iGEM 2018 — Team DTU-Denmark

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# Team:DTU-Denmark/Notebook

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* [![](https://static.igem.org/mediawiki/2018/3/38/T--DTU-Denmark--menu-icon-project.png)
  Project](https://2018.igem.org/Team%3ADTU-Denmark/Description)

  + [Description](https://2018.igem.org/Team%3ADTU-Denmark/Description)
  + [Design](https://2018.igem.org/Team%3ADTU-Denmark/Design)
  + [InterLab](https://2018.igem.org/Team%3ADTU-Denmark/InterLab)
  + [Results](https://2018.igem.org/Team%3ADTU-Denmark/Results)
  + [Choice of Organism](https://2018.igem.org/Team%3ADTU-Denmark/Results-choosing-organism)
  + [amilCP Expression](https://2018.igem.org/Team%3ADTU-Denmark/Results-amilCP)
  + [melA Expression](https://2018.igem.org/Team%3ADTU-Denmark/Results-melA)
  + [nat1 Expression](https://2018.igem.org/Team%3ADTU-Denmark/Results-NAT-1)
  + [Ganoderma Protoplastation](https://2018.igem.org/Team%3ADTU-Denmark/Ganoderma-protoplastation)
  + [Demonstrate](https://2018.igem.org/Team%3ADTU-Denmark/Demonstrate)
* [![](https://static.igem.org/mediawiki/2018/8/86/T--DTU-Denmark--menu-icon-model.png)
  Modeling](https://2018.igem.org/Team%3ADTU-Denmark/Model)

  + [Model](https://2018.igem.org/Team%3ADTU-Denmark/Model)
  + [Growth Modeling](https://2018.igem.org/Team%3ADTU-Denmark/GrowthModelling)
  + [Design of Experiments](https://2018.igem.org/Team%3ADTU-Denmark/DesignOfExperiments)
  + [Modeling the Design](https://2018.igem.org/Team%3ADTU-Denmark/ModellingTheDesign)
  + [Structural Integrity](https://2018.igem.org/Team%3ADTU-Denmark/StructuralIntegrity)
  + [Hardware](https://2018.igem.org/Team%3ADTU-Denmark/Hardware)
* [![](https://static.igem.org/mediawiki/2018/9/94/T--DTU-Denmark--menu-icon-parts.png)
  Parts](https://2018.igem.org/Team%3ADTU-Denmark/Parts)
* [![](https://static.igem.org/mediawiki/2018/f/f3/T--DTU-Denmark--menu-icon-journal.png)
  Journal](https://2018.igem.org/Team%3ADTU-Denmark/Notebook)

  + [Notebook](https://2018.igem.org/Team%3ADTU-Denmark/Notebook)
  + [Experiments](https://2018.igem.org/Team%3ADTU-Denmark/Experiments)
* [![](https://static.igem.org/mediawiki/2018/c/c9/T--DTU-Denmark--menu-icon-logo.png)](https://2018.igem.org/Team%3ADTU-Denmark)
* [![](https://static.igem.org/mediawiki/2018/6/61/T--DTU-Denmark--menu-icon-people.png)
  People](https://2018.igem.org/Team%3ADTU-Denmark/Team)

  + [Team Members](https://2018.igem.org/Team%3ADTU-Denmark/Team)
  + [Attributions](https://2018.igem.org/Team%3ADTU-Denmark/Attributions)
* [![](https://static.igem.org/mediawiki/2018/1/18/T--DTU-Denmark--menu-icon-hp.png)
  Human Practices](https://2018.igem.org/Team%3ADTU-Denmark/Human_Practices)

  + [Human Practices](https://2018.igem.org/Team%3ADTU-Denmark/Human_Practices)
  + [Education and Engagement](https://2018.igem.org/Team%3ADTU-Denmark/Public_Engagement)
* [![](https://static.igem.org/mediawiki/2018/4/4a/T--DTU-Denmark--menu-icon-collab.png)
  Collaboration](https://2018.igem.org/Team%3ADTU-Denmark/Collaborations)

  + [Collaborations](https://2018.igem.org/Team%3ADTU-Denmark/Collaborations)
  + [BioBrick Tutorial](https://2018.igem.org/Team%3ADTU-Denmark/BioBrickTutorial)
* [![](https://static.igem.org/mediawiki/2018/f/f5/T--DTU-Denmark--menu-icon-safety.png)
  Safety](https://2018.igem.org/Team%3ADTU-Denmark/Safety)

[Hyphae Hackers](https://2018.igem.org/Team%3ADTU-Denmark)

Project

[Description](https://2018.igem.org/Team%3ADTU-Denmark/Description)
[Design](https://2018.igem.org/Team%3ADTU-Denmark/Design)
[InterLab](https://2018.igem.org/Team%3ADTU-Denmark/InterLab)
[Results](https://2018.igem.org/Team%3ADTU-Denmark/Results)
[Choice of Organism](https://2018.igem.org/Team%3ADTU-Denmark/Results-choosing-organism)
[amilCP Expression](https://2018.igem.org/Team%3ADTU-Denmark/Results-amilCP)
[melA Expression](https://2018.igem.org/Team%3ADTU-Denmark/Results-melA)
[nat1 Expression](https://2018.igem.org/Team%3ADTU-Denmark/Results-NAT-1)
[Ganoderma Protoplastation](https://2018.igem.org/Team%3ADTU-Denmark/Ganoderma-protoplastation)
[Demonstrate](https://2018.igem.org/Team%3ADTU-Denmark/Demonstrate)

Modeling

[Model](https://2018.igem.org/Team%3ADTU-Denmark/Model)
[Growth Modeling](https://2018.igem.org/Team%3ADTU-Denmark/GrowthModelling)
[Design of Experiments](https://2018.igem.org/Team%3ADTU-Denmark/DesignOfExperiments)
[Modeling the Design](https://2018.igem.org/Team%3ADTU-Denmark/ModellingTheDesign)
[Structural Integrity](https://2018.igem.org/Team%3ADTU-Denmark/StructuralIntegrity)
[Hardware](https://2018.igem.org/Team%3ADTU-Denmark/Hardware)

[Parts Overview](https://2018.igem.org/Team%3ADTU-Denmark/Parts)
Journal

[Notebook](https://2018.igem.org/Team%3ADTU-Denmark/Notebook)
[Experiments](https://2018.igem.org/Team%3ADTU-Denmark/Experiments)

People

[Team Members](https://2018.igem.org/Team%3ADTU-Denmark/Team)
[Attributions](https://2018.igem.org/Team%3ADTU-Denmark/Attributions)

[Safety](https://2018.igem.org/Team%3ADTU-Denmark/Safety)
Human Practices

[Human Practices](https://2018.igem.org/Team%3ADTU-Denmark/Human_Practices)
[Education & Engagement](https://2018.igem.org/Team%3ADTU-Denmark/Public_Engagement)

Collaborations

[Collaborations](https://2018.igem.org/Team%3ADTU-Denmark/Collaborations)
[BioBrick Tutorial](https://2018.igem.org/Team%3ADTU-Denmark/BioBrickTutorial)

![](https://static.igem.org/mediawiki/2018/0/0c/T--DTU-Denmark--toppernotebook.jpg)

# Notebook

Week 5 (January 29 - February 4)

* ![](https://static.igem.org/mediawiki/2018/2/2d/T--DTU-Denmark--logbookall1icon.png)

  #### 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 (April 9 - April 15)

* ![](https://static.igem.org/mediawiki/2018/2/2d/T--DTU-Denmark--logbookall1icon.png)

  #### Team Overview

  The annual [BioBrick Tutorial](https://2018.igem.org/Team%3ADTU-Denmark/BioBrickTutorial) was held and 89 exited and talented iGEM participants were gathered at DTU for an exciting weekend.

  [![](https://static.igem.org/mediawiki/2018/3/3d/T--DTU-Denmark--BBT-group.jpeg)](https://2018.igem.org/Team%3ADTU-Denmark/BioBrickTutorial)

Week 17 (April 23 - April 29)

* ![](https://static.igem.org/mediawiki/2018/2/2d/T--DTU-Denmark--logbookall1icon.png)

  #### Team Overview

  The team participated in [Science in Forum](https://2018.igem.org/Team%3ADTU-Denmark/Public_Engagement#eduscienceinforum), 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 (April 30 - May 6)

* ![](https://static.igem.org/mediawiki/2018/2/2d/T--DTU-Denmark--logbookall1icon.png)

  #### 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](https://2018.igem.org/Team%3ADTU-Denmark/Description).

Week 23 (June 4 - June 10)

* ![](https://static.igem.org/mediawiki/2018/2/2d/T--DTU-Denmark--logbookall1icon.png)

  #### 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.

  ![NiC poster](https://static.igem.org/mediawiki/2018/0/01/T--DTU-Denmark--logbook-poster-NiC.png)

  ×
  ![]()

  Needless to say that everyone had fun seeing and discussing the other groups' projects.

Week 24 (June 11 - June 17)

* ![](https://static.igem.org/mediawiki/2018/2/27/T--DTU-Denmark--logbooksynthicon.png)

  #### 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 modeling)
  2. To multiply our pure species for use in liquid media

  We used different PDA and MEA media for the species. 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.

  More details here

  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 24 (June 11 - June 17)

* ![](https://static.igem.org/mediawiki/2018/2/27/T--DTU-Denmark--logbooksynthicon.png)

  #### 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)

* ![](https://static.igem.org/mediawiki/2018/2/27/T--DTU-Denmark--logbooksynthicon.png)

  #### SynthLab Overview

  **26/6/2018**
  ![](https://static.igem.org/mediawiki/2018/5/57/T--DTU-Denmark--notebook-wetlab-scommune.png)
  *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 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)

* ![](https://static.igem.org/mediawiki/2018/1/1c/T--DTU-Denmark--logbookmycoicon.png)

  #### 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.
* ![](https://static.igem.org/mediawiki/2018/6/63/T--DTU-Denmark--logbookdryicon.png)

  #### 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)

* ![](https://static.igem.org/mediawiki/2018/1/1c/T--DTU-Denmark--logbookmycoicon.png)

  #### Mycolab Overview

  **9/7/2018**
  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](https://2018.igem.org/Team%3ADTU-Denmark/Experiments#sporesuspension).
  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.
* ![](https://static.igem.org/mediawiki/2018/6/63/T--DTU-Denmark--logbookdryicon.png)

  #### 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)

* ![](https://static.igem.org/mediawiki/2018/2/27/T--DTU-Denmark--logbooksynthicon.png)

  #### 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 [protocol](%E2%80%9Chttps%3A//2018.igem.org/Team%3ADTU-Denmark/Experiments#sporesuspension”).
  Using part 1 and 2 from the BioBrick tutorial (Later adapted together with the [resuspension](https://2018.igem.org/Team%3ADTU-Denmark/Experiments#resuspensionofdna) the parts were digested and ligated with the backbone from *RFP* (BBa\_J04450)
* ![](https://static.igem.org/mediawiki/2018/1/1c/T--DTU-Denmark--logbookmycoicon.png)

  #### 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](%E2%80%9Chttps%3A//2018.igem.org/Team%3ADTU-Denmark/Experiments#protoandtransfofasper”).

  **19/7/2018**
  Protoplastation and transformation: Protoplastation was initialised according to the [Protoplastation and Tranformation protocol](%E2%80%9Chttps%3A//2018.igem.org/Team%3ADTU-Denmark/Experiments#protoandtransfofasper”).

  **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.

  ![](https://static.igem.org/mediawiki/2018/1/16/T--DTU-Denmark--logbook-myco-2007.png)
* ![](https://static.igem.org/mediawiki/2018/6/63/T--DTU-Denmark--logbookdryicon.png)

  #### 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](https://2018.igem.org/Team%3ADTU-Denmark/Hardware). 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)

---

* ![](https://static.igem.org/mediawiki/2018/2/27/T--DTU-Denmark--logbooksynthicon.png)

  #### Synthlab Overview

  **23/7/2018**
  Experiment: First insertion of genes into a backbone
  Competent cells were transformed using the [transformation protocol](%E2%80%9Chttps%3A//2018.igem.org/Team%3ADTU-Denmark/Experiments#adaptedassemblyofcomposite”). After the transformation, the cells were plated on LB amp plates (which in retrospect is the wrong kind of plates) and left overnight. The plates from Monday 23rd were not successful, as we used amp plates instead of cam plates. The protocol has been rewritten accordingly.
  The leftover cells were plated on some new LB cam plates.

  **25/7/2018**
  Experiment: Preparation of 2nd round of genes
  The purpose of this experiment is to insert the DNA delivered from IDT into plasmids for progapagation and further use. The genes that are to be inserted are; *pGpdA*, *pTrpC*, *pTrpC-GFP* and *ScGPa*. This will be accomplished as per the [Adapted assembly protocol](%E2%80%9Chttps%3A//2018.igem.org/Team%3ADTU-Denmark/Experiments#adaptedassemblyofcomposite”).

  **26/7/2018**
  New Gene have arrived: CaMV 35S Terminator, *TS1* holology region, Hygromycin B. These were digested following the [adapted protocol](%E2%80%9Chttps%3A//2018.igem.org/Team%3ADTU-Denmark/Experiments#adaptedassemblyofcomposite”).

  The ligation results was transformed and plated on LB cam plates O/N.

  **27/7/2018**
  Experiment: First insertion of genes into a backbone
  The prepared O/N cultures were miniprepped (see [miniprep protocol](%E2%80%9Chttps%3A//2018.igem.org/Team%3ADTU-Denmark/Experiments#QIAprep”)) in order to have the purified plasmids.

  Experiment: Preparation of 2nd round of genes
  The O/N plates produced 3 transformants and a correct positive control, but the 4 older digestions produced no colonies, which suggests that digestions was unsuccesful. Consequently, the digestions, ligation and transformation was redone. The transformation used 35 µL competent cells instead of the 50 µL the protocol requires. The liquid media was scaled down acordingly.

  **28/7/2018**
  Experiment: Preparation of 2nd round of genes
  The plates containing transformants was moved to the fridge.
* ![](https://static.igem.org/mediawiki/2018/1/1c/T--DTU-Denmark--logbookmycoicon.png)

  #### Mycolab Overview

  **23/7/2018**
  Boxes: New boxes made with different combinations of rice, saw dust and coffee. Tests with different kinds of treated rice initialised: porridge rice cooked, raw or grounded were autoclaved and so were jasime rice, raw (but cleaned), normally cooked, overcooked and grounded.

  **24/7/2018**
  Another spore suspension of **A. oryzae** made according to the [spore suspension protocol](%E2%80%9Chttps%3A//2018.igem.org/Team%3ADTU-Denmark/Experiments#QIAprep”).

  **25/7/2018**
  Small bricks: More small bricks made. This time from differently treated rice: porridge rice, normally cooked (1), jasmine rice, overcooked (2) and jasmine rice, normally cooked (3).
  Rice mixed with spore inoculated medium and put into the wells in following pattern:

  ![](https://static.igem.org/mediawiki/2018/a/ad/T--DTU-Denmark--logbook-myco1.png)

  Incubated at 28°C dark.

  **26/7/2018**
  Small bricks: Same procedure for inoculating medium as 25/7. Other rice used:
  1. Ground jasmine rice
  2. Porridge rice
  3. Ground porridge rice
  4. Washing jasmine rice
  5. Finely ground rice
  6. 10 ml rice

  Added to two trays. First 5 ml inoculated rice was added, in the second inoculated rice was added until full.

  **27/7/2018**
  Test of contaminations found in boxes: Contaminated saw dust and PDA plated to ID them. The contaminated PDA might be **A. oryzae** and the contaminated saw dust might be *S. commune*. This is interesting because we might have found a way to get spores from *S. commune*.
* ![](https://static.igem.org/mediawiki/2018/6/63/T--DTU-Denmark--logbookdryicon.png)

  #### 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)

* ![](https://static.igem.org/mediawiki/2018/2/27/T--DTU-Denmark--logbooksynthicon.png)

  #### Synthlab Overview

  **30/7/2018**
  Experiment: Preparation of 2nd round of genes
  O/N cultures have been prepaired. 2 for each plate
  Experiment: Extraction of *RFP* from the kit
  The purpose of this experiment is to transform the *RFP* gene (BBa\_J04450) from the distribution kit into an DH-5-α compent E.coli and use a miniprep to extract it again.
  Using the transformation (subpart 3) from the [Adapted assembly protocol](%E2%80%9Chttps%3A//2018.igem.org/Team%3ADTU-Denmark/Experiments#adaptedassemblyofcomposite”), four transformations were made; 1 *RFP*, 1 negative and 2 positive with (2 µL) *A1*. The transformants were plated on LB cam plates and cultivated overnight.

  **31/7/2018**
  Experiment: Preparation of 2nd round of genes
  Overnight cultures were Miniprepped.
  Experiments were performed by Joen, David and Jacob.
  This concludes the preparation of 2nd round of genes experiment.

