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

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.

Team Overview

The annual BioBrick Tutorial was held and 89 exited and talented iGEM participants were gathered at DTU for an exiting weekend.

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.

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.

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.

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:

1. To illustrate an examine the growth rate (for modelling)

2. 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.

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.

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.

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.

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.

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.

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:

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: