of them, the DTU BioBuilders have now finally come to an agreement. This year the team will work on making a general tool box for uses of fungi. This entails ways of creating mycotextures to be used in the colonization of Mars.
Week 23 (June 4 - June 10)
-
Team Overview
The team attended the Nordic iGEM Conference hosted by Team Lund. Here they presented their project and showed off the amazing poster specially made for this event.
×Needless to say that everyone had fun seeing and discussing the other groups' projects.
Week 24 (June 11 - June 17)
-
Synthlab Overview
The first day in the lab was used for creating media of different types and concentrations for the species to be grown on for two reasons:
To illustrate an examine the growth rate (for modelling)
To multiply our pure species for use in liquid media
We used the media created in the "PDB (or PDA) and MEA media for species" protocol. It was also the plan to create Vogel's medium as well as a mineral salt medium as certain species had shown in the previous study to grow well. However, we did not have the proper materials to create those two and discarded the idea. We also came to this conclusion as we were recommended not to spend time on specific media just yet.
All media was prepared before 11 am to be autoclaved. The actual weights of the different media was:
MEA Media Materials Weight (g) Weight (g) Weight (g) Malt extract 47.6 49.9 52.5 ZnSO4·7H2O 0.01 0.012 0.01 CuSO4·5H2O 0.05 0.049 0.051 Agar 14.99 14.99 15.04 PDB Media Potato dextrose extract Weight (g) Weight (g) Weight (g) Malt extract 19.54 39.12 58.56 ZnSO4·7H2O 0.012 0.012 0.0107 CuSO4·5H2O 0.0056 0.0053 0.0049 Agar 15.02 15.04 15.04 All media were stirred with a magnetic stirrer at around 110 degrees Celsius and 1000 rpm until homologous.
The species available (Pleuratus ostreatus) was inoculated on the solid PDA and MEA agar plates with a sterile toothpick under a fume hood and left at 30 degrees Celsius. It was on Monday discovered that the sample given was of mycelia without any spores and the process was redone.
Week 25 (June 18 - June 24)
-
SynthLab Overview
20/6/2018
Pleuratus ostreatus was re-inoculated on the solid agar plates by placement of around 8 mycelia "balls" under a fume hood and left at 30 degrees Celsius. As to save time, fewer plates were inoculated and a YBD plate was included.
Today, we had gotten another species, Schizophyllum commune, in two forms: a wildtype as well as a mutant (Δsc3) which were also inoculated on PDB, MEA and YBD plates.
22/6/2018
The first sign of growth in our species was showing and pictures were taken of every plate as to count pixels and indicate the growth rate. Pictures were taken every morning for 5 days.
Week 26 (June 25 - July 1)
-
SynthLab Overview
26/6/2018
Aspergillus oryzae was received and inoculated on MEA, PDB and YBD plates (5 days of photographs will follow this
species's growth in the same manner as the other species).
Today we decided that our species had grown enough and the samples were then removed and stored at a cooler temperature excluding those used for later liquid media.
Liquid PDB (PDA) was prepared for growth in our "brick"-molds (see further down) using the "PDB and MEA media for species" protocol without the addition of agar. 500 mL was created for each species. The PDB concentration chosen was the middle concentration in the table as these showed the strongest growth. At the end of this process, we should be ready to start with the actual growth of our building material.
Week 27 (July 2 - July 8)
-
Mycolab Overview
2/7/2018
Boxes: Initialisation of tests to determine best substrates and conditions for formation of a brick made of fungi. Saw dust was autoclaved.
Spore suspension: Spore suspensions are made for easy storage of spores. The spores are used for protoplastation and inoculation of boxes and plates. A. oryzae did not sporulate. It was incubated from June 26th to June 29th. Three days is not enough time for it to sporulate. P. ostreatus and S. commune did not sporulate either.
Reinoculation: Fungi strains were reinoculated onto new plates that all contained the same medium. Plates were checked for best growing conditions for the different fungi. PDA was in general the most promising medium and this was chosen as the medium used going forward. Three plates were inoculated for each strain with three-point inoculation. Depending on strain, different light and temperature conditions were used in incubation. The conditions were chosen based on knowledge of growth from research:S. commune (wildtype and ΔSC3): Two plates for 30°C light and one for 27°C light.
P. ostreatus: Two plates for 28°C dark and for for 25°C light.
A. oryzae: Two plates for 28°C dark and one for 30°C dark.
3/7/2018
Boxes: A. oryzae and S. commune were used for inoculation of boxes. PDA and mycelium with attached agar mixed. Inoculated medium mixed well with saw dust in boxes and incubated at 30°C dark for a week.
5/7/2018
Boxes: Pipette boxes inoculated with fungus/hay mixture from Skyttegaarden. 4 boxes inoculated, two with parafilm and 2 with the lid. Inoculation of boxes with species of our own.
6/7/2018
Photos of fungi: Photos were taken of the fungi every day to assess the growth. All species show growth although not all plates did. A. oryzae showed signs of sporulation.
Boxes: S. commune show growth regardless of medium concentration and volume. However, higher concentration of medium means more growth. A. oryzae boxes moved to 28°C dark. S. commune all moved to 30°C light as they seemed to thrive there. -
Drylab Overview
Up until this week, we have had a normal semester. Now all members, of drylab, are able to be working full time in the iGem project, not counting scheduled personal holidays. This week Mathies worked on Poisson HMM (Hidden Marcow model) and investigating binomial distributions with regards to simulating hyphal growth and branching probabilities. Nicolai is manufacturing/welding growth boxes for the Wetlab group. They need these to be able to make consistently shaped fungi squares for drylab compression testing. Lau and Mathias are investigating other modeling possibilities; how to grow Mycelium on different substrates, and how we, as a group, can enhance our productivity and usefulness for the other subgroups. Hannah collected a lot of data, to be used for more precise modeling parameters, and is working on writing a program that will be able to calculate growth speed using image recognition. Collectively we made a safety assessment and handed for review for us to be able to gain access to proper material characterizations machines at the mechanical department at DTU.
Week 28 (July 9 - July 15)
-
Mycolab Overview
9/7/2018
All through week pictures of plates were taken everyday to follow growth and sporulation.
Spore suspension: Spore suspension made from plates from last week following the spore suspension protocol. Spore suspension ended up with a concentration of 1.69 * 108 spores/ml.
13/7/2018
Boxes: New substrates used: white and brown rice and coffee grounds. Rice are natural substrates for A. oryzae and coffee grounds are supposed to add to the texture of the mycelia. All substrates autoclaved before inoculation of boxes. Pipette boxes inoculated and incubated at 30°C dark.
Baking of boxes: The baking or autoclavation of the boxes were to find a way to kill the mycelia, so the boxes could be brought outsite the laboratory. Boxes inoculated from Skyttegaarden and saw dust boxes were autoclaved. The saw dust boxes were very crumbly regardless of concentration before autoclavation.
Weighing of boxes: Boxes were weighed everyday to follow the growth of the mycelia and to see if there was a difference over time. -
Drylab Overview
We began the week with a meeting to set the course for the rest of the week. This week Nicolai is on holiday. On the modeling side of things, the work on the HMM is coming along nicely but it is not clear how it can be related to real life. The model outputs the probabilities of branching for individual hyphae. The bigger picture is not clear for this model. Hannah is working on codon optimization after having dropped the imaging programme. The reason being that the color variation of the sample pictures was too great and writing a program to be able to take this into consideration proved to be too hard. An alternative program was used to do this, called VGG Image Annotator (VIA) (http://www.robots.ox.ac.uk/~vgg/software/via/), where the radius of all the colonies were manually annotated. The area of each fungal colony was calculated and visualized in plots.
Mathias and Lau made good progress in investigating live imaging of Mycelia. The purpose is to nail down real parameters spaces for use in simulations. We handed in the safety assessment to DTU Byg (Mechanical Department) for review/verification and got a short tour of the material lab. Many different people were contacted in regards to material supply and modeling/simulation. More growth boxes were manufactured.
Week 29 (July 16 - July 22)
-
Synthlab Overview
20/7/2018
Experiment: First insertion of genes into a backbone
The purpose of this experiment is twofold. Firstly, to gain experience in using the basic techniques associated with molecular biology. Secondly, the scientific focus of the experiment is to perform an insertion of the MelA gene () and the CaMV 35S promoter (BBa_K1825004) into seperate backbone for amplification in E. coli This is followed by a miniprep to extract the plasmids. The DNA delivered by IDT was suspended in EB buffer (see protocol) Using part 1 and 2 from the BioBrick tutorial (Later adapted together with protocol 2) the parts were digested and ligated with the backbone from RFP (BBa_J04450) -
Mycolab Overview
16/7/2018
Reinoculation of plates: A. oryzae inoculated on new plates in three point inoculation with spores from spore suspension. So far, inoculation has been done with mycelia from earlier plates. Three plates were incubated as normally in 28°C and one at 4°C to see how the fungus fares at lower temperatures.
Boxes: Rice boxes were quite hard after only two days in the incubator, which was viewed as a very good sign.