  **1/8/2018**
  Experiment: Extraction of *RFP* from the kit
  Two colonies from the *RFP* transformant plate were picked and placed in a liquid O/N culture.

  **2/8/2018**
  Experiment: Extraction of *RFP* from the kit
  Overnight culture were miniprepped to extract the plasmids.
  This concludes the Extraction of *RFP* from the kit experiment.
  Experiment: Gibson assembly of basic toolkit
  The purpose of this experiment is to assemble what we can of out basic toolkit using Gibson assembly. The parts to be assembled are; *TS1*, *pTrpC*, *hph*, CaMV 35S terminator and *TS2*. The Gibson assembly will be performed according to the [Hifi assembly protocol](%E2%80%9Chttps%3A//2018.igem.org/Team%3ADTU-Denmark/Experiments#hifiDNAassembly”). As the second homology region, *TS2*, had not arrived at this point, we began assembling the other genes into a composite.
  Part 1 of the protocol was done (PCR amplification). Experiments performed by David, Joen and Jacob.

  ![](https://static.igem.org/mediawiki/2018/0/0b/T--DTU-Denmark--logbook-synth.png)
* ![](https://static.igem.org/mediawiki/2018/1/1c/T--DTU-Denmark--logbookmycoicon.png)

  #### Mycolab Overview

  **30/7/2018**
  Reinoculation of plates: Cold **A. oryzae** grown to spores. Probably because of the cold incubator being broken. Temperature was around 20°C instead of 4°C.

  Baking of boxes: Ice cube trays from 20/07/2018 photographed and baked. Baked at 70°C for 4 hours and 40 minutes and then photographed. Put bad to bake O/N.
  Before baking:

  ![](https://static.igem.org/mediawiki/2018/b/b5/T--DTU-Denmark--logbook-myco-2.png)

  After 4 hours and 40 minutes:

  ![](https://static.igem.org/mediawiki/2018/f/f2/T--DTU-Denmark--logbook-myco-3.png)

  **31/7/2018**
  Baking of boxes: Ice tray taken out after being baked at 100°C for roughly one hour.
  Small bricks: New ice tray inoculated with spores suspension. Same method as last time. Incubated in fridge at 4°C.

  **2/8/2018**
  Death test: After baking we need to make sure the mycelia in the bricks are actually dead. That's what we're doing here.
  For the large bricks: Tape is touched onto brick to catch potentially alive mycelia or spores and touched onto plate afterwards.
  For the small bricks: Blue inoculation needles are touched onto the brick and inoculated on plates according to marks from 20/07.
  Test of contamination found in boxes: Turned out to be nothing of interest.

  **3/8/2018**
  Baking of boxes: As the large boxes from 20/07 had been unprotected while they were in the office (after their baking) spores from the office may have landed on them and started growing. They are baked again at 100°C for two and a half hours.
  Death test: Another death test in which the small bricks are broken in half and the inside is touched slightly on a PDA plate to see if the inside is dead as well.
* ![](https://static.igem.org/mediawiki/2018/6/63/T--DTU-Denmark--logbookdryicon.png)

  #### Drylab Overview

  This week Mathias returned and Hannah went on holiday. The [DIY hydraulic press](https://2018.igem.org/Team%3ADTU-Denmark/Hardware) 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.

Week 32 (August 6 - August 12)

* ![](https://static.igem.org/mediawiki/2018/2/27/T--DTU-Denmark--logbooksynthicon.png)

  #### Synthlab Overview

  **6/8/2018**
  Experiment: Gibson assembly of basic toolkit
  Adding the homology regions to *pTrpC*, CaMV Terminator and Melanin for assembly into the basic plasmid after it's contruction. This is done via PCR amplification with primers containing and extra 20 bp upstream from the binding part of the primer.

  **7/8/2018**
  Experiment: Gibson assembly of basic toolkit
  PCR products was gel-purified and nano-dropped

  ![](https://static.igem.org/mediawiki/2018/a/ad/T--DTU-Denmark--logbook-synth-1.png)

  [note: the difference between "top *TS2*" and "bottom *TS2*" is that the gel had two bands for *TS2*]
  The Gibson mix arrived and we followed the [Gibson protocol](%E2%80%9Chttps%3A//2018.igem.org/Team%3ADTU-Denmark/Experiments#hifiDNAassembly”) to assemble the basic plasmid. We assembled two different plasmids. One with TS2u and one with *TS2b*. The difference is from the gel electroforesis. One band was slightly further down than the other.We did not know which was the right one, so we collected both.

  **8/8/2018**
  Experiment: Extraction of *amilCP* from distribution kit
  A liquid O/N culture were prepared from the plated *amilCP* transformants. Addionally, replating from the leftover media from 7/8 were performed, as well as a clean streaking of a sucessfull transformant (from the LB Cam plates), as there were a low number of transformanants on the plates.

  **9/8/2018**
  Experiment: Gibson assembly of basic toolkit
  Only one of the Mix 2 plates had colonies. We will continue to grow these plates. One colony was cleanstreaked and one was incubated in an overnight culture.
  We will retry the Gibson assembly. This time we will start with 70 ng backbone and work from there.
  We increased the incubation time to ~4 hours to maximize our chances of a successful assembly
  Experiment: Extraction of *amilCP* from distribution kit
  The liquid O/N culture was miniprepped and subsequently concentrations were determined by nanodrop. This concludes the Extraction of *amilCP* from distribution kit experiment.

  **11/8/2018**
  Experiment: Gibson assembly of basic toolkit
  Gibson assembly products was transformed and plated. Different amounts of cell culture were used in the hopes of getting more colonies. The first batch of gibson products had grown and exhibited a red color, reminiscent of *RFP*. AN overnight culture was miniprepped.

  **10/8/2018**
  Experiment: Investigation of unsuccessful Gibson assemblies
  The attempted Gibson assemblies have thus far been unsuccessful yielding only red, *RFP*-expressing, colonies. This indicates either: i) wrong PCR amplification of the backbone from BBa\_E1010 (*A1* henceforth) or ii) that the assembly has not worked and that left-over BBa\_E1010 is still present in the mix. Thus, *A1* were amplified again according to the PCR . This was then run on a 1% agarose gel together with the unsuccessful Gibson assemblies and BBa\_E1010.
  Conclusions from the gel:
  1. The new PCR amplifications seems to have worked producing a clear bands, however some *A1* seems to be present in the mix still, thus calling for more stringent cutting during gel purification.
  2. The Gibson assembly have not been sucessfull at assembling the total fragment
  3. *A1* seems to present in the Gibson assembly - explaining the red transformants
* ![](https://static.igem.org/mediawiki/2018/1/1c/T--DTU-Denmark--logbookmycoicon.png)

  #### Mycolab Overview

  **6/8/2018**
  Baking of trays: Tray from 26/7 baked for two hours at 100°C. Both trays from 25/7 and 26/7 baked at 70°C O/N.
  Inoculation of boxes: As the mycelia has not been able to grow all the way to the bottom of the large boxes a hypothesis of air flow being the reason was raised. A box was supplyed with an air tube in the bottom of the box and inoculated as normally. Incubated at 28°C dark.

  **7/8/2018**
  Small bricks: An ice cube tray was inoculated with different ratios of rice and sawdust to see how that affected the texture of the bricks. See the detailed notebook on Tueday 7/8 for further details.

  **8/8/2018**
  Baking of trays:
  Tray from 25/7: In the bricks made with overcooked jasmine rice a strong and tough air pocket had been formed. It was tough to break by force with tools. This pocket was filled with air. For the porrigde rice it was filled with liquid and much more wet that the overcooked jasmine rice.
  Tray from 26/7: Dry ground rice from first tray seems to have grown well and is dry after baking. Second tray show less growth with many spores.
  All from 25/7 taken out of tray and baked individually because htye were not yet dry. This is attributed to them being made from cooked rice already contained a high amount of water.

  **9/8/2018**
  We did more protoplasts according to the [Protoplastation and Tranformation protocol](%E2%80%9Chttps%3A//2018.igem.org/Team%3ADTU-Denmark/Experiments#protoandtransfofasper”), they were stored at -80°C.

  **10/8/2018**
  Small bricks: We wanted to test difference rice with sawdust. From earlier test (7/8) it seemed that 50% rice and 75% rice gave the best bricks. These ratios will be used with different kinds of rice. White rice, granulated rice, porridge rice, grounded porridge rice were used and one tray was with 50% and another was with 75%. See detailed notebook for Friday 10/8 for schematic of the trays. Incubated at 28°C dark.
  Death test: Test from 3/8 showed no signs of mycelia growth.

  ![](https://static.igem.org/mediawiki/2018/e/e5/T--DTU-Denmark--logbook-myco-4.png)

  Test from 8/8 showed signs of growth, although not mycelial. Plates put back in incubator for further inspection later. It seems like we are good with the baking. This means that the bricks that needed further drying should be dead as well - especially because they have had even longer in the oven than these tests.
* ![](https://static.igem.org/mediawiki/2018/6/63/T--DTU-Denmark--logbookdryicon.png)

  #### Drylab Overview

  **6/8/2018**
  It was decided today that we are going to pick up the mycelium growth simulations, but approaching it differently than the previous attempt involving hidden markov models. It started out with a discussion about how a mycelium develops over time and discussing parameters based on experimental measurements, such as branching frequency and branching extension rates. Work was begun on two complementary growth models: a detailed micro growth model with a focus on hyphal growth and a meso growth model based on a system of partial differential equations describing the change in biomass density as a function of time, nutrients, space and species characteristics.
  Nicolai and Lau collaborated in the sense that the brick design was finalized and Lau started simulating structures constructed using the brick design. Mathias and Nicolai tried to get a hold on the DTU Byg technician who could teach us to use their material properties machines. But he was still not available even though his schedule said otherwise. The first preliminary test was done using the “home built” hydraulic press. It initially showed a rough link between dryness and toughness. The more dry samples were generally more resistant to compression. 2 different kinds of foams were also tested to see if they were comparable to the material properties of the fungi. They were not, since they were actually tougher.

Week 33 (August 13 - August 19)

* ![](https://static.igem.org/mediawiki/2018/2/27/T--DTU-Denmark--logbooksynthicon.png)

  #### Synthlab Overview

  **13/8/2018**
  Experiment: Gibson assembly of basic toolkit
  After reviewing the available literature for troubleshooting Gibson assembly we have come to the conclusion that the problem probably arises from trying to assemble more than 5 fragments at once. Thus, we have decided to assemble the plasmid sequentially. Thus, Gibson assembly were performed of the *TS1* homology regions, the *pTrpC* promoter and *hph*, the Hygromycin B resistance gene (This product referred to as TH) Additionally assembly of *TS2* homology region and the CaMV terminator were performed (this product referred to as CT).
  The assembly were performed according to the [Hi-Fi assembly protocol](%E2%80%9Chttps%3A//2018.igem.org/Team%3ADTU-Denmark/Experiments#hifiDNAassembly”).

  **14/8/2018**
  Experiment: Gibson assembly of basic toolkit
  Another round of Gibson were performed according to the specifications given 13/8.
  The resulting products from both rounds of assembly were amplified by PCR, utilizing primers a forward gibson-primer matching on end of the product and a reverse primer matching the other end, I.E. D0008, the forward primer for *TS1* and D0009 the reverse primer for *hph* were used.
  The gibson assembled fragments were then run on a 1% agarose gel (the first number specifies round and the second number specifies duplicate):

  ![](https://static.igem.org/mediawiki/2018/c/c8/T--DTU-Denmark--logbook-synth-2.png)

  PCR products have been inserted following the headline

  **15/8/2018**
  Experiment: Geleletrophoresis of PCR produts of Gibson assembly
  After yesterdays nice looking gelelectrophoresis, we wanted to do a new electrophoresis (replicate):
  This time we loaded 20 uL instead of 5 uL to get a sufficient amount of DNA for gel purification

  ![](https://static.igem.org/mediawiki/2018/d/d5/T--DTU-Denmark--logbook-synth-3.png)

  PCR products have been inserted following the headline.
  Given how vague the ladder is and how smeared the bands are, we assume the gel to be bad (given that is was also the very last drops of the gel bottle.
  The second gel electrophoresis was run on machine RunOne #8. We loaded 25 uL of DNA.
  Based on online discussion we have identified overloading as the most likely problem

  After the unsuccessful gelelectrophoresis, we realised that we would run out of PCR product of the 14/8 gibson assembly product after the next gel. For this reason we decided to make another 50 μL of TH1, TH2, CT1 and CT2. Due to the double banding the product, we decided to increased the cycle time to 5 min and 30 seconds (30 seconds is the minimum cycle, where 30 sec should be added for every kb. We added 5 mins).

  **16/8/2018**
  Experiment: Gel Electrophoresis of 15/8 PCR products
  Due to the hypothesis that too high DNA concentrations were the cause of smearing in the two previous gel electrophoresis, we decided to reduce the loaded amount to 5 μL

  **18/8/2018**
  *amilCP* PCR products.
  11-12-21-22

  ![](https://static.igem.org/mediawiki/2018/9/94/T--DTU-Denmark--logbook-synth-4.png)

  PCR products have been inserted following the headline
  PCR composite 18/8
  *amilCP*, *GfaA*, *MelA*, A0, *hygB*

  ![](https://static.igem.org/mediawiki/2018/a/a9/T--DTU-Denmark--logbook-synth-5.png)

  PCR products have been inserted following the headline

  **19/8/2018**
  Tried to run gel for purification, unsuccessful, ran out of PCR products.
* ![](https://static.igem.org/mediawiki/2018/1/1c/T--DTU-Denmark--logbookmycoicon.png)

  #### Mycolab Overview

  **14/8/2018**
  Death test: Test from 8/8: no sign of mycelial growth. Any growth present looks like bacteria or yeast.

  ![](https://static.igem.org/mediawiki/2018/b/b3/T--DTU-Denmark--logbook-myco-5.png)

  Baking of trays: Ice cube tray from 7/8 ready for baking. Put in for baking at 100°C for four hours. Turned down to 70°C for O/N baking.
  Boxes: Pipette boxes with tips for air checked: Only the rice/saw dust mix box had any noteworthy growth around the tips but not much growth in the bottom of the box.

  **15/8/2018**
  Baking of trays: Bricks taken out and put in sterile pipette boxes for storage. Both completely sawdust and completely rice were not coherent. The mixes became more coherent the more rice there was.
  Boxes: Drylab made new boxes.

  **17/8/2018**
  Checked pipette boxes and ice trays. Rice + saw dust and rice + coffee should be baked in three days. All of them lacked strength which means no coherent results.
  Boxes: Drylab made new boxes.
* ![](https://static.igem.org/mediawiki/2018/6/63/T--DTU-Denmark--logbookdryicon.png)

  #### Drylab Overview

  **13/8/2018**
  This week Mathias, Hannah, and Mathies were successful in completing some initial growth simulations, in which mycelium grew from one spore, using their new models. After these results, the code was optimized in order to simulate growth from multiple spores and see their progression. A gif, visualizing the growth using the micro model, was made and it was **stunning**.
  Nicolai and Mathias managed to get a hold of the DTU Byg technician and received training in the machinery in order to make really good compressional tests. The compression tests that our foam-like material is not really yielding the type of results we wanted e.g. we were expecting to see a clear “snap” when the sample was under sufficient compression. We are not seeing any “snap/crack”, only an increasing amount of compressional force, until the machine stage bottoms out. What we can do is to stop measuring at a given deformation, and look at HOW that acts.
  This is also taken into consideration for the simulation of the “hut design”. Since we have a finished design, it only needs to get realistic inputs. Lau applied physics (gravity, winds etc.) to the 3d model, but had problems with convergence.

Week 34 (August 20 - August 26)

* ![](https://static.igem.org/mediawiki/2018/2/27/T--DTU-Denmark--logbooksynthicon.png)

  #### Synthlab Overview

  **21/8/2018**
  Experiment: 3A of genes w. camV Terminator
  The following genes, terminator and backbone have been digested:
  + *MelA*
  + *MelA*
  + *A.oryzae*
  + *GfaA*
  + *amilCP*
  + *hygB*
  + The CaMV 35S terminator
  + *A1* backboneOf these, the *amilCP*, CaMV 35S terminator and *A1* came as pladsmids. The others were linearized fragments from IDT.
  The resulting digestion mix had a concentration of 10 ng/µl.

  **22/8/2018**
  Experiment: 3A of genes w. camV Terminator
  Ligation: *MelA*,A0,*GfaA*,*amilCP*,*hph*.
  Transformation: The ligations from yesterday and the ligations from the failed 3A PCR experiment were transformed into *E. coli* and placed at 37 C overnight.
  Colony PCR: In parallel, we tested the colony using the [colony PCR protocol](%E2%80%9Chttps%3A//2018.igem.org/Team%3ADTU-Denmark/Experiments#colonypcr”). It seemed like diluting the colonies doesn't give good results.

  **23/8/2018**
  Experiment: 3A of genes w. camV Terminator:
  The transformants was a success.
  Colony PCR: 3 colonies from each plate was suspended in water. A small fraction of this suspension was used for a liquid overnight culture, that can be used for a miniprep. The rest was heat-treated for 10 min and used for a colony PCR afterwards.
  The results show that the ligation products have the correct length.
  Experiment: Second round of colony PCR (multicolony PCR)
  Explanation of procedure. 5 transformants plated on a new plate - and further transferred to the same tube.
  Two PCR reactions - one using VR/VF primers, the other using [Gene]\_fw/CaMV\_Term\_bw primers.
  Backup experiments: As a backup, we digested *MelA*, *MelA* - A.oryzae, *hph* and GfaA and prepaired the ligation.
  New experiment: 3A of *amilCP*+CaMV Terminator with. promoters
  *amilCP* digestion: Digestion of: *pGpdA*, *pTrpC*, *PcamV*, *amilCP* and *A1*
  *amilCP* ligation: Ligated with *pGpdA*, *pTrpC* and *PcamV*

  **24/8/2018**
  Experiment: 3A of genes w. camV Terminator
  Created gel of multicolony PCR. In this experiment the forward sequencing primers were used together with the backward sequencing primer of CaMV. It was conducted to test whether a multicolony PCR approach would work.
  Backup Experiments: The backup genes were ligated and transformed into *E. coli*. The colonies were plated and left overnight.
  Delivered assembly: We have recieved an assembly from IDT cocsisting of '*pTrpC-HygB-CaMV*- 35S term'. This assembly was digested and ligated to be transformed tomorrow. When the gene is ready and amplified, it can be handed over to Mycolab for transformation into *A. oryzae* and *G. lucidum*.
  Experiment: 3A of *amilCP*+CaMV Terminator with. promoters
  Assembly of *amilCP* and promoters. The successful ligation and colony PCR of the '*amilCP*-CaMV 35S term' assembly was digested and ligated with 3 different promoters: '*pGpdA*', '*pTrpC*' and '*pCaMV 35S*'. The ligation will be transformed tomorrow

  **25/8/2018**
  Experiment: 3A of *amilCP*+CaMV Terminator
  Colony PCR of backup: The colony PCR was not successful. It is not clear why the gel showed no significant answers. The problems could include a failed ligation, failed PCR, failed gel or some other factor.
  PCR amplification of delivered assembly: The delivered assembly was amplified with verification primers. The product has run on a gel and the results was mediocre (at best). The amplification is probably a success, but it cannot be confirmed from the gel.
  Experiment: 3A of *amilCP*+CaMV Terminator with. promoters
  Tranformation: The *amilCP* + promoter assemblies and the delicered assembly has been transformed and plated on cam plates

  **26/8/2018**
  Experiment: 3A of *amilCP*+CaMV Terminator with. promoters
  The different transformants (*amilCP* + promoter and *hph* marker) were colony PCR'ed.
* ![](https://static.igem.org/mediawiki/2018/1/1c/T--DTU-Denmark--logbookmycoicon.png)

  #### Mycolab Overview

  **20/8/2018**
  Baking of bricks: Bricks baked last weeked seemed to still be alive; they were put back into the oven at 70°C.
  Baking the pipette box of rice + saw dust; mycelia can be seen everywhere. It has a strong and spongy strucutre so far. O/N 70°C.
  Spore suspension: A new spore suspension was made from plates from 060818.

  **21/8/2018**
  Protoplastation and transformation: Inoculated plates containing Hygromycin B to check if the concentration of Hygromycin B is enough too inhibit growth.
  Baking of bricks: Small bricks taken out and put back in containers. Large brick put back for more baking. Small part of large brick taken out.
  Reinoculation of plates: New plates were inoculated and incubated at 4°C to assess growth at low temperatures.
  Small bricks: Drylab made more bricks.

  **22/8/2018**
  Baking of trays: Drylab baked trays.
  Protoplastation and transformation: Hygromycin B plates available will not work. **A. oryzae** not inhibited by hygromycin B. We try different approaches.

  **23/8/2018**
  Protoplastation and transformation: We try NTC as selection antibiotic instead. We try inoculating plates with different concentrations: 100 µg/ml, 200µg/ml, 400µg/ml, 600µg/ml and 800µg/ml.
  Could also try an in-house auxotroph of **A. oryzae**.

  **24/8/2018**
  Protoplastation and transformation: Growth on all plates with NTC although the higher concentration of NTC in media, the slower growth rate. **Ganoderma** received from Ecovative.
* ![](https://static.igem.org/mediawiki/2018/6/63/T--DTU-Denmark--logbookdryicon.png)

  #### Drylab Overview

  **20/8/2018**
  Great steps have been taken since last week, where we now have an approach to estimating the density of the mycelium.
  Still trouble with 3d convergence.

  **21/8/2018**
  Try to create much simpler model to just try out the software, now have trouble visualizing the deformation.

  **24/8/2018**
  Used the knowledge and confidence from simpler model to locate, that the error was in the visualization. Successfully created a test video, of a combination of 6 bricks(see drylab folder), created a design with round edges to allow rotation

Week 35 (August 27 - September 2)

* ![](https://static.igem.org/mediawiki/2018/2/27/T--DTU-Denmark--logbooksynthicon.png)

  #### Synthlab Overview

  **27/8/2018**
  Experiment: 3A of genes w. camV Terminator
  The colony PCR products was examined by gel elecroforesis and the following *amilCP* assemblies was identified as successful:

  G3 (or G4 & G7), C3, C5

  Furthermore, we chose H2 and H4 to be an overnight culture. We chose G8 as a control.

  ![](https://static.igem.org/mediawiki/2018/3/32/T--DTU-Denmark--logbook-35-synth1.png)

  GelDoc 2018-08-27 13hr 06min - C1 -C2 -C3-C4-C5-C6-C7-C8-C9-C10-H1-H2-H3-H4-NEG

  ![](https://static.igem.org/mediawiki/2018/9/90/T--DTU-Denmark--logbook-35-synth2.png)

  GelDoc 2018-08-27 G1-G2-G3-G4-G5-G6-G7-G8-G9-G10

  ![](https://static.igem.org/mediawiki/2018/f/f1/T--DTU-Denmark--logbook-35-synth3.png)

  GelDoc 2018-08-27 T1-T2-T3-T4-T5-T6-T7-T8-T9-T10

  The colonies were cultivated further in a liquid overnight cultre for miniprepping tomorrow. Lastly, we redid the gel for the *hph* selection marker. From this, it seems like H2 is the most viable selection marker.

  ![](https://static.igem.org/mediawiki/2018/4/4d/T--DTU-Denmark--logbook-35-synth4.png)

  GelDoc 2018-08-27 16hr 13min H1 - H2 - H3 - H4 - Neg

  Based on these gels, we isolated specific cultures.

  It seems like the backup digestion and ligation does not work. We will therefore redo that experiment, this time with linear inserts.
  We think that if the inserts excist as plasmids, there is a posibility that they can aquire only the terminator and not the combined ligation. Therefore, only linear fragments will be used. This ensures that a colony is either;

  1) red and contains the *RFP* gene or

  2) white and contains the correctly assembled plasmid.

  **28/8/2018**
  Experiment: 3A assembly of CaMV terminator onto genes of interest
  The CaMV terminator was digested as downstream part, while the genes of interest, namely: GfaA, *MelA* and *MelA* optimized for **A. oryzae** were digested as upstream part.
  They were inserted into *pSC1C3* (the *RFP* expressing variant was used for ease of identifying background transformants).

  Experiment: 3A assembly of *hph* selection marker and *amilCP* constructs (containing)
  The *amilCP* constructs containing promoter and terminator as determined by the cPCR from 26-27/8 were digested as downstream part, while the *hph* selection marker from IDT were digested as upstream part. *pSC1C3* was used as backbone.

  **29/8/2018**
  Experiment: 3A of genes w. camV Terminator:
  Based on the Gel Tuesday, we isolated the C3, C5, H2, H4, G3, G4, G7 and G8 cultures, I wanted to confirm the construct we have made. Given the differing migration lengths from the 27/8 gel electrophoresis, we are currrently not sure whether it is the C1 or the C3 that is the successful assembly of P. CaMV-*amilCP*-CaMV term or whether any of them actually are. Likewise for G3, G4 and G7.
  In this experiment I am to verify the sequences by amplifying the gene regions overlapping two adjacent gene fragments.Tomorrow the products from this PCR will be run on a gel, to hopefully either verify or disprove the existance of expxected gene fragments.
  The PCR used a ONETAQ polymerase. The PCR was run with an anealing temperature of 49C and a elongation time of 1 min.

  **30/8/2018**
  Experiment: 3A of genes w. camV Terminator
  Today we performed gel electrophoresis on the 29/9 verification PCR.

  ![](https://static.igem.org/mediawiki/2018/b/be/T--DTU-Denmark--logbook-35-synth5.png)

  G31-G32-G41-G42-G71-G72-G81-G82-C31-C32-C51-C52

  ![](https://static.igem.org/mediawiki/2018/7/7d/T--DTU-Denmark--logbook-35-synth6.png)

  H21-H22-H41-H42-NEG3-NEG4

  Experiment: Gibson assembly of gene cassettes
  PCR extention with homology regions to allow for Gibson assembly were performed on the genes of interest. This was done using specific primers and Q5 PCR.
  After the PCR the products were run on a gel. The lanes corresponds to the above chart in terms of product (So lane 1 contaings *pTrpC* with homology regions for *MelA* and so forth):

  ![](https://static.igem.org/mediawiki/2018/1/1b/T--DTU-Denmark--logbook-35-synth7.png)

  **31/8/2018**
  The following genes, with their respective Gibson overhangs, were purified using the [gel extraction protocol](%E2%80%9Chttps%3A//2018.igem.org/Team%3ADTU-Denmark/Experiments#gelextraction”) as they were the only sucessfull PCR amplified genes:
  + *pTrpC* (*MelA*)
  + *pTrpC* (GfaA)
  + *MelA*
  + *MelA* (**A. oryzae**)
  + GfaA
  + CaMV terminator (*MelA*)
  + CaMV terminator (GfaA)Experiment: 3A assembly of *hph* selection marker and *amilCP* constructs (containing)
  Transformants containing the 3A assembled *hph* selection marker were screened for the correct inserts by cPCR with the verification primers, VF2 and VR. 5 Colonies were chosen from each of the CaMV promoter containing transformants and 8 colonies were chosen from the *pGpdA* containin transformants, due to higher background (estimated by amount of *RFP*-expressing cells). A one-taq PCR amplification were then performed and the products run on a gel:

  ![](https://static.igem.org/mediawiki/2018/d/de/T--DTU-Denmark--logbook-35-synth8.png)

  ![](https://static.igem.org/mediawiki/2018/6/64/T--DTU-Denmark--logbook-35-synth9.png)

  Based on the gel none of the screened mutants contained the right insert, which should be about 3200 bp, though g34 seem to containg some sort of additional assembly. Thus, this should be investigated.
* ![](https://static.igem.org/mediawiki/2018/1/1c/T--DTU-Denmark--logbookmycoicon.png)

  #### Mycolab Overview

  **27/8/2018**
  Protoplastation and transformation
  Transformation media prepared. See specifications in the [protocol for fungi media](%E2%80%9Chttps%3A//2018.igem.org/Team%3ADTU-Denmark/Experiments#mediaforfungi”).

  Statistical study: Tray 2, 7, 8, 6 were placed into the oven.

  **28/8/2018**
  Statistical study: Tray 6 and 8 were removed from oven.

  Protoplastation and transformation
  Plates with different concentrations of NTC prepared: 200, 400 and 800 and a positive. *E. coli* O/N cultures prepared with strains for MiniPrep.

  **29/8/2018**
  Protoplastation and transformation: Mini-prepped *E. coli* cultures from yesterday according to the  [QIAprep Spin Miniprep protocol](%E2%80%9Chttps%3A//2018.igem.org/Team%3ADTU-Denmark/Experiments#QIAprep”) and nanodropped the minipreps.

  **30/8/2018**
  Protoplastation and transformation: Transforming mini-prep into protoplasts to test protoplasts and miniprep. 5 transformations in 5 tubes. Each plated onto different plates with different NTC concentrations. Using protoplasts from 9/8.

  Statistical study: Tray 1 and trayy 5 placed in oven at 70°C. Tray 2 and 6 placed in oven at 100°C.
* ![](https://static.igem.org/mediawiki/2018/6/63/T--DTU-Denmark--logbookdryicon.png)

  #### Drylab Overview

  The investigation for design of the dome have now been expanded into investigation of internal structures. That means Lau has also decided on what kind of brick forms should be used, as the result of his work revealed that he had to have at least two types of bricks designs. In regards of simulation microscopic mycelium growth, this week was spent investigating how to make the branch extension dependent on growth kinetics of the specific fungi.
  The mechanical compression tests were carried out, where Mathias and Mathies have been crushing many bricks. The mycelium bricks have a lot of moisture of them which influenced the bricks quite a lot in terms of pressure resistance.

Week 36 (September 3 - September 9)

* ![](https://static.igem.org/mediawiki/2018/2/27/T--DTU-Denmark--logbooksynthicon.png)

  #### Synthlab Overview

  **3/9/2018**
  Experiment: Verification of PCR amplified camV terminator
  The camV. T. PCR amplificants were run on a gel to verify that the amplification had been successful. It seems that the amplificants seem successful.

  ![](https://static.igem.org/mediawiki/2018/a/a5/T--DTU-Denmark--logbook-36-synth1.png)

  The colonies were cultivated further in a liquid overnight cultre for miniprepping tomorrow. Lastly, we redid the gel for the *hph* selection marker. From this, it seems like H2 is the most viable selection marker.

  ![](https://static.igem.org/mediawiki/2018/4/4d/T--DTU-Denmark--logbook-35-synth4.png)

  Transformation of all the ligates from today were transformed into Dh5-α-competent cells following the [3A transformation protocol](%E2%80%9Chttps%3A//2018.igem.org/Team%3ADTU-Denmark/Experiments#proto3aassembly”)
  Experiment: Gibson Assembly of gene casettes.

  Since the previous PCR amplification with Gibson primers didn't yield any backbone to assemble into, as well as lacking some other amplifications, these were attempted again. This time both plasmid-inserted and linearized PCR amplification was performed. As can be seen from the gel below the plasmid amplifed backbone (PCR product 4 in the freezer) yielded an amplfied backbone.
  The products ampflied are *pTrpC*-*MelA* (**A. oryzae**), *GfaA*, *CaMV-GfaA*, backbone from plasmid and backbone. The last three are from an investigation of a construct from the *hph*-selection marker 3a assembly:

  ![](https://static.igem.org/mediawiki/2018/4/40/T--DTU-Denmark--logbook-36-synth2.png)

  **4/9/2018**
  Experiment: Gibson Assembly of gene cassettes
  As seen on the gel picture from Monday (September 3rd), we have two clear bands. These were gel extracted using the [protocol](%E2%80%9Chttps%3A//2018.igem.org/Team%3ADTU-Denmark/Experiments#gelextraction”). The resulting DNA concentrations are 38.1 and 38.6 ng/µl respectivly for band.

  Experiment: Colony PCR
  5 successful transformants from each plate was extracted and used for a colony PCR following the [colony PCR protocol](%E2%80%9Chttps%3A//2018.igem.org/Team%3ADTU-Denmark/Experiments#colonypcr”). We used the verification primers for this PCR to examine if length of the transformed constructs. This will be used to verify the digestion and ligation. In parallel, we experimented with an alternative technique for colony PCR. We scraped multiple colonies from the same plate off and treated it as if it was a colony. By doing it this way, we hope that we can screen plates faster, as we are looking for a band of the correct size in the gel. A possible limitation of using this technique with the verification primers is that other plasmids, such as the *RFP* will show bright bands, while our gene of interest might be very dim. This can be avoided by using other primers, but we will discuss this at a later point.

  **5/9/2018**
  Experiment: Gel results of colony PCR
  The colony PCR from 4/9 was run on a gel and gave rather dissapointing results

  ![](https://static.igem.org/mediawiki/2018/b/b1/T--DTU-Denmark--logbook-36-synth3.png)

  GelDoc 2018-09-05 15hr 10min Colony PCR 11- 41

  ![](https://static.igem.org/mediawiki/2018/c/c8/T--DTU-Denmark--logbook-36-synth4.png)

  GelDoc 2018-09-05 15hr 12min Colony PCR 42 - 72

  ![](https://static.igem.org/mediawiki/2018/8/81/T--DTU-Denmark--logbook-36-synth5.png)

  GelDoc 2018-09-05 15hr 14min Colony PCR 73 - 95 - Pos - Neg1 - Neg2

  ![](https://static.igem.org/mediawiki/2018/3/36/T--DTU-Denmark--logbook-36-synth6.png)

  GelDoc 2018-09-05 15hr 08min Colony PCR 1-9

  Troubleshooting:
  + Premixing a 'master-mix' might be problematic?
  + The annealing temperature was too high
  + The extension time too long
  + Too long amplification sequences
  + An error was made mixing the master mixes for the colony PCR
  + The lysated contain insuficient DNA or otherwise make for a bad templatePCR to test the lysates:
  We have made a small PCR to test the viability of the lysates as template. In this PCR we have the colony lysates 1,2,3, a positive control using *RFP* (0023) and a negative control. We have reduced the annealing temperature to 49\*C and otherwise we have tried to keep the parameters the same. Should we get good results, we will retry the colony PCR using the lysates and the new conditions. Should the results fail us, we can consider to try to use Q5 PCR on the lysates or make a new batch.

  **6/9/2018**
  Gel results from the lysate PCR test: The PCR results from the experiment on the 5/9 were run on a gel. We can see that there are some results, which suggests the lysates are not the problem. This could mean that it was the annealing temperature which was too high, that was causing the problems.

  **9/9/2018**
  Experiment: Colony PCR of transformants.
  A colony PCR of the Gibson transformants was prepared, with 5 colonies picked from each plate. This was done early in the day, which may not have allowed the *RFP*-expressing tranformants to show yet, thus increasing risk of picking a wrong transformant. A one Taq-PCR was performed with verificiation primers at 53 degrees celcius.
* ![](https://static.igem.org/mediawiki/2018/1/1c/T--DTU-Denmark--logbookmycoicon.png)

  #### Mycolab Overview

  **3/9/2018**
  Protoplastation and transformation
  Transformation plates had already sporulated. They were replica plated onto minimum media plates each containing the same NTC concentration as the original transformation plate.

  **5/9/2018**
  Protoplastation and transformation
  Replicate plates phtographed and put in fridge. **A. oryzae** plates incubated at 20°C.

  **7/9/2018**
  Protoplastation and transformation
  *Ganoderma resinaceum* protoplastation experiments started according to [protocol](%E2%80%9Chttps%3A//2018.igem.org/Team%3ADTU-Denmark/Experiments#ganodermaprotocol”). For incubation until Wednesday 12/9. 20°C **A. oryzae** plates checked. There is growth but it is very little.

  **9/9/2018**
  Protoplastation and transformation.
  Transformation of colour genes according to [protocol](https://2018.igem.org/Team%3ADTU-Denmark/Experiments#protoandtransfofasper).
* ![](https://static.igem.org/mediawiki/2018/6/63/T--DTU-Denmark--logbookdryicon.png)

  #### Drylab Overview

  This week the statistical DOE implemented into the compressive strength experiments by Mathias and Mathies. The pilot study was conducted for a screening of multiple substrates in the fungal lab by Mathias and Mathias. One conclusion was that the samples should definitely be dried for a longer time in the oven. Probably found explanation of the math behind geodesic domes.

Week 37 (September 10 - September 16)

* ![](https://static.igem.org/mediawiki/2018/2/27/T--DTU-Denmark--logbooksynthicon.png)

  #### Synthlab Overview

  **10/9/2018**
  Experiment: Gibson Assembly of gene casettes
  After two days in the cabinet incubator, the red colonies (*RFP*) were easily distinguishable from the white colonies (maybe succesful transformants). In total 12 colonies were to be found of the 3 agar plates, and these were isolated for a second round of colony PCR on the gibson transformants, Gel of 1st and 2nd round of colony PCT:

  ![](https://static.igem.org/mediawiki/2018/1/1d/T--DTU-Denmark--logbook-37-synth1.png)

  ![](https://static.igem.org/mediawiki/2018/2/26/T--DTU-Denmark--logbook-37-synth2.png)

  The colony PCR of the white colonies from the 10th of september (today) all seem to be succesful gibson transformants. Colony 1-4 have been prepared as an O/N culture for miniprep tomorrow.

  **11/9/2018**
  Experiment: Gibson Assembly of gene cassettes.
  Colony 1-4 had been grown O/N and 0.5 ml was transfered to a glycerol stock and placed at -80 C. The rest of the O/N culture was miniprepped. The resulting concentration of plasmids can be seen here. Plasmid 0063 has been sent to sequencing.
  Experiment: 3A assembly of composite parts.
  We PCR amplified sequnces of 0056, 0057 and 0058 to increase their concentration for sequencing. In addition, we have prepaired an O/N culture of 0056, 0057 and 0058, but it is not certain we will need them. The plasmids have been sent to sequencing to be verified.

  ![](https://static.igem.org/mediawiki/2018/7/7a/T--DTU-Denmark--logbook-37-synth3.png)

  **12/9/2018**
  Experiment: 3A assembly of NTC resistance gene
  As the *hph* selection marker may not be a viable option in **A. oryzae**, we have decided to trying NTC as a selection marker. The *Nat1* gene has been PCR amplified to contain the BioBrick prefix and suffix in a previous experiment. Now we will assemble it into a plasmid with a promoter and terminator. As with a lot of the consructs, we are testing different promoters and we will also be doing that here.

  Experiment: Digestion.
  We started with assembling the *Nat1* gene together with *PcamV* (0028), *pTrpC* (0018) or *pGpdA* (0008). We are using *RFP* (0024) as the backbone. The reason for starting with the promoter, is that this unfinished construct could have some limited use in the finished product, while the gene and terminator has no direct application.

  Experiment: Gibson Overhang Extension PCR
  Homology regions for Gibson assembly was added via PCR extensions to specific fragments. This time the 5x round of the PCR were done at the intial annealing temperature of the primers and afterwards the annealing temperature was increased to account for the elongated DNA molecules present which can then bind the full primer (with the extension on). The products was run on a 1% agarose gel producing the following bands. Thus, all fragments was succesfully extended except for *GfaA*, which continues to be a problem. This could very well indicate a need for redesign of primers.
* ![](https://static.igem.org/mediawiki/2018/1/1c/T--DTU-Denmark--logbookmycoicon.png)

  #### Mycolab Overview

  **10/9/2018**
  Protoplastation and transformation.
  Performed another transformation of colour genes. The [protocol for protoplastation of the **A. oryzae** was initialised](%E2%80%9Chttps%3A//2018.igem.org/Team%3ADTU-Denmark/Experiments#protoandtransfofasper”).

  **12/9/2018**
  Protoplastation and transformation
  New spore suspension. **A. oryzae** protoplastation according to [protocol](%E2%80%9Chttps%3A//2018.igem.org/Team%3ADTU-Denmark/Experiments#protoandtransfofasper”). Next *Ganoderma* protoplast step taken. There was a human error and the samples were centrifuged instead of vortexed.

  **14/9/2018**
  Protoplastation and transformation
  Osmotic buffer and sorbitol solution made and autoclaved. This is the day that the [*Ganoderma* protocol](%E2%80%9Chttps%3A//2018.igem.org/Team%3ADTU-Denmark/Experiments#ganodermaprotocol”) was attempted changed for the first time. With no Driselase available we had to think of other ways to protoplast *Ganoderma*. After consulting Ecovative we tried the first time with higher concentrations of Glucanex instead of both Glucanex and Driselase as originally in the protocol.
* ![](https://static.igem.org/mediawiki/2018/6/63/T--DTU-Denmark--logbookdryicon.png)

  #### Drylab Overview

  The sample from the pilot study was burning at different time intervals according to the optimal DOE. the samples were after the burning carried over to byg where the hydraulic testing of the cubes were commenced. Following the test resulted in the replicated DOE which was implemented into the DOE script and an optimal design was found which was carried out the same week.
  Doing the pilot sampling study, a huge part of the data (the fungus) were mushy and thus a lot of substrates were left out as optimal conditions. Another interesting results were that fungal cubes gets more brittle at higher temperatures.
  The first thoughts of implementing the Ganodermerma as a second brick option was considered due to the results of the pilot study.

Week 38 (September 17 - September 23)

* ![](https://static.igem.org/mediawiki/2018/2/27/T--DTU-Denmark--logbooksynthicon.png)

  #### Synthlab Overview

  **18/9/2018**
  Experiment: 3A assembly of NTC resistance gene
  The plasmids containing a promoter and the *Nat1* gene were PCR amplified using verification primers and run on a gel to ensure a correct ligation.

  ![](https://static.igem.org/mediawiki/2018/9/91/T--DTU-Denmark--logbook-38-synth1.png)

  These results look promising and we will continue with these plasmids. We are using the first of the four plasmids from each promoter construct.
  The digestions were ligated and will be ready for transformation tomorrow.

  Experiment: Gibson Assembly.
  The Gibson products from 17/9 were transformed, plated and incubated at 37 C O/N.

  Experiment: Existential Crisis
  Have you ever considered that the parking lots built in your lifetime will still be there, when you are long gone and forgotten?

  **19/9/2018**
  Experiment:3A assembly of NTC resistance gene
  The ligated products were transformed into E.coli and plated on LB cam plates.

  Experiment: Gibson Assembly
  The plated colonies may have been contaminated. We are solving this in two ways. We have redone the transformaiton and plated everything again We have spred the liquid culture on another set of plates.

  Experiment: Gel of PCR amplified gene fragments for Gibson.
  The PCR amplified gene fragments for gibson from yesterday (18/9) were run on a gel. The resulting gel:

  ![](https://static.igem.org/mediawiki/2018/f/f3/T--DTU-Denmark--logbook-38-synth2.png)

  Given this result, samples 3, 4 & 5 (The three *pTrpC*s with the right prefix) were isolated for gel extraction (performed 21/9).

  Experiment: Preparation of *amilCP* for mycolab
  For the transformaiton mycolab needs a steady supply of *amilCP*. We will provide that by producing multiple O/N cultures and puryfing the plasmids. We are also interested in discovering if the transformation is easier with linear DNA or with plasmids, so we will provide both. Multiple O/N cultures of C3 and G3 (See week 35) have been made from a glycerol stock.

  **21/9/2018**
  Experiment: 3A assembly of NTC resistance gen and Gibson Assembly
  The colony PCR products were run on a gel. The results were rather disappointing.

  ![](https://static.igem.org/mediawiki/2018/9/95/T--DTU-Denmark--logbook-38-synth3.png)

  Test of the colony PCR lysates: Due to the dissapointing results from the previous PCR, we wanted to test whehter the lysates were the problem, for this reason we selected the following lysates as a probe: F1 (*RFP* contaminated?), F2, C1, C2 and a positive control POS (*RFP* plasmid 0024).

  ![](https://static.igem.org/mediawiki/2018/5/57/T--DTU-Denmark--logbook-38-synth4.png)

  **22/9/2018**
  Experiment: 3A assembly of NTC resistance gene and Gibson Assembly.

  ![](https://static.igem.org/mediawiki/2018/2/2f/T--DTU-Denmark--logbook-38-synth5.png)

  ![](https://static.igem.org/mediawiki/2018/d/dd/T--DTU-Denmark--logbook-38-synth6.png)

  The only interesting results from this Gel electrophoresis were C4,D1-D4,E2-E4.
  The cultures for C4, (D2, D3?), E2, E3 where set to incubate O/N.
  + C: *pCaMV+Nat1+CamC Term*
  + D: *pTrpC+Nat1+CamC Term*
  + E: *pGpdA+Nat1+CamC Term*Given the migration patterns on the gel, I suspect that the the C,D,E constructs are too short to have both the promoter, the *NAT1* gene and the terminator. This can be investigated from the O/N cultures.
  Concluding remarks: It seems that this round of gibson transformations was unsuccessful.

  Experiment: PCR on the **A. oryzae** Lysates.
  The PCR on the **A. oryzae** lysates didn't yield any good results.
  **23/9/2018**
  Experiment: Tranformation of **A. oryzae** with *amilCP*.
  **A. oryzae** was tranformed with *amilCP* cassettes, either containing the pCaMV promoter or the *pGpdA* promoter. Both cirucular and linear versions of the *PcamV* promoter constructs were used. 4 replicates of each was made and placed at 28 degrees celicus for growth.
* ![](https://static.igem.org/mediawiki/2018/1/1c/T--DTU-Denmark--logbookmycoicon.png)

  #### Mycolab Overview

  **17/9/2018**
  Protoplastation and transformation
  *Ganoderma* protoplastation: No Driselase found anywhere in the lab so trying out with different concentraions of Glucanex:
  + 20 mg/ml
  + 30 mg/ml
  + 40 mg/mlThe 40 mg/ml is the same concentration as in the [*Aspergillus oryzae* protocol](%E2%80%9Chttps%3A//2018.igem.org/Team%3ADTU-Denmark/Experiments#protoandtransfofasper”) which is what prior knowledge we had to draw from. Transformation with chromoprotein: No colour colonies which would indicate that there has been no transformation. But! The transformed spores are very green compared to the yellow-green spores of the wildtype which indicates some change.

  **18/9/2018**
  Protoplastation and transformation
  First part of the process for isolating protoplast done. Tubes labelle 1, 2 and 3 put in the fridge for finishing up the next day.

  **19/9/2018**
  Protoplastation and transformation
  Samples were centrifuged but no pellet was isolated. Samples were thrown out and the [protoplasting and transformation protocol](%E2%80%9Chttps%3A//2018.igem.org/Team%3ADTU-Denmark/Experiments#protoandtransfofasper”) was replanned.

  **20/9/2018**
  Protoplastation and transformation
  Inoculated 15 PDA plates with *A. oryzae*. [Ecovative *Ganoderma* protoplastation protocol](%E2%80%9Chttps%3A//2018.igem.org/Team%3ADTU-Denmark/Experiments#protoandtransfofasper”) restarted. Next step is on Tuesday.

  **21/9/2018**
  Protoplastation and transformation
  Transformation of *PcamV* and PgdgA of *A. oryzae*.

  **23/9/2018**
  Protoplastation and transformation
  Transformation of *A. oryzae* with circular and linear fragments. Used 10 µl for all transformations.
* ![](https://static.igem.org/mediawiki/2018/6/63/T--DTU-Denmark--logbookdryicon.png)

  #### Drylab Overview

  Confirmed source found earlier was an explanation of the math behind geodesic domes. We will use the same math for calculating the size and placement of the pieces of the structure that should be made from fungi. Turns out it geodesic domes are based on a icosahedron, split into smaller triangles with corner points projected onto a circle, to ensure a more circular structure which ensures more equal distribution of structural load.
  Grow *Ganoderma* from the Ecovative kit To crush.
  Mathias spoke to Risø. They are going to irradiate them and see how resistant(growth rate) the spores are to gamma radiation. we are going to get a certificate as proof of all the technicalities of the test and results. they’ll test three strands. *Ganoderma*, Oreacce.
  we had a meeting to determine the deadlines and assigned names to each wiki part.
  Results from the replicated experiment of the best substrates from the pilot study came in and seemingly the lower temperatures of design was too low and so these were changed. Also seemingly pure rice gave the best results with regards to compression strength. The design was carried out once more with the new lower temperature. Also the *Ganoderma* experiments started using the same DOE as the *Aspergillus* fungi.

Week 39 (September 24 - September 30)

* ![](https://static.igem.org/mediawiki/2018/2/27/T--DTU-Denmark--logbooksynthicon.png)

  #### Synthlab Overview

  **24/9/2018**
  Experiment: PCR amplification of *amilCP* constructs (for transformation).
  Anealing temperature of verification primers: 53 Celsius. We are amplifying *pGdp-amilCP-CaMV* and *pCaMV-amilCP-CaMV* which have a length of ~1600 bases and 1300 bases. We are running the PCR triplets. We are using 1 µl og DNA for each reaction. In total, we have 8 *amilCP* reactions, a positive with *RFP* and a negative.

  Experiment: Prepartion of *RFP* in *pSCB1A3*.
  *RFP* was aquired from the kit and transformed into *E. coli* and plated on ampicillin-containing plates.

  Experiment: Miniprep of G3 and C3.
  4 cultures of each plasmid was miniprepped. They still need to be nano-dropped.

  **25/9/2018>**
  Experiment: Melanin 3A assembly.
  We are very interested in testing our *MelA* and *MelA*-A.oryzae constructs work in a fungi. We will therefore try to assemble them using 3A assembly again.

  Experiment: Purification of promoters for E.coli
  BBa\_J23119 is a strong constittutive promoter for E.coli from the Anderson promoter library. We want a promoter for E.coli to quickly verify that our *MelA* genes. We will be using the brick in two different backbones, *pSB1C3* and *pSB1A2*. These part are found on the distribution kit.
  The following parts have been transformed into E.coli and plated on their respective plates:
  + BBa\_J23119 - *pSB1C3*: Plate 3, well 17O
  + BBa\_J23119 - *pSB1A2*: Plate 4, well 17B
  **26/9/2018**
  Experiment: Purification of promoters for E.coli.
  A 17O culture has been transfered to LB cam liquid media and is being incubated O/N. The 17B transformants where wrongfully plates (as these contain the Amp resistance), but a new LB amp plate was prepared and the 17E cultures replated and let to incubate O/N.

  Experiment: Melanin 3A assembly.
  The amp resitance backbone pSB1A3 where minipreped and digested as backbone for the assembly.

  **27/9/2018**
  Experiment: Melanin 3A assembly.
  The ligations were transformed into E.coli and plated on LB amp plates. *RFP* (0025) was used for as a positive control and was plated on a LB cam plate.

  **28/9/2018**
  Experiment: Melanin 3A assembly.
  Onetaq protocol using verification primers and a annealing time of 3 minutes per cylce.

  **30/9/2018**
  Experiment: Gibson fragments.
  The fragments intended for Gibson assembly showed terrible results on the gel. We need to retry the PCR to get usable results.

  Experiment: Melanin 3A assembly.
  Some of the colonies were transfered to an O/N culture and we can hopefully extract successful plasmids tomorrow.
* ![](https://static.igem.org/mediawiki/2018/1/1c/T--DTU-Denmark--logbookmycoicon.png)

  #### Mycolab Overview

  **24/9/2018**
  Protoplastation and transformation.
  Samples from transformatinos (21/09/18) taken for colony PCR. See synthlab notebook from this week for rest of this. *A. oryzae* protoplastation initialized.

  **25/9/2018**
  Protoplastation and transformation.
  No PDB available, so the [Ganoderma protocol](https://2018.igem.org/Team%3ADTU-Denmark/Experiments#ganodermaprotocol) was postponed to tomorrow to make the PDB.

  **26/9/2018**
  Protoplastation and transformation.
  Funnels prepared for protoplastation of *A. oryzae*. Digestion of *A. oryzae* was postponed to tomorrow. So the first couple of steps were done and then put in the fridge to be finished tomorrow. First steps of [Ganoderma protocol](https://2018.igem.org/Team%3ADTU-Denmark/Experiments#ganodermaprotocol) carried out as well.

  **27/9/2018**
  [Ganoderma protocol](https://2018.igem.org/Team%3ADTU-Denmark/Experiments#ganodermaprotocol) continued. This time with varying concentrations of Glucanex, shaking speeds during digestion and digestion times. For details on this, see the protocol for protoplastation of *Ganoderma*.
  No cell strainers are available so the samples that were only given 2-3 hours of digestion were not filtered correctly. Henceforth sterile funnels with miracloth were used instead.
  We continued working on the [protoplastation and transformation of A. oryzae](https://2018.igem.org/Team%3ADTU-Denmark/Experiments#protoandtransfofasper).

  **28/9/2018**
  [*Ganoderma* protoplastation](https://2018.igem.org/Team%3ADTU-Denmark/Experiments#ganodermaprotocol) of samples that were given 19-24 hours of digestion continued. Following the protocol, all samples (Both 8 19-24 hour and 8 2-3 hour samples) were put into the fridge until Sunday for finalization of the protocol.

  **30/9/2018**
  Finalised [Ganoderma protocol](https://2018.igem.org/Team%3ADTU-Denmark/Experiments#ganodermaprotocol) by centrifuging. No pellet, so no protoplast. Back to drawing board.
* ![](https://static.igem.org/mediawiki/2018/6/63/T--DTU-Denmark--logbookdryicon.png)

  #### Drylab Overview

  Succeeded in visualising that the corner points of the pieces placement projected onto a circle was calculated correctly. Growth model: A model of the fungi biomass. The top is going a bit down but that’s okay. Risø gave three days of gamma radiation while fungi where grown. They grew as normal under radiation. Mathies have been creating the last bricks and the last tests on those will happen next week.*Ganoderma* tested as well.

Week 40 (October 1 - October 7)

* ![](https://static.igem.org/mediawiki/2018/2/27/T--DTU-Denmark--logbooksynthicon.png)

  #### Synthlab Overview

  **1/10/2018**
  Experiment: Amplified Insert Assembly of *MelA*.
  The promoter-*MelA* assemblies seem to be successfully assembled, so we will try to add a terminator to the constructs.
  We are using 0113-0116 as the *MelA* parts and 0014 was used as the terminator and backbone. We adapted the [3A assembly protocol](%E2%80%9Chttps%3A//2018.igem.org/Team%3ADTU-Denmark/Experiments#proto3aassembly”) to just using digestion of the parts and then ligating them. If this doesn't work, we may have to try using PCR amplification to produce enough insert.The products were ligated using the [3A protocol](%E2%80%9Chttps%3A//2018.igem.org/Team%3ADTU-Denmark/Experiments#proto3aassembly”) with the minor adjustment of just using two parts instead of three. The ligated DNA were then transformed into E.coli and plated 150 µl on LB cam plates.

  Experiment: PCR verification of *NAT1* and Beta-tubulin in *A. oryzae*.
  A Onetaq PCR was performed on the *A. oryzae* lysates PC1, PC2, PC3, PC4, PG1, PG2, PG3 and PG4 to verify the presence of *NAT1* and Beta-tubulin. Each of the lysate samples were amplified using the Seq\_*NAT1*\_in\_fw/Seq\_*NAT1*\_in\_bw or seqP\_betatub\_in\_fw/seqP\_betatub\_in\_bw primers. The resulting gels were dissapointing and the lysates declared unsuccesful End of Experiment: PCR verification of *NAT1* and Beta-tubulin in *A. oryzae*.

  Experiment: Sequencing of biobricks.
  Sequencing results:
  + Confirmed the 0092,0093,0094 plasmids.
  + Showed that 0101,0102,0103 does not contain *NAT1* nor terminator. Only the promoters were present in the plasmids.
  **3/10/2018**
  Experiment: Amplified Insert Assembly of *MelA*.
  The plated cultures have grown overnight and 3 plates showed growth. We have therefore made liquid O/N cultures of 0113, 0114 and 0116. The plates continues to incubate at 37 degrees.

  Experiment: Melanin 3A assembly.
  Two cultures that showed positive results on the gel were grown O/N and miniprepped. They have been added to the spredsheet as 0127 and 0128.

  Experiment: Sequencing of biobricks.
  Some of our promoter-melanin constructs were sent for sequencing. We hope to determine if it is worth it to continue with this line of experiments, and we will therefore verify that we have the correct constructs.

  **4/10/2018**
  Experiment: Amplified Insert Assembly of *MelA*.

  Experiment: Assembly of *Nat1* plasmid.
  The *Nat1* resistence gene has to be inserted into a backbone to be submitted to the registry. We will therefore cut it out and insert it into the backbone of *RFP* (0023).
  The *Nat1* gene, with the prefix and suffix, and *RFP* (0023) was digestied with EcoRI and PstI. Following that they were ligated and transformed into E.coli.

  Experiment: Sequencing of BioBricks.
  The miniprepped promoter-*MelA*-terminator constructs were sent for sequencing.

  **5/10/2018**
  Experiment: Assembly of *Nat1* plasmid.
  The plated colonies didn't look great, so we chose the least red colonies and ran them on a colony PCR.
  In parallel, since the results seemed very poor, we have redone the digestion and ligation, but this time we are using other enzymes. We suspect that PstI may not be able to cut our *Nat1* fragment, as our primer design hadn't allowed for extra bases following the cut site. We therefore used EcoRI and SpeI as our enzymes. In addition to using other enzymes, we also tried to use a linearized backbone from the distribution kit to see if that could increase the success-rate.

  Experiment: Colony PCR on overnight cultures.
  A total of 4 white colonies (N1,N2,N31 & N32) were discovered on the three plates for *NAT1* transformants. A standard verification primer Onetaq was performed on these cultures and they were run on a gel.

  ![](https://static.igem.org/mediawiki/2018/2/28/T--DTU-Denmark--logbook-40-synth1.png)

  Suprisingly, cultures N31 and N32 seem rather interesting and should be grown O/N.

  **6/10/2018**
  Experiment: Colony PCR of the overnight.
  A colony PCR was performed on the overnight *NAT1* transformants. The PCR used was a standard verification primer PCR with 60 seconds extension time.

  ![](https://static.igem.org/mediawiki/2018/c/cf/T--DTU-Denmark--logbook-40-synth3.png)

  None on the colonies seem promising and looks mostly like selfligated backbones.
  Experiment: PCR on N31 and N32.
  A PCR with primers was performed on the N31 and N32 lysates from yesterday (5/10) using the primers.

  Suprising something worked. However the results a both encouraging and deconsolate, as the two first samples shows promising 400 bp bands, however the negative test gives more or less a band in the same height, for which reason I believe we should run another gel tomorrow.

  **7/10/2018**
  Experiment: Rerun of PCR on N31 and N32.
  The verification PCR from yesterday was repeated using 51 C as annealing temperature instead of 64\*C. The results are like those of yesterday and sadly rather inconclusive.

  Experiment: Digestion, Ligation & Transformation of *NAT1*.
  A new digestion of *NAT1* was performed using EcoRI & PstI was made. Each of the *NAT1* digests (N1,N2,N3) were ligated with both a digested backbone and a linear backbone resulting in the six ligates (N1B,N1L,N2B,N2L,N3B,N3L). These we subsequently transformed and plated alongside with the gibson transformants.
  Experiment: Gibson assembly of *NAT1* into backbone.
  Using the prefix/suffix homologous ends of the linear backbone and our *NAT1* PCR extracts, we performed gibson assembly for insertion of the *NAT1* gene into the backbone. These were transformed and plated for growth O/N.

  Experiment: Overnight cultures of *NAT1*.
  Two addtional O/N culture of N31 and N32 were prepared in liquied LB cam media and left of incubate.
* ![](https://static.igem.org/mediawiki/2018/1/1c/T--DTU-Denmark--logbookmycoicon.png)

  #### Mycolab Overview

  **1/10/2018**
  [*Ganoderma* protocol](%E2%80%9Chttps%3A//2018.igem.org/Team%3ADTU-Denmark/Experiments#ganodermaprotocol”) started.
  *Ganoderma* was inoculated on PDA plates and incubated at 30°C.

  **3/10/2018**
  We inoculated 12 plates of *A. oryzae* on PDA and incubated them at 28°C and prepared sorbitol solution and funnels for the aforementioned [*Ganoderma* protocol](%E2%80%9Chttps%3A//2018.igem.org/Team%3ADTU-Denmark/Experiments#ganodermaprotocol”).

  **5/10/2018**
  [*Ganoderma* protocol](%E2%80%9Chttps%3A//2018.igem.org/Team%3ADTU-Denmark/Experiments#ganodermaprotocol”) continued. This time with twice as many samples to try an up the amount of mycelium in the tubes. 1 ml og PDB was put into 32 tubes and inoculated with *Ganoderma*. Incubated at 30°C and at either 85 rpm and 150 rpm for 48 hours instead of 24, again to get more mycelium. Protoplastation of *A. oryzae* continued.

  **7/10/2018**
  [*Ganoderma* protocol](%E2%80%9Chttps%3A//2018.igem.org/Team%3ADTU-Denmark/Experiments#ganodermaprotocol”) continued. The digest and start of isolation were done for the samples that were only given 2-3 hours and the digestion was started for the samples that need 19-24 hours.
* ![](https://static.igem.org/mediawiki/2018/6/63/T--DTU-Denmark--logbookdryicon.png)

  #### Drylab Overview

  Continued work on python code to calculate placement and size of pieces. A substrate grid was implemented to investigate the mycelium development dependent on substrate availability or depletion. Statistical modeling was implemented based on the DOE for both the replicated and unreplicated design. So far a Gamma model or a random effect model is seemingly the best choices.

Week 41 (October 8 - October 14)

* ![](https://static.igem.org/mediawiki/2018/2/27/T--DTU-Denmark--logbooksynthicon.png)

  #### Synthlab Overview

  **8/10/2018**
  Colony PCR and Nat1 transformants were run. a miniprep of N31 and N32 was performed.

  We had failed miniprep of A4 and A5 melA constructs as the A4 and A5 were incubated in media with a wrong antibiotic.

  **10/10/2018**
  Miniprep of O/N cultures was performed and samples were sent to sequencing.

  **11/10/2018**
  Results of the sequencing showed that all nat1 attempts have been unsuccessful.

  **14/10/2018**
  Experiment: PCR Verification of A. oryzae transformants.
  New gDNA was purified from 7 different A. oryzae plates. 4 plates contains a (probably) CAC from linear DNA transformation, There are 2 transformants from a GAC transformation and 1 MelA transformant.

  Experiment: Transformation of A. oryzae with melA genes.
  A. oryzae protoplasts were transformed with pcamV-melA-tcamv constructs (0131) and (0130) and left to incubated on transformations media containing 0,6 mg/mL tyrosine.
* ![](https://static.igem.org/mediawiki/2018/1/1c/T--DTU-Denmark--logbookmycoicon.png)

  #### Mycolab Overview

  **8/10/2018**
  *Ganoderma* protocol continued: The 19-24 hours samples taken out and started the isolation procedure. Put in fridge with the other samples.

  **9/10/2018**
  Looked at transformation plates with Kyle to see if something might have transformed: He thinks that the media being coloured and the spores looking greener than usual on the transformants might indicate that something has been transformed. New bricks were made to see if they secrete the colour into the bricks.

  **10/10/2018**
  The [*Ganoderma* protocol](%E2%80%9Chttps%3A//2018.igem.org/Team%3ADTU-Denmark/Experiments#ganodermaprotocol”) was finished by centrifuging for 10 minutes at 10000 rpm. There was no pellet.
  Liquid cultures of the suspected transformants were inoculated and incubated at 28°C and 150 rpm. The cultures were inoculated with both green spores and white spores to see if they secreted any kind of colour.

  **11/10/2018**
  As a last try, the [*Ganoderma* protocol](%E2%80%9Chttps%3A//2018.igem.org/Team%3ADTU-Denmark/Experiments#ganodermaprotocol”) was restarted, this time with driselase as we hand received some the same day. There was no time to make new plates to incubate for 5 days, so we had to try with some of the old plates. Three tubes with 1 ml PDB was inoculated for continuation the next day.

  **12/10/2018**
  The three tubes were taken through the digestion steps and put in the incubator at 30°C, 85 rpm.

  **13/10/2018**
  The samples were taken out of the incubator. The incubator had been changed to 28°C, 100 rpm when we returned to the samples. The samples were run through the first steps for isolation of protoplasts.

  **14/10/2018**
  Samples were centrifuged according to protocol but there was no pellet.
* ![](https://static.igem.org/mediawiki/2018/6/63/T--DTU-Denmark--logbookdryicon.png)

  #### Drylab Overview

  Decided that too many complications had arisen in calculating all side lengths and placements and implementing in comsol. Instead we approximate the structure as half a sphere, which is also a good baseline for future work and is more inline with the inaccuracies of our material properties.

Week 42 (October 15 - October 21)

* ![](https://static.igem.org/mediawiki/2018/2/27/T--DTU-Denmark--logbooksynthicon.png)

  #### Synthlab Overview

  **15/10/2018**
  Experiment:PCR Verification of A. oryzae transformants.
  The purified gDNA was PCR amplified using the AmilCO Forward primer and the tCaMV Backwards primer. This should reveal bands around 230 bp. (0092) was used as a positive control.

  We see bands around 200 bp, but it is not certain if this is our gene of interest or not.

  ![](https://static.igem.org/mediawiki/2018/f/fc/T--DTU-Denmark--NotebookSynth42Week1.png)

  We ran the PCR products on a 2% agerose gel and we got the following:

  ![](https://static.igem.org/mediawiki/2018/3/3e/T--DTU-Denmark--NotebookSynth42Week2.png)

  Here it could seem like we have a band on the first sample that matches the positive control. Another PCR was made to verify the results, but this showed signs of contamination, as the negative also showed bands that was alsom identical to the positive.

  ![](https://static.igem.org/mediawiki/2018/9/9a/T--DTU-Denmark--NotebookSynth42Week3.png)

  **16/10/2018**
  Experiment: Confirmation of amilCP construct integration.
  Another round gDNA preparation and PCR was performed to confirm integration of amilCP constructs in apparent transformants. Extra care was taken in order to ensure no leakage from wells as this had been a problem earlier. These gel can be seen in the [results page for *amilCP*](https://2018.igem.org/Team%3ADTU-Denmark/Results-amilCP).

  **17/10/2018**
  Experiment: Sequencing of gDNA.
  We would like to know where the inserted fragments are in the genome o A. oryzae. Therefore, we have send a sample of the most successful transformants to sequencing with primers that point out of the cassette.
  We tried to ethanol percipitate our previous samples, but we got very low DNA concentrations, which means we cannot expect resonable results. We have therefore performed a new extraction of gDNA from the same spot on the plate and disolved the DNA in 50, 100 and 200 µl of Milli-Q water.
* ![](https://static.igem.org/mediawiki/2018/6/63/T--DTU-Denmark--logbookdryicon.png)

  #### Drylab Overview

  Completed the simplified simulations in comsol and finished the corresponding wiki site writing.
  The meso-growth model and the statistical model were finalized, plots were generated and the wiki was completed.

Week 43 (October 22 - October 28)

---

* ![](https://static.igem.org/mediawiki/2018/2/2d/T--DTU-Denmark--logbookall1icon.png)

  #### Team Overview

  The DTU BioBuilders are ready and thrilled for Boston. Good luck to everyone.

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# Team:DTU-Denmark/Experiments

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# Experiments

## Synthlab protocols

Resuspension of DNA/primer

DNA from IDT will typically be delivered in a white flaky substance, which needs to be resuspended before the DNA is ready for use.

#### Materials

* Table centrifuge
* EB/TE buffer or Milli-Q Water
* Genes/Primers from IDT or other DNA provider

#### Procedure

##### Resuspension

1. Quickly spin the DNA down in the table centrifuge.
2. Calculate the amount of EB buffer needed to dilute the gBlocks to the desired concentration.
   **Important**: gene fragments and primers are not diluted to the same concentration:
   The concentration of gene fragments is usually 25 ng/μL and for primers it is 100 μM.
   * Note 1: DNA from IDT usually comes in dried flakes of 500 or 1000 ng of DNA. To achieve the desired concentration (usually 25 ng/μL) the needed amount of EB buffer is 40 μL (for 1000 ng samples) or 20 μL (for 500 ng samples).
   * Note 2: In order to calculate the molar amount of primer, use the [NEB calculator.](https://nebiocalculator.neb.com/#!/ssdnaamt)
3. Add the calculated amount of EB buffer/Milli-Q water.
4. Store the resuspended DNA at -20°C.

Adapted Assembly of Composite Parts Into Backbone

**Timeline for procedure:**
Day 1: Minimum 4 ½ hours from start to finish
Day 2: Overnight culture from plating
Day 3: Culture ready for MiniPrep

#### Materials

##### Digestion materials

* Milli-Q water
* Buffer (e.g. cut-smart)
* Gene or plasmid
* Restriction enzyme (e.g. EcoRI)
* Restriction enzyme (e.g. PstI)

##### Ligation materials

* Milli-Q water
* T4 DNA ligase Buffer
* Plasmid 3 kB
* Cut part
* T4 DNA ligase

##### Transformation materials

* Competent cells
* Ice

#### Procedure

##### Digestion

1. Calculate the amount of gene fragment and plasmid backbone (A1) used for the digestion.
   * The recommended amount is 100 ng gene fragment and given that the standard concentration of resuspended DNA is 25 ng/μL, the needed amount is 4 μL.
   * Beware: For each digested gene fragment, one should prepare 50 ng of digested backbone (e.g. for 4 gene fragments one should prepare 200 ng of backbone).
2. Combine the contents of the table in PCR tubes:

   | Reagent | Value |
   | --- | --- |
   | For each gene fragment | |
   | Milli-Q water | Up to 10 ul |
   | Buffer | 1 µl |
   | Gene or A1 | \* |
   | Restriction enzyme | 0,2 µl |
   | Restriction enzyme | 0,2 µl |
   | Total Volume | 10 µl |
3. Digest at 37°C for 1 hour.
4. Heat inactivate at 80°C for 20 min.
5. Store in the freezer or use for ligation.

##### Ligation

6. Calculate the volume of Digested DNA and Digested Backbone in the table below.
   **Volume of Digested DNA:** Open the [NEB calculator](https://nebiocalculator.neb.com/#!/ligation):
   * Use the Digested DNA-to-Plasmid-ratio 3:1.
   * We are using 50 ng backbone, as per a [NEB protocol](https://international.neb.com/Protocols/0001/01/01/dna-ligation-with-t4-dna-ligase-m0202)
   * Digestion concentration is 10 ng/µl

   | Reagent | Volume |  | |
   | --- | --- | --- | --- |
   | For each of Digested DNA fragment | | Standard DNA digest volumes\* | |
   | Milli-Q water | up to 20 µl | Length of digested part | Volume |
   | T4 DNA ligase Buffer | 2.0 µl | 500 bp | 3.75 µl |
   | Plasmid  3 kB | 50 ng | 1000 bp | 7.5 µl |
   | Digested part | x | 1500 bp | 11.25 µl |
   | T4 DNA ligase | 1  µl | 2000 bp | 15 µl |
   | Total Volume | 20  µl |

   \* using the 2 kb A1 backbone.
7. Mix the contents of the table above in a PCR tube.
8. Incubate at 25°C for 30 min.
9. Inactivate at 65°C for 20 min.
10. Put the ligation in the freezer or use it for transformation.

##### Transformation

11. 5 µl of the ligated parts is transferred to an Eppendorf tube.
12. 1 µl of the positive control is transferred to an Eppendorf tube.
13. 50 µl competent cells is transferred to each tube. Competent cells must be held on ice!
14. Mix gently by rolling the tubes by hand.
15. Put the tubes back on ice for 30 minutes.
16. Make sure the heating block is heated up to 42°C and heat-shock the tubes for 30 seconds.
17. Place the tubes back on ice for 5 minutes.
18. Pipette 500 ul of sterile LB media to each tube.
19. Incubate for 2 hours for 37°C with shaking (this step can be shortened if an amp-backbone is used).
20. Shake the tube with the cells gently. Pipette 200 µl cell culture on each plate.
21. Incubate at 37°C O/N.

3A Assembly

*Adapted from [iGEM 3A assembly and [iGEM Victoria 3A protocol](http://igem-victoria.pbworks.com/w/page/7992050/3A%20Assembly%20Protocol).](http://parts.igem.org/Help%3AProtocols/3A_Assembly)*

#### Materials

##### Digestion

* Cutsmart buffer
* SpeI, XbaI, PstI and EcoRI restriction enzymes
* Biobrick compatible genes
* Milli-Q water
* 2 DNA fragments with verification primers
* PCR tubes (2 per gene fragment: 1 for digestion, 1 for ligation)

##### Ligation

* T4 ligase buffer
* T4 ligase

##### Transformation

* Competent cells
* Ice

#### Procedure

##### Digestion

1. Mix the assembly parts following their respective tables a PCR tube.

   \*100 ng is needed.

   **Upstream fragment**

   | Reagent | Volume |
   | --- | --- |
   | For each gene fragment | |
   | Milli-Q water | Up to 10 ul |
   | Cutsmart buffer | 1 µl |
   | Gene | \* |
   | SpeI | 0,2 µl |
   | EcoRI | 0,2 µl |
   | Total Volume | 10 µl |

   **Downstream fragment**

   | Reagent | Volume |
   | --- | --- |
   | For each gene fragment | |
   | Milli-Q water | Up to 10 ul |
   | Cutsmart buffer | 1 µl |
   | Gene | \* |
   | SpeI | 0,2 µl |
   | EcoRI | 0,2 µl |
   | Total Volume | 10 µl |

   **Backbone**

   | Reagent | Volume |
   | --- | --- |
   | For every other gene fragment | |
   | Milli-Q water | Up to 10 ul |
   | Buffer | 1 µl |
   | Gene | \* |
   | EcoRI | 0,2 µl |
   | PstI | 0,2 µl |
   | Total Volume | 10 µl |
2. Digest at 37°C for 1 hour.
3. Heat inactivate at 80°C for 20 min.
4. Store in the freezer or use for ligation.

##### Ligation

5. Mix the following in a PCR tube.

   | Reagent | Volume |
   | --- | --- |
   | **For each of Digested DNA fragments** | |
   | Milli-Q water | up to 20 µl |
   | T4 DNA ligase Buffer | 2.0 µl |
   | Backbone | 2 µl |
   | Upstream fragment | 2 µl |
   | Downstream fragment | 2 µl |
   | T4 DNA ligase | 1  µl |
   | Total Volume | 20  µl |
6. Incubate at 25°C for 30 min.
7. Inactivate at 65°C for 20 min

##### Transformation

8. 5 µl of the ligated parts is transferred to a chilled Eppendorf tube.
9. 1 µl of the positive control is transferred to an Eppendorf tube.
10. 50 µl competent cells is transferred to each tube.
    Competent cells must be held on ice!
11. Mix gently by rolling the tubes by hand.
12. Put the tubes back on ice for 30 minutes.
13. Make sure the heating block is heated up to 42°C and heat-shock for 30 seconds.
14. Place the tubes back on ice for 5 minutes.
15. Pipette 500 µl of sterile LB media to each tube.
16. Incubate for 2 hours for 37°C with shaking (this step can be shortened if an amp-backbone is used).
17. Next step is to plate it on to the plates.
18. Shake the tube with the cells gently. Pipette 100 µl cell culture on each plate.
19. Incubate at 37°C O/N.

QIAprep® Spin Miniprep Kit

*Adapted from Qiagen's [QIAprep® Spin Miniprep Kit](https://www.qiagen.com/us/resources/download.aspx?id=56b0162c-23b0-473c-9229-12e8b5c8d590&lang=en)*
This protocol assumes using a centrifuge, and not vacuum manifold processing.

#### Materials

##### Buffers

* P1 buffer
* P2 buffer
* N3 buffer
* LyseBlue reagent
* PB buffer
* PE buffer
* EB buffer

##### Tubes

* Centrifuge tubes
* 1.5 mL Eppendorf tubes

#### Procedure

##### Quick-start protocol

1. Pellet 1–5 ml bacterial overnight culture by centrifugation at >8000 rpm (6800 x g) for 3 min at room temperature (15–25°C).
2. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube.
3. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min. If using LyseBlue reagent, the solution will turn blue.
4. Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times. If using LyseBlue reagent, the solution will turn colorless.
5. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
6. Apply 800 μl supernatant from step 5 to the QIAprep 2.0 spin column by pipetting. Centrifuge for 30–60 s and discard the flow-through.
7. Recommended: Wash the QIAprep 2.0 spin column by adding 0.5 ml Buffer PB. Centrifuge for 30–60 s and discard the flow-through.
8. Wash the QIAprep 2.0 spin column by adding 0.75 ml Buffer PE. Centrifuge for 30–60 s and discard the flow-through. Transfer the QIAprep 2.0 spin column to the collection tube.
9. Centrifuge for 1 min to remove residual wash buffer.
10. Place the QIAprep 2.0 column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM TrisCl, pH 8.5) or water to the center of the QIAprep 2.0 spin column, let stand for 1 min, and centrifuge for 1 min.
11. If the extracted DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.

Hifi DNA Assembly and Transformation Protocol

*Adapted from IDT's [HiFi assembly protocol](https://international.neb.com/-/media/catalog/datacards-or-manuals/manuale2621.pdf)*

#### Materials

##### Consumables

X is the number of reactions.

* X PCR tubes for each reaction + 1 for positive control
* X Eppendorf tube for each reaction + 1 for positive control
* X selection plate for each reaction
* 1 Amp plate for positive control

##### Chemicals

* Hifi DNA assembly Master mix
* Milli-Q water
* Competent *E. coli* (e.g. DH5α)
* Prepared DNA fragments for assembly (See information on primer construction)

#### Procedure

##### Assembly protocol

1. Set up the following reaction on ice:

   |  | Recommended amount of fragments used for assembly | | |
   | --- | --- | --- | --- |
   |  | **2-3 Fragments\*** | **4-6 Fragments** | **Positive control** |
   | **Recommended DNA Molar Ratio** | Vector:insert = 1:2 | Vector:insert = 1:1 |  |
   | **Total amount of DNA fragments** | 0.03-0.3pmols X uL | 0.2-0.5 pmols X uL | 10 uL |
   | **NEB Hifi Assembly master mix** | 10 uL | 10 uL | 10 uL |
   | **Milli-Q water** | 10-X uL | 10-x uL | 0 uL |
   | **Total volume** | 20 uL\*\* | 20 uL\*\* | 20 uL |

   \* If the inserts are less than 200 bp, use a 5-fold excess of inserts instead of a 2-fold excess.
   \*\* If a greater number of fragments are assembled, increase the volume of the reaction and use additional HiFi DNA assembly master mix.
2. Incubate the reaction samples in a thermocycler at 50°C for 15 minutes (when 2-3 fragments are assembled) or 60 minutes (when 4-6 fragments are assembled). Following incubation, store the reaction samples at -20°C for subsequent transformation.
   Note: Extended incubation up to 60 minutes can in some cases improve transformation efficiency.

##### Transformation protocol

3. Thaw chemically-competent cells on ice.
4. Add 2 µL of the chilled assembly product to the competent cells. Mix gently by pipetting up or down or by flicking the tube 4-5 times. Do NOT vortex.
5. Place the mixture on ice for 30 minutes. Do not mix.
6. Heat shock at 42°C for 30 seconds. Do not mix.
7. Transfer tubes to ice for 2 minutes.
8. Add 950 µL of room temperature SOC media to the tubes.
9. Incubate the tube for 37°C for 60 minutes. shake vigorously (250 rpm) or rotate.
10. Warm selection plates to 37°C.
11. Spread 100 µL of the cells onto the selection plates.
    Note: Use Amp plates for the positive control.
12. Incubate overnight at 37°C.

Gel Extraction Protocol

*Adapted from The Chen Laboratory's [QIAquick Gel Extraction Kit Protocol](http://www.indiana.edu/~lchenlab/protocol_files/agarose_gel_extraction.pdf).*

#### Materials

##### Consumables

N is the number of reactions.

* 2\*Ν 1.5 mL Eppendorf tubes
* Ν spin columns with collection tubes

##### Chemicals

* Buffer QG
* Buffer PE
* Buffer EB
* Isopropanol
* (potentially: 3 M sodium acetate - see procedure step 4)

##### Instruments

* Thermocycler/thermoshaker
* Scalpel
* Table centrifuge for 1.5 mL microcentrifuge/Eppendorf tubes.

#### Procedure

##### Gel extraction

1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel. Minimize the size of the gel slice by removing extra agarose.
2. Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg ~ 100 µl).
   For example, add 300 µl of Buffer QG to each 100 mg of gel. For >2% agarose gels, add 6 volumes of Buffer QG. The maximum amount of gel slice per QIAquick column is 400 mg; for gel slices >400 mg use more than one QIAquick column.
3. Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). To help dissolve gel, mix by vortexing the tube every 2–3 min during the incubation. IMPORTANT: Solubilize agarose completely. For >2% gels, increase incubation time.
4. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10 µl of 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn to yellow.
   Note: The adsorption of DNA to the QIAquick membrane is efficient only at pH ≤7.5. Buffer QG contains a pH indicator which is yellow at pH ≤7.5 and orange or violet at higher pH, allowing easy determination of the optimal pH for DNA binding.
5. Add 1 gel volume of isopropanol to the sample and mix.

   For example: if the agarose gel slice is 100 mg, add 100 µl isopropanol. This step increases the yield of DNA fragments <500 bp and >4 kb. For DNA fragments between 500 bp and 4 kb, the addition of isopropanol has no effect on yield. Do not centrifuge the sample at this stage.
6. Place a QIAquick spin column in a provided 2 ml collection tube.
7. To bind DNA, apply the sample to the QIAquick column, and centrifuge for 1 min at ≥10,000 x g (~13,000 rpm). The maximum volume of the column reservoir is 800 µl. For sample volumes of more than 800 µl, simply load and spin again.
8. Discard flow-through and place QIAquick column back in the same collection tube.
9. Optional: Add 0.5 ml of Buffer QG to QIAquick column and centrifuge for 1 min ≥10,000 x g (~13,000 rpm). This step will remove all traces of agarose. It is only required when the DNA will subsequently be used for direct sequencing, in vitro transcription or microinjection.
10. To wash, add 0.75 ml of Buffer PE to QIAquick column and centrifuge for 1 min ≥10,000 x g (~13,000 rpm). Note: If the DNA will be used for salt-sensitive applications, such as blunt-end ligation and direct sequencing, let the column stand 2–5 min after the addition of Buffer PE, before centrifuging.
11. Discard the flow-through and centrifuge the QIAquick column for an additional 1 min at ≥10,000 x g (~13,000 rpm). IMPORTANT: Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifugation.
12. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
13. To elute DNA, add 50 µl of Buffer EB (10 mM Tris·Cl, pH 8.5) or H2O to the center of the QIAquick membrane and centrifuge the column for 1 min at maximum speed. Alternatively, for increased DNA concentration, add 30 µl elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge for 1 min.
    **IMPORTANT**: Ensure that the elution buffer is dispensed directly onto the QIAquick membrane for complete elution of bound DNA. The average eluate volume is 48 µl from 50 µl elution buffer volume, and 28 µl from 30 µl. Elution efficiency is dependent on pH. The maximum elution efficiency is achieved between pH 7.0 and 8.5. When using water, make sure that the pH value is within this range, and store DNA at –20°C as DNA may degrade in the absence of a buffering agent. The purified DNA can also be eluted in TE (10 mM Tris·Cl, 1 mM EDTA, pH 8.0), but the EDTA may inhibit subsequent enzymatic reactions.

Q5 PCR amplification with Standard Primers

*Adapted from NEB Q5 [High-Fidelity 2X Master Mix](https://international.neb.com/protocols/2012/12/07/protocol-for-q5-high-fidelity-2x-master-mix-m0492)*

#### Materials

##### Tubes

* PCR tubes (1 per reaction)

### Reagent

* Milli-Q water

##### DNA

* VR/VF primers
* Ligation mix

#### Procedure

##### Q5 PCR amplification with standard primers

1. Determine the needed amount (x) of template DNA via the table below. Added volumes of about 1 µl are preferred, so dilute the sample if necessary with Milli-Q water.

   **Template amounts**

   | Template source | Template amount |
   | --- | --- |
   | Genomic | 1 ng - 1 μg |
   | Plasmid or viral | 1 pg - 1 ng |
2. Mix the reagents in a PCR tube.

   **PCR reagents**

   | Reagent | Volume |
   | --- | --- |
   | For each of Digested DNA fragment | |
   | Milli-Q water | up to 50 µl |
   | VF primer | 2.5 µl |
   | VR primer | 2.5 µl |
   | Template sample | x |
   | Q5 2X Master Mix | 25 µl |
   | Total Volume | 50  µl |
3. For PCR machines without heated lid: overlay the sample with mineral oil.
4. Place in thermocycler using the following routine:

   **Thermocycler routine**

   | Step | Temperature (°C) | Time |
   | --- | --- | --- |
   | Initial Denaturation | 98 | 30 sec |
   | 35 cycles | 98 | 5-10 sec |
   | 66 | 10-30 sec |  |
   | 72 | 20-30 sec/kb\* |  |
   | Final extension | 72 | 120 sec |
   | Hold | 4-10 |  |

   \*The recommended extension temperature is 72°C. Extension times are generally 20–30 seconds per kb for complex, genomic samples, but can be reduced to 10 seconds per kb for simple templates (plasmid, *E. coli*, etc.) or complex templates < 1 kb. Extension time can be increased to 40 seconds per kb for cDNA or long, complex templates, if necessary.
   \*\*When amplifying products > 6 kb, it is often helpful to increase the extension time to 40–50 seconds/kb.
5. After thermocycling the product can be stored at -20°C or be used for gel electrophoresis.

Colony PCR

*By David Faurdal, adapted in part from NEB's one-taq protocol.*
The purpose of this protocol is to confirm correct insertion of fragments after 3A assembly/GIBSON assembly into the pSB1C3 backbone. This is done by PCR amplifying the insert one is interested in and confirming that it's the correct size (by gel electrophoresis). As the backbone comes equipped with verification primer-binding sites, these primers, VF2/VR (sequences can be found [here](http://parts.igem.org/Primers/Catalog)), can simply be used if one does insertion into it. The protocol can, however, be used with different primers for different backbones. As the fragments run on the gel won't be used for cloning purposes there is no reason to use high-fidelity polymerases on this, just use one-taq.

#### Materials

* Transformants from whatever assembly method you fashion
* Eppendorf tubes
* Sterile toothpicks/inoculation lops/pipette tips for transferring colonies
* Milli-Q water
* LB media with appropriate antibiotics

#### Procedure

##### Preparing the template DNA from the transformants

1. Pick a number of transformants, typically 3-10, from each plate of interest and mark them on the back of the plate.
2. Set up 2 Eppendorf tubes for each colony and mark them accordingly:
   1. Fill the first one (1) with 15 µl Milli-Q water.
   2. The other one (2) remains empty for now.
3. Transfer each colony to the Eppendorf containing 15 µl water using a sterile toothpick, inoculation loop or autoclaved pipette tip.
4. Transfer 5 µl of the water from (1) to the empty (2) tube. This tube (2) is now for safekeeping in case the colony PCR shows that the transformant in question contains the correct insertion.
5. Boil the (1) tubes for 10 minutes at 98 °C. Prepare the PCR mastermix, while the colonies are boiling.

##### Setting up the PCR itself

1. Set up a 25 µl reaction for each colony to be screened, as per NEB'S one-taq PCR protocol (see the [protocol](https://international.neb.com/protocols/2012/10/11/onetaqdnapolymerasem0480) for troubleshooting).

   | Component | 25 µl reaction |
   | --- | --- |
   | 5x OneTaq Standard Reaction Buffer | 5 µl |
   | 10 mM dNTP | 0,5 µl |
   | 10 µM Forward Primer | 0,5 µl |
   | 10 µM Reverse Primer | 0,5 µl |
   | OneTaq  DNA Polymerase | 0.125 µl |
   | Template | 1 µl from the (1) tube |
   | Nuclease-Free Water | up to 25 µl |
2. Run the PCR in the thermocycler (use this [website](https://tmcalculator.neb.com/) to calculate temperatures used based upon the primers and polymerase used).
3. Run the products on a gel to check for correct insertion.
4. Prepare an O/N culture from the (2) tubes that have the correct insertions by adding 1 ml LB media to the tube, mixing it and transfer a W-tube containing 4 ml LB media.

gDNA purification from *Aspergillus*

*Adapted by Jacob Mejlsted*

#### Materials

##### Consumables

N is the number of samples.

* Ν 2 mL Fastprep tubes
* Ν 1.5 mL microcentrifuge/Eppendorf tube

##### Chemicals

* Lysis buffer **or** breaking buffer + LiAc
* Small glass beads
* 5 M NaCl
* Icecold 96% ethanol
* Milli-Q water

##### Instruments

* FastPrep machine
* Table centrifuge for 1.5 mL microcentrifuge/Eppendorf tubes.
* Heating block

#### Procedure

##### gDNA purification

1. Mix the following in a Fastprep tube: Scrape from plate colony and 500 µL lysis buffer **or** 500 µL breaking buffer + LiAc and 200 µL small glass beads.
2. Put in FastPrep machine at speed 4 for 40 seconds.
3. Spin down with a table centrifuge and transfer 150 µL of the supernantant to a new microcentrifuge tube.
4. Add 15 µL 5 M NaCl and 400 µL icecold 96% ethanol and mix
5. Spin 3 minutes at 10,000xg
6. Remove supernantant
7. Dry tubes on a heating block at 50 °C
8. Add 200 µL Milli-Q water and vortex
9. **Optional:** Spin down and transfer 150 µL to a new tube. (This can give a cleaner solution)

## Mycolab protocols

Growth of Fungi on Agar Plates

Basic protocol on plating of the fungi.

#### Materials

* Agar plates with media of interest
* Sterile toothpicks

#### Procedure

Plating using mycelia

1. Open the plate containing the mycelia from the species of interest
2. With a sterile toothpick, scratch the surface of the mycelia.
3. Spike the new agar plate with the toothpick.
4. Repeat steps 1-3 times. Three inoculation points should be made in the agar forming a triangle.
5. Put the plate into a plastic growth bag.
6. Place growing bag into the incubator.

Plating using spores

1. Touch the sterile tooth pick in the spore suspension.
2. Spike the new agar plate with the toothpick.
3. Repeat steps 1-3 times. Three inoculation points should be made in the agar forming a triangle.
4. Put the plate into a plastic growth bag.
5. Place growing bag into the incubator.

Media for Fungi

Minimal media used for protoplastation of *A. oryzae*. This is for 1 L of media.

#### Materials

##### Minimal media

* 50 mL D-glucose (20% w/V)
* 50 mL nitrate salts (20x)
* 1 mL trace elements (1000x)
* 1 mL thiamine (1%)
* 20 g agar (SO.BI.GEL, solid)

##### Transformation media

* 342,30 g sucrose (solid)
* 50 mL nitrate salts (20x)
* 1 mL trace elements (1000x)
* 1 mL thiamine (1%)
* 20 g agar (SO.BI.GEL, solid)

#### Procedure

1. Mix in a blue cap flask.
2. Add water until the volume is 1 L.
3. Autoclave.

Minimal Medium Supplemented

Minimal medium: *Schizophyllum commune* SMM agar (1L) needed for *S. commune* plates

#### Materials

* Glucose - 20g
* Monopotassium phosphate KH2PO4 - 0.46g
* Potassium phosphate dibasic K2HPO4 3H2O - 1.28g
* Magnesium sulfate MgSO4 7H2O - 0.5g
* Trace elements solution - 1mL
* FeCl3 solution - 1mL
* L- Asparagin - 1.5g
* Agar - 20g
* Thiamine (10mg/100mL) - 1.2mL

#### Procedure

Preparation of the media

1. Weigh and add the required solid components (except agar).
2. Add the required liquid components in a bottle.
3. Dissolve in demineralized water until the total volume is 900ml.
4. Stir the liquid using a magnet (no pH adjustment is required).
5. Add the required quantity of agar.
6. Add demineralized water until the desired total volume (1L).
7. Autoclave
8. After sterilization add 1.2 ml of filter sterilized thiamine.

Protoplastation and Transformation of *Aspergillus* spp.

Protocol provided by Fabiano Jares from DTU Bioengineering. The protocol is for species of *Aspergillus*. We used it for *Aspergillus oryzae*.

#### Materials

* *Aspergillus* protoplastation buffer (APB) 1L solution

+ Final conc: 1.1 M MgSO4 and 10 mM Na-phosphate buffer. (Hint: use 1 M solutions of Na2HPO4 and NaH2PO4 to prepare the sodium phosphate buffer). pH is adjusted with 2 N NaOH to 5.8.

* APB with Glucanex (40 mg Glucanex/ml APB)
* *Aspergillus* transformation buffer (ATB) 1 L solution

+ Final conc: 1.2 M Sorbitol; 50 mM CaCl2·2 H2O; 20 mM Tris; and 0.6 M KCl. pH is adjusted with 2 N HCl to 7.2.

* PCT (200 mL stock solution)

+ Final conc: 1.2 M Sorbitol; 50 mM CaCl2·2 H2O; 20 mM Tris; and 0.6 M KCl. pH is adjusted with 2 N HCl to 7.2.
+ Final conc: 50 % w/vol PEG 8000; 50 mM CaCl2; 20 mM Tris; and 0.6 M KCl. pH is adjusted with 2 N HCl to 7.5. Store PCT at 4 °C.

#### Procedure

Protoplastation

1. Collect the conidia from a plate by adding 5 mL of sterile liquid MM (or YPD for *A. niger*) with required supplements and firmly rubbing the colonies with a sterile Drigalski spatula. The conidial suspension is withdrawn from the plate and added to the shake flask containing 100 mL of media.
2. The culture is incubated over night (or 2 days for *A. niger* and other strains growing slower) at appropriate temperature and 150 rpm.
3. Harvest the mycelia and germlings by using Mira cloth.
4. Wash the mycelia with *Aspergillus* protoplastation buffer (APB) to remove the liquid media from the mycelia.
5. Resuspend the mycelium in 10-20 mL APB solution containing 40 mg Glucanex/mL APB. Homogenize mycelial and enzyme suspension gently to obtain the best possible digestion of the fungal cell wall.
6. Shake at the 30°C and 150 rpm for 2-3 hours.
7. Filter through Mira cloth and collect the flow through.
8. Add APB up to the total volume 40 mL.
9. Carefully make an overlay with 5 mL of 2 fold diluted *Aspergillus* transformation buffer (ATB). Dilute with sterile Milli-Q H2O.
10. Centrifuge at 3000xg (acceleration 9, deceleration 4) for 12 min.
11. Upon a successful protoplastation, a halo of white protoplast slurry is caught just below the surface. Collect protoplast slurry and transfer it to new tube.
12. Add ATB up to the total volume 40 mL.
13. Centrifuge at 3000xg (acceleration 9, deceleration 9) for 12 min. Discard supernatant.
14. Resuspend the protoplasts in approx. 1 mL ATB.

Transformation

15. Gently mix DNA and protoplast in an eppendorf tube (for a simple transformation 50μL protoplast is enough).
16. Add 150 μL PCT.
17. Mix by inversion of the tube.
18. Incubate 10 min at room temperature.
19. Add 250 μL ATB and mix.
20. Plate on osmotic stabilized, selective media.

#### Notes

1. The volume of DNA in water should preferably be kept below 25 % of the total volume of DNA-protoplast mix to avoid osmotic stress in protoplasts. The amount of DNA needed for a successful transformation varies with the type of DNA substrate.

* For linear DNA, add ≈10 μL linearized plasmid
* Use self-replicating plasmids as positive controls to test the competence of the protoplasts and evaluate the success of transformation. AMA1 plasmids (pLAT4-3): add ≈2 μL miniprep.
* Remember to include a negative control plate, where protoplasts are plated without any DNA.

Protoplastation of *Ganoderma*

Protocol for protoplastation of *Ganoderma resinaceum* provided by Ecovative Designs. This is the original protocol that we received from Ecovative. Modifications have been made every single time it has been used due to limitations in our own lab.

#### Materials

* Glucanex (Sigma L1412)
* Driselase (Sigma D9515)
* Potato Dextrose Agar (pre-mix) + 5g/L Bacteriological Agar
* Potato Dextrose Broth (pre-mix)
* Sterile Millipore Water
* Osmotic Buffer (500mL)

+ 74.04g MgSO4 (0.6M)
+ 1.05g MOPS (10mM)
+ Bring volume to 500mL with MilliQ water
+ Autoclave

* Sorbitol Solution (500mL)

+ 91.1g D-sorbitol (1M)
+ 1.05g MOPS (10mM)
+ Bring volume to 500mL with Millipore water
+ Autoclave

* Lysing Solution (10mL)

+ Add 100mg of Driselase to 5mL osmotic buffer in a 50mL centrifuge tube.
+ Allow to dissolve for 15-20min at room temperature with gentle swirling.
+ Add 100mg of Lysing Enzyme from *Trichoderma harzianum* (Glucanex) to 5mL osmotic buffer in a 50mL centrifuge tube.
+ Allow to dissolve for 15-20min at room temperature with gentle swirling.
+ Centrifuge the Driselase solution at 10,000xg for 1min to pellet the starch component.
+ Pour the supernatant into the tube with the Glucanex solution.
+ Attach a 30mm diameter 0.22um syringe filter to a 5mL syringe.
+ Fill the syringe with the lysing solution and filter into a 15mL centrifuge tube.
+ Pour the rest of the lysing solution into the syringe and filter.
+ Final, sterile lysing solution is 10mg/mL Driselase and 10mg/mL Glucanex in osmotic buffer.

#### Procedure

Tissue Generation

1. Inoculate potato dextrose agar plates with a single piece of tissue from a stock plate.
2. Parafilm and incubate at 30°C for 5 days.
3. Scrape tissue from the agar surface and place into 1mL of potato dextrose broth in a microcentrifuge tube.
4. Avoid bringing any agar into the tube.
5. Vortex the tubes at full speed for 30-60sec.
6. Place tubes in a tube rack and secure with tape.
7. Place the tube rack in a shaker incubator so that the tubes are completely horizontal.
8. Incubate at 30°C and 85rpm for 24hrs.

Enzymatic digest

9. Centrifuge tubes at 10,000xrcf for 5min.
10. Gently discard supernatant.
11. Re-suspend tissue pellet in 1mL sterile water.
12. Invert tube several times and vortex briefly. Do not vortex too long or mycelium will begin to shear.
13. Centrifuge tubes at 10,000xrcf for 5min.
14. Gently discard supernatant.
15. Re-suspend tissue pellet in 1mL osmotic buffer.
16. Invert tube several times and vortex briefly.
17. Centrifuge at 10,000xrcf for 5min.
18. Gently discard supernatant.
19. Re-suspend tissue pellet in 1mL osmotic buffer.
20. Invert tube several times and vortex briefly.
21. Centrifuge at 10,000xrcf for 5min.
22. Gently discard supernatant.
23. Re-suspend tissue pellet in 1mL lysing solution.
24. Invert tube several times, but do NOT vortex.
25. Place tubes in a tube rack and secure with tape.
26. Place the tube rack in a shaker incubator so that the tubes are completely horizontal.
27. Incubate at 30°C and 85rpm for 19-24hrs.

Isolation of Protoplasts

28. Invert tube several times and vortex to detach and protoplasts from the central cell mass.
29. Place a 40µm cell strainer in a new 50mL centrifuge tube.
30. Use a 1000µL pipette to further suspend the lysate. Pipette the lysate into the cell strainer and allow to drain fully.
31. Pipette 1mL of the sorbitol solution into the microcentrifuge tube used in the digestion and invert for 1min to rinse any additional protoplasts.
32. Pipette this volume through the same cell strainer and allow to drain fully. Cell strainer can be gently spun to remove more of the solution.
33. Repeat steps 3-5 for remaining protoplast preps (up to 5 additional 1mL preps).
34. Pour an additional 6-10mL of the sorbitol solution into the cell strainer and allow to fully drain.
35. Remove the cell strainer from the centrifuge tube.
36. Gently invert the tube 5-10 times to thoroughly mix the solutions.
37. Place the tube in a tube rack at 4 °C and incubate for at least 24hrs.
38. Centrifuge at 10,000xg for 10min.
39. Carefully pour the supernatant into a new 50mL centrifuge tube. Try to avoid disturbing the pellet along the side and bottom of the tube. The supernatant can be stored at 4 °C and more protoplasts may be able to be isolated at a later date, if needed.
40. Re-suspend the pellet into 1mL of sorbitol solution and transfer to a new 1.5mL microcentrifuge tube.
41. Prepare a 1:10 dilution in sorbitol solution and count protoplasts via hemocytometer.
42. Store protoplast suspension at 4 °C.

#### Modifications and notes

1. There were no Driselase in our lab, so we did the protocol with three different concentrations of Glucanex: 20 mg/mL, 30 mg/mL and 40 mg/mL for the lysing solution after consulting Ecovative.
2. Second time we tried combining the protoplastation protocol we already had for *A. oryzae*. We used different conditions in three places with all in all 16 samples: 8 of them were incubated with a shaking speed of 85 rpm and 8 at a speed of 150 rpm. Of these 8, 4 of them were incubated for 2-3 hours and the 4 others for 19-24 hours. Each of the four samples had a different concentration of Glucanex for the digestion: 10 mg/mL, 20 mg/mL, 30 mg/mL and 40 mg/mL.
    As there were no cell strainers in the lab, autoclaved funnels with mira cloth were used instead.
3. The third time we worked with 32 samples instead of 16. The conditions were still the same as the previous run but for an increased amount of mycelium we ran 2 two samples for each unique combination of conditions. Aside from this we also let the samples incubate for twice as long in the tissue generation part.
4. The fourth and last time we tried the protocol we had acquired Driselase and were, therefore, able to do the lysing solution according to the protocol. We still had no cell strainers, though, so funnels were still used. Aside from this 3 samples were made, all the same, and they were pooled together during the isolation of the protoplasts.

Posted with permission from Ecovative Designs

Spore Suspension

To obtain the spores generated from a plate culture in agar.

#### Materials

* Milli-Q water
* Mira-cloth
* Autoclaved Funnels
* Falcon tubes (50 mL)
* Drigalski spatula

#### Procedure

Spore suspension

1. Pour Milli-Q water into the fungal culture with plates.
2. Rub the plate with the Drigalski spatula. Make sure the spores get into solution. Spores are hydrophobic and can spread easily into the surroundings.
3. After spores are in suspension, if more plates with the same spores are to be harvested, pour water and spores to the next plate. Repeat steps 1-3 for as many plates you have.
4. Set a filter (funnel + mira cloth) on a Falcon tube.
5. Pour Milli-Q water + spores onto the mira cloth and filter solution. Make sure all liquid goes through the filter.
6. Do a second filtration on a new Falcon tube and filter. Make sure all liquid goes through.

Spore counting

1. Make 1:100 dilution with the spore suspension (only 10 µL needed).
2. With a counting chamber under the optical microscope, put 5 µL of the dilution into the center of the chamber. Count spores in one of the squared cells.
3. Calculate spore concentration.

## Building Experiments

Fungal brick protocol

The complete guide how to make and break fungal bricks for compression experiments

#### Materials

* Vortex
* PDA
* 50 ml falcon tubes
* Slicone ice-cube tray
* Spore suspension
* Substrate of interest
* Incubator
* Hydralic press

#### Procedure

##### Creating the bricks

1. Fill 20 microliter sporesuspension in falcon tube
2. Mix sporesuspension with 50ml PDA.
3. Transfer 10 ml PDA/spore mix to 30 ml sustrate mix
4. Vortex the substrate PDA/spore mix for 30 seconds
5. Transfer mixture to the silicon tray
6. Place tray in incubator for at least 1 week

##### Compression of bricks

1. Wrap trays in aluminum foil
2. Poke holes all around the foil
3. Place the wraped trays in an oven high temperature for atleast 12 hours
4. Remove trays and press each cube with a Hydralic press

##### Useful links

[Home](https://2018.igem.org/Team%3ADTU-Denmark)
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[Project description](https://2018.igem.org/Team%3ADTU-Denmark/Description)
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[Modeling](https://2018.igem.org/Team%3ADTU-Denmark/Model)
•
[Parts overview](https://2018.igem.org/Team%3ADTU-Denmark/Parts)
•
[Notebook](https://2018.igem.org/Team%3ADTU-Denmark/Notebook)
•
[Team](https://2018.igem.org/Team%3ADTU-Denmark/Team)
•
[Safety](https://2018.igem.org/Team%3ADTU-Denmark/Safety)
•
[Human practices](https://2018.igem.org/Team%3ADTU-Denmark/Human_Practices)

##### About us

* dtubiobuilders@gmail.com
* [Anker Engelunds Vej 1 Bygning 101A, 2800 Kgs. Lyngby, Denmark](https://www.google.dk/maps/%4055.7854419%2C12.5196676%2C16z)

Contact us

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# Team:DTU-Denmark/Results

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* [![](https://static.igem.org/mediawiki/2018/3/38/T--DTU-Denmark--menu-icon-project.png)
  Project](https://2018.igem.org/Team%3ADTU-Denmark/Description)

  + [Description](https://2018.igem.org/Team%3ADTU-Denmark/Description)
  + [Design](https://2018.igem.org/Team%3ADTU-Denmark/Design)
  + [InterLab](https://2018.igem.org/Team%3ADTU-Denmark/InterLab)
  + [Results](https://2018.igem.org/Team%3ADTU-Denmark/Results)
  + [Choice of Organism](https://2018.igem.org/Team%3ADTU-Denmark/Results-choosing-organism)
  + [amilCP Expression](https://2018.igem.org/Team%3ADTU-Denmark/Results-amilCP)
  + [melA Expression](https://2018.igem.org/Team%3ADTU-Denmark/Results-melA)
  + [nat1 Expression](https://2018.igem.org/Team%3ADTU-Denmark/Results-NAT-1)
  + [Ganoderma Protoplastation](https://2018.igem.org/Team%3ADTU-Denmark/Ganoderma-protoplastation)
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  Modeling](https://2018.igem.org/Team%3ADTU-Denmark/Model)

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  + [Modeling the Design](https://2018.igem.org/Team%3ADTU-Denmark/ModellingTheDesign)
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  Journal](https://2018.igem.org/Team%3ADTU-Denmark/Notebook)

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  People](https://2018.igem.org/Team%3ADTU-Denmark/Team)

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  Human Practices](https://2018.igem.org/Team%3ADTU-Denmark/Human_Practices)

  + [Human Practices](https://2018.igem.org/Team%3ADTU-Denmark/Human_Practices)
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* [![](https://static.igem.org/mediawiki/2018/4/4a/T--DTU-Denmark--menu-icon-collab.png)
  Collaboration](https://2018.igem.org/Team%3ADTU-Denmark/Collaborations)

  + [Collaborations](https://2018.igem.org/Team%3ADTU-Denmark/Collaborations)
  + [BioBrick Tutorial](https://2018.igem.org/Team%3ADTU-Denmark/BioBrickTutorial)
* [![](https://static.igem.org/mediawiki/2018/f/f5/T--DTU-Denmark--menu-icon-safety.png)
  Safety](https://2018.igem.org/Team%3ADTU-Denmark/Safety)

[Hyphae Hackers](https://2018.igem.org/Team%3ADTU-Denmark)

Project

[Description](https://2018.igem.org/Team%3ADTU-Denmark/Description)
[Design](https://2018.igem.org/Team%3ADTU-Denmark/Design)
[InterLab](https://2018.igem.org/Team%3ADTU-Denmark/InterLab)
[Results](https://2018.igem.org/Team%3ADTU-Denmark/Results)
[Choice of Organism](https://2018.igem.org/Team%3ADTU-Denmark/Results-choosing-organism)
[amilCP Expression](https://2018.igem.org/Team%3ADTU-Denmark/Results-amilCP)
[melA Expression](https://2018.igem.org/Team%3ADTU-Denmark/Results-melA)
[nat1 Expression](https://2018.igem.org/Team%3ADTU-Denmark/Results-NAT-1)
[Ganoderma Protoplastation](https://2018.igem.org/Team%3ADTU-Denmark/Ganoderma-protoplastation)
[Demonstrate](https://2018.igem.org/Team%3ADTU-Denmark/Demonstrate)

Modeling

[Model](https://2018.igem.org/Team%3ADTU-Denmark/Model)
[Growth Modeling](https://2018.igem.org/Team%3ADTU-Denmark/GrowthModelling)
[Design of Experiments](https://2018.igem.org/Team%3ADTU-Denmark/DesignOfExperiments)
[Modeling the Design](https://2018.igem.org/Team%3ADTU-Denmark/ModellingTheDesign)
[Structural Integrity](https://2018.igem.org/Team%3ADTU-Denmark/StructuralIntegrity)
[Hardware](https://2018.igem.org/Team%3ADTU-Denmark/Hardware)

[Parts Overview](https://2018.igem.org/Team%3ADTU-Denmark/Parts)
Journal

[Notebook](https://2018.igem.org/Team%3ADTU-Denmark/Notebook)
[Experiments](https://2018.igem.org/Team%3ADTU-Denmark/Experiments)

People

[Team Members](https://2018.igem.org/Team%3ADTU-Denmark/Team)
[Attributions](https://2018.igem.org/Team%3ADTU-Denmark/Attributions)

[Safety](https://2018.igem.org/Team%3ADTU-Denmark/Safety)
Human Practices

[Human Practices](https://2018.igem.org/Team%3ADTU-Denmark/Human_Practices)
[Education & Engagement](https://2018.igem.org/Team%3ADTU-Denmark/Public_Engagement)

Collaborations

[Collaborations](https://2018.igem.org/Team%3ADTU-Denmark/Collaborations)
[BioBrick Tutorial](https://2018.igem.org/Team%3ADTU-Denmark/BioBrickTutorial)

![](https://static.igem.org/mediawiki/2018/d/d5/T--DTU-Denmark--topperbasicb.png)

# Results

# What we accomplished

* Assembled and transformed a cassette for expression of *amilCP* in *A. oryzae** Assembled constructs for expression of *melA* in *A. oryzae** Demonstrated *nat1* as a selectable marker in *A. oryzae** Thoroughly characterised and modeled the properties of our manufactured material
      * Simulated the stability of a structure created from fungal mycelium
      * Create an optimal design of experiments for fungal bricks testing
      * Modeling the design of experiment results, coupling compressive strength and process factors

[## Choice of organism

Many fungal species were considered to achieve our goals of creating biobricks with tunable properties. To familiarize ourselves with cultivation of fungal cultures and to investigate what species were optimal for our research, *Pleurotus ostreatus*, *Aspergillus oryzae* and *Schizophyllum commune* were tested. The species were grown on Malt Extract Agar (MEA), Potato Dextrose Agar (PDA) or Yeast-extract-Peptone-Dextrose agar (YPD) at different concentrations. In addition, sporulation was assessed for each of the species, although it was only successful for *A. oryzae*.

Key achievements:

* Experimental determination of the best fungal species to generate a toolbox to form brick structures.
* Successfully set the growth conditions for *A. oryzae* needed for the formation of spores.
* Familiarised ourselves with cultivation of fungi.](https://2018.igem.org/Team%3ADTU-Denmark/Results-choosing-organism)
[## *amilCP* expression

One aim of our projects was to produce colored bricks. To achieve this we assembled constructs for expression of AmilCP, a blue chromoprotein in *A. oryzae*.

Key achievements:

* Assembled and submitted multiple constructs for expression of *amilCP* in fungi
* Demonstrated integration of the construct into the genome of *A. oryzae*
* Produced evidence indicating change in spore coloration by expression of *amilCP*](https://2018.igem.org/Team%3ADTU-Denmark/Results-amilCP)
[## *melA* expression

In order to provide our fungal materials with increased resistance to UV radiation we aimed to engineer *A. oryzae* to overexpress melanin. To achieve this we constructed an expression cassette containing *melA*, which encodes a tyrosinase utilized in the first step of the biosynthetic pathway of melanin.

Key achievements:

* Assembled and submitted bricks containing constructs for expression of *melA*.](https://2018.igem.org/Team%3ADTU-Denmark/Results-melA)
[## *nat1* selectable marker

In order to generate a genetic toolbox for this host organism there was a need for a transformant selection. However, *A. oryzae* has proved to be resistant to the most commonly used antimycotics, geneticin (G418) and hygromycin-B. In order to establish a selection for our plasmids, we tested a less common antibiotic called Nourseothricin (NTC).

Key achievements:

* Transformed pDIV079 into *A. oryzae* and demonstrated selection via NTC.](https://2018.igem.org/Team%3ADTU-Denmark/Results-NAT-1)
[## Ganoderma protoplastation

Protoplast-mediated transformation is a widely used transformation method for fungi. Here, we attempted to generate protoplasts for the species *Ganoderma resinaceum*. Benefiting from Ecovative Design, who shared their protoplastation protocol with us, we aimed to improve their protocol by making it more affordable and efficient. For that, we avoided using Driselase, a costly lysis enzyme and tried several different concentrations of Glucanex. Although with the variations in the protocol that we tried we did not have any successful protoplasts, we pathed the way to future improvement in this area.

Key achievements:

* Testing of a wide range of Glucanex concentrations.
* Testing of different incubation times that would make the protoplast protocol more efficient.](https://2018.igem.org/Team%3ADTU-Denmark/Ganoderma-protoplastation)
[## Design of Experiments

When testing the many factors involved in the processing of the fungal bricks several combinations are involved. The pilot design involved testing 8 substates, 2 burning temperatures, 2 burning times and 2 incubation times. From a full factorial design this would lead to 216 combinations with no replicates. Applying the optimal design of the fedorov exchange algorithm which utilizes the A-criteria, D-criteria and G-criteria, the design was reduced into 64 trials.
From the optimal substrates which produced the highest compressive strength bricks, the replicated design was created. This involved the testing of substates, 2 burning temperatures, 2 burning times, 2 incubation times and 3 mixing ratios. This would lead to 164 combinations replicated 4 times. The same algorithm was applied and the design was reduced into 17 trials replicated 4 times.

Key achievements:

* Successful reduction of pilot design from 216 trials to 64 trials.
* Successful reduction of replicated design from 164 trials with 4 replicated to 17 trials with 4 replicates.](https://2018.igem.org/Team%3ADTU-Denmark/DesignOfExperiments)
[## Modelling the design

With the data obtained in the pilot study a fixed main effect model was created for the purpose of screening the optimal substrates. From this model post-hoc analysis was conducted and it was identified that the optimal substrates candidates for further testing was blended jasmine rice and a mixture of jasmine rice and sawdust.
From the replicated design the relation between process factors and compressive strength was modeled. It was identified a gamma mixed effect model was the best choice involving the significant factors of incubation time, substrate and burning temperature. It was revealed that the optimal level of the process factors was low incubation time (1 week), substrate 1 (blended jasmine rice) and high burning temperature of 100° celsius.

Key achievements:

* Successful identifying the optimal substrate candidates via screening modelling
* Successful coupling process factors to gamma mixed effect model
* Successfully identifying the optimal levels of substrate, incubation time and burning temperature](https://2018.igem.org/Team%3ADTU-Denmark/ModellingTheDesign)
[## Simulation of structure

We used the data from our material tests and COMSOL to simulate the stresses on our structure on Mars. This simulation took into account the stress from gravity and the stress from pressure difference between the inside and the outside of the structures. We wanted to study the structural integrity of the material and not the junctures between the individual bricks. Therefore, we modelled it as one cohesive element. Since we saw large variability in the properties during our measurements, it makes sense to make a simple model. We chose the half sphere, which we have shown can let us use approximately ⅓ the material. For the sphere we have shown that a wall thickness of approximately 0.34 m is enough to ensure structural integrity. Thereby, we have also shown that choosing *Aspergillus oryzae* is strong enough for our use case. This gives us grounds for choosing this for being a model organism over for instance *Ganoderma resinaceum* which is used by Ecovative.

Key achievements:

* A 0.34 m thick *A. oryzae* dome is structurally sound
* The lowest livable pressure for humans results in a needed thickness of ½
* The reduction of stress with increased thickness decreases with added thickness](https://2018.igem.org/Team%3ADTU-Denmark/StructuralIntegrity)

##### Useful links

[Home](https://2018.igem.org/Team%3ADTU-Denmark)
•
[Project description](https://2018.igem.org/Team%3ADTU-Denmark/Description)
•
[Modeling](https://2018.igem.org/Team%3ADTU-Denmark/Model)
•
[Parts overview](https://2018.igem.org/Team%3ADTU-Denmark/Parts)
•
[Notebook](https://2018.igem.org/Team%3ADTU-Denmark/Notebook)
•
[Team](https://2018.igem.org/Team%3ADTU-Denmark/Team)
•
[Safety](https://2018.igem.org/Team%3ADTU-Denmark/Safety)
•
[Human practices](https://2018.igem.org/Team%3ADTU-Denmark/Human_Practices)

##### About us

* dtubiobuilders@gmail.com
* [Anker Engelunds Vej 1 Bygning 101A, 2800 Kgs. Lyngby, Denmark](https://www.google.dk/maps/%4055.7854419%2C12.5196676%2C16z)

Contact us