iGEM 2019 — Team DTU-Denmark

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:DTU-Denmark/Notebook

[![logo](https://static.igem.org/mediawiki/2019/f/fe/T--DTU-Denmark--happylogotemp.png)](https://2019.igem.org/Team%3ADTU-Denmark)

* [![Awards](https://static.igem.org/mediawiki/2019/c/c0/T--DTU-Denmark--medallogo.svg)](https://2019.igem.org/Team%3ADTU-Denmark/Awards)
* [Project](https://2019.igem.org/Team%3ADTU-Denmark/Description)

  [Description](https://2019.igem.org/Team%3ADTU-Denmark/Description)
  [Design](https://2019.igem.org/Team%3ADTU-Denmark/Design)
  [Promoter Design](https://2019.igem.org/Team%3ADTU-Denmark/Design_Promoter)
  [Vector Design](https://2019.igem.org/Team%3ADTU-Denmark/Design_Plasmid)
  [Measurement Design](https://2019.igem.org/Team%3ADTU-Denmark/Design_Measurement)
  [Results](https://2019.igem.org/Team%3ADTU-Denmark/Results)
  [Demonstrate](https://2019.igem.org/Team%3ADTU-Denmark/Demonstrate)
  [Safety](https://2019.igem.org/Team%3ADTU-Denmark/Safety)
  [Characterization](https://2019.igem.org/Team%3ADTU-Denmark/Contribution)
* [Modelling](https://2019.igem.org/Team%3ADTU-Denmark/Model)

  [Model](https://2019.igem.org/Team%3ADTU-Denmark/Model)
  [Software](https://2019.igem.org/Team%3ADTU-Denmark/Software)
* [Parts](https://2019.igem.org/Team%3ADTU-Denmark/Parts)

  [Parts Overview](https://2019.igem.org/Team%3ADTU-Denmark/Parts)
  [Basic Parts](https://2019.igem.org/Team%3ADTU-Denmark/Basic_Part)
  [Composite Parts](https://2019.igem.org/Team%3ADTU-Denmark/Composite_Part)
  [Part Collection](https://2019.igem.org/Team%3ADTU-Denmark/Part_Collection)
* [Journal](https://2019.igem.org/Team%3ADTU-Denmark/Notebook)

  [Notebook](https://2019.igem.org/Team%3ADTU-Denmark/Notebook)
  [Experiments](https://2019.igem.org/Team%3ADTU-Denmark/Experiments)
* [People](https://2019.igem.org/Team%3ADTU-Denmark/Team)

  [Team Members](https://2019.igem.org/Team%3ADTU-Denmark/Team)
  [Collaboration](https://2019.igem.org/Team%3ADTU-Denmark/Collaborations)
  [Attributions](https://2019.igem.org/Team%3ADTU-Denmark/Attributions)
* [Human Practices](https://2019.igem.org/Team%3ADTU-Denmark/Integrated_Human_Practices)

  [Integrated Human Practices](https://2019.igem.org/Team%3ADTU-Denmark/Integrated_Human_Practices)
  [Human Practices](https://2019.igem.org/Team%3ADTU-Denmark/Human_Practices)
  [Education and Engagement](https://2019.igem.org/Team%3ADTU-Denmark/Public_Engagement)
  [Biobrick Tutorial](https://2019.igem.org/Team%3ADTU-Denmark/BioBrick_Tutorial)

[LEAP](https://2019.igem.org/Team%3ADTU-Denmark)

Project

[Description](https://2019.igem.org/Team%3ADTU-Denmark/Description)
[Design](https://2019.igem.org/Team%3ADTU-Denmark/Design)
[Promoter Design](https://2019.igem.org/Team%3ADTU-Denmark/Design_Promoter)
[Vector Design](https://2019.igem.org/Team%3ADTU-Denmark/Design_Plasmid)
[Measurement Design](https://2019.igem.org/Team%3ADTU-Denmark/Design_Measurement)
[Results](https://2019.igem.org/Team%3ADTU-Denmark/Results)
[Demonstrate](https://2019.igem.org/Team%3ADTU-Denmark/Demonstrate)
[Characterization](https://2019.igem.org/Team%3ADTU-Denmark/Contribution)

Modelling

[Model](https://2019.igem.org/Team%3ADTU-Denmark/Model)
[Software](https://2019.igem.org/Team%3ADTU-Denmark/Software)

Parts

[Parts](https://2019.igem.org/Team%3ADTU-Denmark/Parts)
[Basic\_Part](https://2019.igem.org/Team%3ADTU-Denmark/Basic_Part)
[Composite\_Part](https://2019.igem.org/Team%3ADTU-Denmark/Composite_Part)
[Part\_Collection](https://2019.igem.org/Team%3ADTU-Denmark/Part_Collection)

Journal

[Notebook](https://2019.igem.org/Team%3ADTU-Denmark/Notebook)
[Experiments](https://2019.igem.org/Team%3ADTU-Denmark/Experiments)

People

[Team Members](https://2019.igem.org/Team%3ADTU-Denmark/Team)
[Collaborations](https://2019.igem.org/Team%3ADTU-Denmark/Collaborations)
[Attributions](https://2019.igem.org/Team%3ADTU-Denmark/Attributions)

[Safety](https://2019.igem.org/Team%3ADTU-Denmark/Safety)
Human Practices

[Integrated Human Practices](https://2019.igem.org/Team%3ADTU-Denmark/Integrated_Human_Practices)
[Human Practices](https://2019.igem.org/Team%3ADTU-Denmark/Human_Practices)
[Education & Engagement](https://2019.igem.org/Team%3ADTU-Denmark/Public_Engagement)
[BioBrick Tutorial](https://2019.igem.org/Team%3ADTU-Denmark/BioBrick_Tutorial)

![Figure showing a fungus surrounded by pages from a calendar with dates on them](https://static.igem.org/mediawiki/2019/a/ae/T--DTU-Denmark--notebookheader1.svg)

## Notebook

This page provides an overview of the the different stages of the project as well as what we did and when.

### Choose a week

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### Week 7 (Feb 11 - Feb 17)

* ![](https://static.igem.org/mediawiki/2019/7/79/T--DTU-Denmark--notebooko.png)

  #### Team Overview

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

### Week 8 (Feb 18 - Feb 24)

* ![](https://static.igem.org/mediawiki/2019/7/79/T--DTU-Denmark--notebooko.png)

  #### Team Overview

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

### Week 10 (Mar 4 - Mar 10)

* ![](https://static.igem.org/mediawiki/2019/7/79/T--DTU-Denmark--notebooko.png)

  #### Team Overview

  We started the preparations for the annual BioBrick Tutorial and attend the annual DTU Open House where high school students can come and hear about the different fields of study and opportunities at DTU.

  ![At the DTU Open House](https://static.igem.org/mediawiki/2019/8/82/T--DTU-Denmark--BB_openhouse.jpg)

### Week 13 (Mar 25 - Mar 31)

* ![](https://static.igem.org/mediawiki/2019/7/79/T--DTU-Denmark--notebooko.png)

  #### Team Overview

  We made dinner together and played board games.

  ![The team is seated around a table with food, champaign and ice cream.](https://static.igem.org/mediawiki/2019/6/63/T--DTU-Denmark--DinnerPhilip.jpg)

### Week 14 (Apr 1 - Apr 7)

* ![](https://static.igem.org/mediawiki/2019/7/79/T--DTU-Denmark--notebooko.png)

  #### Team Overview

  We attended a talk about bioethics in genetically modified insects to be better prepared for working with GMO and sharing our work with the general public.

  ![Mickey Gjerris is standing in front of the DTU logo and a giant, colorful cricket](https://static.igem.org/mediawiki/2019/0/0a/T--DTU-Denmark--lbtb2.jpg)

### Week 15 (Apr 8 - Apr 14)

* ![](https://static.igem.org/mediawiki/2019/7/79/T--DTU-Denmark--notebooko.png)

  #### Team Overview

  The annual BioBrick Tutorial took place with 69 guests from Sweden, Denmark, Norway, and Finland. The meetup offered both an introduction to lab work and to iGEM in general.

  ![All of the participants at the BioBrick Tutorial posing on a grass field in front of the fountain at D.T.U.](https://static.igem.org/mediawiki/2019/1/1d/T--DTU-Denmark--bbgroup.jpg)

### Week 17 (Apr 22 - Apr 28)

* ![](https://static.igem.org/mediawiki/2019/7/79/T--DTU-Denmark--notebooko.png)

  #### Team Overview

  The things needed for the Science EXPO were prepared.
  We attempted an escape room with a laboratory theme and excelled in solving the different puzzles and finished with 29 minutes and 16 seconds to spare - one of the fastest teams ever!

  ![The team are posing, holding a blackbord stating that they completet the escape room mystery with 29 minutes and 16 seconds to spare.](https://static.igem.org/mediawiki/2019/d/d7/T--DTU-Denmark--lbtb3.jpg)

### Week 18 (Apr 29 - May 5)

* ![](https://static.igem.org/mediawiki/2019/7/79/T--DTU-Denmark--notebooko.png)

  #### Team Overview

  We attended the annual Science EXPO competition for primary school and high school students to spread the word about iGEM and talk about our project.

  Students were given the opportunity to come and try pipetting droplets of water with food coloring onto paper.

  ![Two petri dishes with green and blue water, ready to be pipeted.](https://static.igem.org/mediawiki/2019/8/88/T--DTU-Denmark--lbscienceexpo.jpg)

### Week 19 (May 6 - May 12)

* ![](https://static.igem.org/mediawiki/2019/7/79/T--DTU-Denmark--notebooko.png)

  #### Team Overview

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

  ![](https://static.igem.org/mediawiki/2019/f/f2/T--DTU-Denmark--lbg1.jpg)
* ![](https://static.igem.org/mediawiki/2019/6/67/T--DTU-Denmark--notebookdl.png)

  #### Drylab

  First meeting of the drylab group with advisor Chris.
  We discussed gene expression and the true meaning of “constitutive” (regulation is always present in some way). Moreover, the first goals were decided: looking for relevant datasets and finding literature.

### Week 20 (May 13 - May 19)

* ![](https://static.igem.org/mediawiki/2019/7/79/T--DTU-Denmark--notebooko.png)

  #### Team Overview

  We attended the Nordic iGEM conference (NiC) hosted by the [UCopenhagen](https://2019.igem.org/Team%3AUCopenhagen) team. Here, we presented the project and a poster to the other Nordic teams.

  ![The team is posing in front of their poster from the NiC](https://static.igem.org/mediawiki/2019/a/a5/T--DTU-Denmark--lbg2.jpg)

### Week 21 (May 20 - May 26)

* ![](https://static.igem.org/mediawiki/2019/8/89/T--DTU-Denmark--notebookwl.png)

  #### Wetlab

  The first days in the lab were spent creating competent cells and the plasmids needed for the experiments.
* ![](https://static.igem.org/mediawiki/2019/6/67/T--DTU-Denmark--notebookdl.png)

  #### Drylab

  The search for relevant literature continues. The
  group members started searching for information on the inducibility and leakiness of the promoters. First datasets to start our tests were found.