17/7/2018
Baking of boxes: After autoclavation the boxes were quite moist and therefore fell apart quite easily. In stead of autoclaving, microwaving or baking normally was suggested to avoid the bricks being too wet. Box from Skyttegaarden was put in the microwave. After 10 min it was still wet.
Protoplastation and transformation: Protoplastation was initialised according to the Protoplastation and Tranformation protocol
19/7/2018
Protoplastation and transformation: Protoplastation was initialised according to the Protoplastation and Tranformation protocol
20/7/2018
Boxes: Regardless of mycelia and media volume the brown rice inoculated with A. oryzae were crumbly and the mycelia had only grown on the stop of the brick. The white rice boxes (also with A. oryzae) were different according to volume of liquid media added to the rice. The less media, the sturdier the brick was to pressure from hands. Adding glucose to S. commune boxes with saw dust did not make a difference. The brick is still very crumbly.
Baking of boxes: Rice boxes baked at 90°C and checked every 15 minutes. After the fire 15 minutes the paper surrounding the brick was very wet. After discussion a new plan was formed: Rice is cooked when in water and surroundings are very hot, so we try to cook them quickly at high temperatures. Therefore: One rice box is baked at 90°C for two hours and then 200°C for one hour. After that the brick is baked 70°C O/N. The other rice brick is only baked at 70°C O/N. Saturday they will be taken out and photographed. See designated photo entry for photos of the bricks.
Small bricks: We have received ice cube trays to make smaller bricks, giving us the opportunity to make more bricks quicker and even different bricks at the same time. First small bricks are made with spores and different ratios of saw dust, white rice and coffee grounds to see how they work together. To avoid contamination every other well was inoculated instead of each well. See picture.
-
Drylab Overview
This week Nicolai has come back. He assessed, based on the team's meetings with DTU Byg last week, that manufacturing metal inoculation boxes for Wetlab would become far too big of a task since it was unearthed that we would need over 100 boxes. It was decided that we order silicone ice-cube trays instead, both for us to focus on other pressing matters and for the consistency of the cubes. He also started work on a mission architecture including a design the brick for us to construct a martian hut. We obtained foam samples through a contact from last week. Hannah and Lau assisted the Myco-lab subgroup since they were struggling with their workload. At the end of the week, Nicolai began working on a DIY hydraulic press. We weren't going to be able to use the DTU byg machinery before a lab technician would return from his holiday in week 32. Mathias started researching if there existed a gene regulating the frequency of hyphal fusion in the fungi. This was based on findings from polymer science, which showed that vulcanized rubber showed increased strength if the network of rubber was more interconnected or more elastic if the rubber was less interconnected. This would be possible if we could find a homolog of a known gene that regulates hyphal fusion.
Week 30 (July 23 - July 29)
-
Wetlab Overview
26/6/2018
Aspergillus oryzae was received and inoculated on MEA, PDB and YBD plates (5 days of photographs will follow this species growth in the same manner as the other species).
Today we decided that our species had grown enough and the samples were then removed and stored at a cooler temperature excluding those used for later liquid media: -
Drylab Overview
This week Hannah and Lau were helping Myco-lab in growing “ice cube fungi”. They are being grown on a various substrates and inoculated at different times and temperatures. Mathias was on holiday. Nicolai was “fighting” with DTU skylab, as their technicians bailed on appointments, making the progression of the DIY hydraulic press slow and wasteful. Late friday, the small project was completed, only missing some teflon tape to seal the thread. Over the weekend Nicolai made a short video showing the manufacturing process. Mathies planned the data structures for the experimental setup for DTU byg. The purpose is to minimize the necessary test that have to be done, to save time and resources.
Week 31 (July 30 - August 5)
-
Wet Lab Overview
26/6/2018
Aspergillus oryzae was received and inoculated on MEA, PDB and YBD plates (5 days of photographs will follow this species growth in the same manner as the other species).
Today we decided that our species had grown enough and the samples were then removed and stored at a cooler temperature excluding those used for later liquid media: -
Drylab Overview
This week Mathias returned and Hannah went on holiday. The DIY hydraulic press was completely finished and ready to use. Nicolai restarted the mission architecture and brick design. Mathias reached out to a polymer expert (Mika Torkkeli, Postdoc) to discuss the role of the network structure/interconnectedness in the strength of polymer networks, it was confirmed that the sparse mycelium network could perhaps be analogous to the network structure of vulcanized rubber. But because of the macroscale of the network and the sparseness of the network the effects might be less drastic than those found in rubber. Another challenge was that the only gene that was identified that might increase hyphal fusioning also affected mycelium density negatively. It was deemed to be too uncertain if we could control these effects away and whether or not the effect size would be worth it.