### Week 22 (May 27 - Jun 2)

* ![](https://static.igem.org/mediawiki/2019/7/79/T--DTU-Denmark--notebooko.png)

  #### Team Overview

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

  ![The team is seated outside, eating salad, sausages, and garlic bread.](https://static.igem.org/mediawiki/2019/e/e5/T--DTU-Denmark--lbg3.jpg)
* ![](https://static.igem.org/mediawiki/2019/6/67/T--DTU-Denmark--notebookdl.png)

  #### Drylab

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

### Week 23 (Jun 3 - Jun 9)

* ![](https://static.igem.org/mediawiki/2019/8/89/T--DTU-Denmark--notebookwl.png)

  #### Wetlab

  Media and solutions for making protoplasts was prepared.
* ![](https://static.igem.org/mediawiki/2019/6/67/T--DTU-Denmark--notebookdl.png)

  #### Drylab

  We discussed the interpretation of the found data and how to handle data noise in datasets. Our supervisor Chris suggested many bioinformatics tools that would be useful in the journey forward. The group members started learning how to make these tools work.

### Week 24 (Jun 10 - Jun 16)

* ![](https://static.igem.org/mediawiki/2019/7/79/T--DTU-Denmark--notebooko.png)

  #### Team Overview

  We went to The Hague to attend the iGEM meetup for teams and supervisors hosted by Netherlands National Institute for Public Health and the Environment. Here, we participated in workshops regarding i.a. safety by design and learned about storytelling and human practices. We also made a lot of contacts and collaboration agreements with other teams.

  ![The team is standing on the stairs in front of the RIVM in The Hague](https://static.igem.org/mediawiki/2019/0/08/T--DTU-Denmark--lbg4.jpg)
* ![](https://static.igem.org/mediawiki/2019/6/67/T--DTU-Denmark--notebookdl.png)

  #### Drylab

  At this point, we made a more specific plan of action.
  Looking at how to identify the best candidate genes based on the expression level in RNA-Seq data.
  Several *Aspergillus* genomes were obtained and we tried to generate the first promoter. The results were analyzed. Stuff makes sense!!!

### Week 25 (Jun 17 - Jun 23)

* ![](https://static.igem.org/mediawiki/2019/7/79/T--DTU-Denmark--notebooko.png)

  #### Team Overview

  We visited the Novozymes site in Lyngby, got a tour at the site, and learned a lot about what the industry is looking for in a promoter library!

  ![The team are situated around two tables with two different kinds of week-old bread and are to tell the difference]( https://static.igem.org/mediawiki/2019/8/89/T--DTU-Denmark--NovozymesBread.jpg "The team tried the difference between bread that was baked with and without baking enzymes")
* ![](https://static.igem.org/mediawiki/2019/8/89/T--DTU-Denmark--notebookwl.png)

  #### Wetlab

  The first attempt at making protoplasts of *A. niger*. This sadly failed. We talked to some professors who gave us advice on what to improve on the protocol. Furthermore, the first four fragments that will make up the initial integration plasmid were PCR amplified.
* ![](https://static.igem.org/mediawiki/2019/6/67/T--DTU-Denmark--notebookdl.png)

  #### Drylab

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

### Week 26 (Jun 24 - Jun 30)

* ![](https://static.igem.org/mediawiki/2019/8/89/T--DTU-Denmark--notebookwl.png)

  #### Wetlab

  The PCR fragments were assembled into the first whole plasmid of our project! Following this, work began on inserting a GFP marker into the basic plasmid.
* ![](https://static.igem.org/mediawiki/2019/6/67/T--DTU-Denmark--notebookdl.png)

  #### Drylab

  At this point, we made a more specific plan of action. The first goal was to identify the best candidate genes based on the expression level.

### Week 27 (Jul 1 - Jul 7)

* ![](https://static.igem.org/mediawiki/2019/7/79/T--DTU-Denmark--notebooko.png)

  #### Team Overview

  We talked to Peter Richard from the VTT Technical Research Centre of Finland. He gave us excellent feedback on our project - we will definitely take this into consideration!
* ![](https://static.igem.org/mediawiki/2019/8/89/T--DTU-Denmark--notebookwl.png)

  #### Wetlab

  The second attempt at making protoplasts of *A. niger*, this time with an improved protocol. This time, the attempt succeeded and we successfully made protoplasts and transformants, even following two different protocols for transformation!
  Furthermore, in preparation for a Gibson assembly, we amplified several fragments by PCR and on Friday they were assembled into one.

  ![Four agar plates can be seen. On the left are two negative controls with no growth and on the right are two positive controls with growth on them.](https://static.igem.org/mediawiki/2019/8/82/T--DTU-Denmark--lbg5.jpg)
* ![](https://static.igem.org/mediawiki/2019/6/67/T--DTU-Denmark--notebookdl.png)

  #### Drylab

  Gene candidates were picked to run in our program.

### Week 28 (Jul 8 - Jul 14)

* ![](https://static.igem.org/mediawiki/2019/8/89/T--DTU-Denmark--notebookwl.png)

  #### Wetlab

  This week, we confirmed that the plasmids had been assembled correctly and we linearised them to be able to transform them into our fungi.
* ![](https://static.igem.org/mediawiki/2019/6/67/T--DTU-Denmark--notebookdl.png)

  #### Drylab

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

### Week 29 (Jul 15 - Jul 21)

* ![](https://static.igem.org/mediawiki/2019/8/89/T--DTU-Denmark--notebookwl.png)

  #### Wetlab

  Fragments were prepared to be assembled into plasmids.

### Week 30 (Jul 22 - Jul 28)

* ![](https://static.igem.org/mediawiki/2019/7/79/T--DTU-Denmark--notebooko.png)

  #### Team Overview

  This weekend, we participated in the Danish iGEM meetup hosted by the SDU team on the south of Funen. We had so much fun and got to discuss how to make our wikis, how to be better at presenting, and we discussed starting a national Danish iGEM community. Thank you so much to SDU iGEM for hosting this great event!

  ![The BioBuilders team posing with the UCopenhagen and the SDU teams]( https://static.igem.org/mediawiki/2019/0/06/T--DTU-Denmark--collabdanishmeet.svg "Posing with UCopenhagen and SDU")
* ![](https://static.igem.org/mediawiki/2019/8/89/T--DTU-Denmark--notebookwl.png)

  #### Wetlab

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

### Week 31 (Jul 29 - Aug 4)

* ![](https://static.igem.org/mediawiki/2019/8/89/T--DTU-Denmark--notebookwl.png)

  #### Wetlab

  We Gibson assembled the plasmid pGIA2P2e which is the foundation for our testing calibration vector. The plasmid was sent for sequencing and PCR verified. From this, we saw that we had successfully assembled the plasmid. Concurrently, assembly of the plasmid pGIA2P2o began but this turned out to be problematic.

  We additionally began to prepare the fragments required for Golden Gate assembly of the next layer of plasmids.

### Week 32 (Aug 5 - Aug 11)

* ![](https://static.igem.org/mediawiki/2019/8/89/T--DTU-Denmark--notebookwl.png)

  #### Wetlab

  The pGIA2P2e plasmid was linearized and prepared for fungal transformation.

  We also received a kanamycin backbone from the [SDU-Denmark](https://2019.igem.org/Team%3ASDU-Denmark) team. This means that we can now continue the assembly process for the pGIA2P2o plasmid. However, the assembled plasmid turned out not to work so we retraced our steps and tried again. While this was going on, we Golden Gate assembled the plasmid pBB006 which we will use in the future for fungal transformation.

### Week 33 (Aug 12 - Aug 18)

* ![](https://static.igem.org/mediawiki/2019/7/79/T--DTU-Denmark--notebooko.png)

  #### Team Overview

  We visited Novozymes for the second time to follow up on some questions.
* ![Marcus, Helena, Jacob, and Louise pose in front of the Novozymes Lyngby building]( https://static.igem.org/mediawiki/2019/2/20/T--DTU-Denmark--NovozymesFront.jpg "The team visited the Novozymes facilities in Lyngby once more")

  ![](https://static.igem.org/mediawiki/2019/8/89/T--DTU-Denmark--notebookwl.png)

  #### Wetlab

  We realized that the second attempt at Gibson assembling the pGIA2P2o plasmid had failed and tried a third time - which also failed :'(.
  On a more positive note, pBB006 was linearised. A fungal promoter was inserted and the plasmid was then linearised successfully!

  Our synthetic promoters arrived and we were all very happy!

### Week 34 (Aug 19 - Aug 25)

* ![](https://static.igem.org/mediawiki/2019/7/79/T--DTU-Denmark--notebooko.png)

  #### Team Overview

  We had dinner together to take a small break from all of the lab work and celebrate how far we'd come.
* ![The team is sitting and eating Tex Mex food]( https://static.igem.org/mediawiki/2019/f/fd/T--DTU-Denmark--NovozymesDinner.jpg "Following a long day in the lab, the team had dinner together to relax a bit")

  ![](https://static.igem.org/mediawiki/2019/8/89/T--DTU-Denmark--notebookwl.png)

  #### Wetlab

  The promoters arrived so we worked like Trojans to get ready to run the Biolector next week. During this, we inserted the promoters into our plasmid backbones using Golden Gate, inoculated liquid cultures, and miniprepped the 40 promoters in triplicate, totaling 120 plasmid samples!

### Week 35 (Aug 26 - Sep 1)

* ![](https://static.igem.org/mediawiki/2019/8/89/T--DTU-Denmark--notebookwl.png)

  #### Wetlab

  Despite setbacks by the fungi's lack of will to grow, we were able to run the first batch in the biolector and got some nice results!

### Week 36 (Sep 2 - Sep 8)

* ![](https://static.igem.org/mediawiki/2019/7/79/T--DTU-Denmark--notebooko.png)

  #### Team Overview

  The semester started again so we were busy following our classes. But there was still time to go to the lab.
* ![](https://static.igem.org/mediawiki/2019/8/89/T--DTU-Denmark--notebookwl.png)

  #### Wetlab

  We tried out our new Opentrons robot and got it to prepare a PCR. The official name of that robot is now Robert.
[
 Your browser does not support the video tag.
](https://static.igem.org/mediawiki/2019/e/e7/T--DTU-Denmark--Opentron1.mp4)

### Week 39 (Sep 23 - Sep 29)

* ![](https://static.igem.org/mediawiki/2019/7/79/T--DTU-Denmark--notebooko.png)

  #### Team Overview

  On Tuesday, we visited Bagsværd Kostskole og Gymnasium to talk about synthetic biology and our iGEM project.
  On Sunday, we gathered and worked on the wiki. We got a lot of stuff done, including, but not limited to; checking the safety page, documenting our results until now, and describing our collaborations with other teams.

  ![Helena and Louise presenting our project at Bagsværd Boarding School and High School](https://static.igem.org/mediawiki/2019/f/f5/T--DTU-Denmark--BagSkol.jpg "Presenting our project at Bagsværd High School")

  ![The team sits by a long table and work on the wiki writing ]( https://static.igem.org/mediawiki/2019/c/c1/T--DTU-Denmark--wikiWriteDay1.jpg "The team is hard at work on the wiki – but also having fun!")

### Week 40 (Sep 30 - Oct 6)

* ![](https://static.igem.org/mediawiki/2019/8/89/T--DTU-Denmark--notebookwl.png)

  #### Wetlab

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

  ![Ready to start shake flask experiment](https://static.igem.org/mediawiki/2019/2/2b/T--DTU-Denmark--ShakeFlasks.jpg "Ready to begin the shake flask experiment")
* ![](https://static.igem.org/mediawiki/2019/6/67/T--DTU-Denmark--notebookdl.png)

  #### Drylab

  The drylab team started to clean up the software so other people could use it and began the analysis of data from the Biolector.

### Week 41 (Oct 7 - Oct 13)

* ![](https://static.igem.org/mediawiki/2019/7/79/T--DTU-Denmark--notebooko.png)

  #### Team Overview

  On Friday, we visited Birkerød High School to talk about the project and synthetic biology. We had a blast and the students were incredibly interested in our presentation!

  ![Marcus is standing in front of a PowerPoing talking about D.N.A. and restriction enzymes.]( https://static.igem.org/mediawiki/2019/f/fb/T--DTU-Denmark--BirkSkol.jpg "Jacob and Marcus presenting our project at Birkerød Gymnasium")
* ![](https://static.igem.org/mediawiki/2019/8/89/T--DTU-Denmark--notebookwl.png)

  #### Wetlab

  While still running the shake flasks and Biolector, we started the bioreactors in order to test the scalability of our synthetic promoters. Additionally, we examined the transformed fungi with our promoters under a confocal microscope, seeing that both the conidia and hyphae were red, proving that our promoters had worked as intended!

  ![Science Expo](https://static.igem.org/mediawiki/2019/7/71/T--DTU-Denmark--fermentors.jpg "A workplace of fermentors and wires, lots and lots of wires.")

  ![Fungal spores with RFP](https://static.igem.org/mediawiki/2019/0/06/T--DTU-Denmark--NotebookConfocal.png "RFP producing fungal spores isolated from minimal media plate")
* ![](https://static.igem.org/mediawiki/2019/6/67/T--DTU-Denmark--notebookdl.png)

  #### Drylab

  A guide for the software and for how to find data for the software was developed. This was sent to the Brown-Stanford-Princeton team for them to test and evaluate the software.

### Week 42 (Oct 14 - Oct 20)

* ![](https://static.igem.org/mediawiki/2019/7/79/T--DTU-Denmark--notebooko.png)

  #### Team Overview

  We spent the entire week writing the wiki and making it perfect :D
* ![](https://static.igem.org/mediawiki/2019/8/89/T--DTU-Denmark--notebookwl.png)

  #### Wetlab

  The samples taken from the shake flasks and the bioreactor were analyzed in multiple ways. The biomass was separated and its proteins purified so we could determine the amount of mCherry produced by the different promoters. Additionally, the samples were also tested for the amount of remaining glucose to indicate how far they were in their growth phases at the different sampling times.
* ![](https://static.igem.org/mediawiki/2019/6/67/T--DTU-Denmark--notebookdl.png)

  #### Drylab

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

### Week 43 (Oct 21 - Oct 27)

* ![](https://static.igem.org/mediawiki/2019/7/79/T--DTU-Denmark--notebooko.png)

  #### Team Overview

  We're almost there and we've worked so very hard on our wiki. Wiki freeze is here and Boston is on the horizon!

[![](https://static.igem.org/mediawiki/2019/f/fe/T--DTU-Denmark--happylogotemp.png)](https://2019.igem.org/Team%3ADTU-Denmark)

##### Useful links

[Home](https://2019.igem.org/Team%3ADTU-Denmark)
·
[Project description](https://2019.igem.org/Team%3ADTU-Denmark/Description)
·
[Modelling](https://2019.igem.org/Team%3ADTU-Denmark/Model)
·
[Parts overview](https://2019.igem.org/Team%3ADTU-Denmark/Parts)
·
[Notebook](https://2019.igem.org/Team%3ADTU-Denmark/Notebook)
·
[Team](https://2019.igem.org/Team%3ADTU-Denmark/Team)
·
[Safety](https://2019.igem.org/Team%3ADTU-Denmark/Safety)
·
[Human practices](https://2019.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/Experiments

[![logo](https://static.igem.org/mediawiki/2019/f/fe/T--DTU-Denmark--happylogotemp.png)](https://2019.igem.org/Team%3ADTU-Denmark)

* [![Awards](https://static.igem.org/mediawiki/2019/c/c0/T--DTU-Denmark--medallogo.svg)](https://2019.igem.org/Team%3ADTU-Denmark/Awards)
* [Project](https://2019.igem.org/Team%3ADTU-Denmark/Description)

  [Description](https://2019.igem.org/Team%3ADTU-Denmark/Description)
  [Design](https://2019.igem.org/Team%3ADTU-Denmark/Design)
  [Promoter Design](https://2019.igem.org/Team%3ADTU-Denmark/Design_Promoter)
  [Vector Design](https://2019.igem.org/Team%3ADTU-Denmark/Design_Plasmid)
  [Measurement Design](https://2019.igem.org/Team%3ADTU-Denmark/Design_Measurement)
  [Results](https://2019.igem.org/Team%3ADTU-Denmark/Results)
  [Demonstrate](https://2019.igem.org/Team%3ADTU-Denmark/Demonstrate)
  [Safety](https://2019.igem.org/Team%3ADTU-Denmark/Safety)
  [Characterization](https://2019.igem.org/Team%3ADTU-Denmark/Contribution)
* [Modelling](https://2019.igem.org/Team%3ADTU-Denmark/Model)

  [Model](https://2019.igem.org/Team%3ADTU-Denmark/Model)
  [Software](https://2019.igem.org/Team%3ADTU-Denmark/Software)
* [Parts](https://2019.igem.org/Team%3ADTU-Denmark/Parts)

  [Parts Overview](https://2019.igem.org/Team%3ADTU-Denmark/Parts)
  [Basic Parts](https://2019.igem.org/Team%3ADTU-Denmark/Basic_Part)
  [Composite Parts](https://2019.igem.org/Team%3ADTU-Denmark/Composite_Part)
  [Part Collection](https://2019.igem.org/Team%3ADTU-Denmark/Part_Collection)
* [Journal](https://2019.igem.org/Team%3ADTU-Denmark/Notebook)

  [Notebook](https://2019.igem.org/Team%3ADTU-Denmark/Notebook)
  [Experiments](https://2019.igem.org/Team%3ADTU-Denmark/Experiments)
* [People](https://2019.igem.org/Team%3ADTU-Denmark/Team)

  [Team Members](https://2019.igem.org/Team%3ADTU-Denmark/Team)
  [Collaboration](https://2019.igem.org/Team%3ADTU-Denmark/Collaborations)
  [Attributions](https://2019.igem.org/Team%3ADTU-Denmark/Attributions)
* [Human Practices](https://2019.igem.org/Team%3ADTU-Denmark/Integrated_Human_Practices)

  [Integrated Human Practices](https://2019.igem.org/Team%3ADTU-Denmark/Integrated_Human_Practices)
  [Human Practices](https://2019.igem.org/Team%3ADTU-Denmark/Human_Practices)
  [Education and Engagement](https://2019.igem.org/Team%3ADTU-Denmark/Public_Engagement)
  [Biobrick Tutorial](https://2019.igem.org/Team%3ADTU-Denmark/BioBrick_Tutorial)

[LEAP](https://2019.igem.org/Team%3ADTU-Denmark)

Project

[Description](https://2019.igem.org/Team%3ADTU-Denmark/Description)
[Design](https://2019.igem.org/Team%3ADTU-Denmark/Design)
[Promoter Design](https://2019.igem.org/Team%3ADTU-Denmark/Design_Promoter)
[Vector Design](https://2019.igem.org/Team%3ADTU-Denmark/Design_Plasmid)
[Measurement Design](https://2019.igem.org/Team%3ADTU-Denmark/Design_Measurement)
[Results](https://2019.igem.org/Team%3ADTU-Denmark/Results)
[Demonstrate](https://2019.igem.org/Team%3ADTU-Denmark/Demonstrate)
[Characterization](https://2019.igem.org/Team%3ADTU-Denmark/Contribution)

Modelling

[Model](https://2019.igem.org/Team%3ADTU-Denmark/Model)
[Software](https://2019.igem.org/Team%3ADTU-Denmark/Software)

Parts

[Parts](https://2019.igem.org/Team%3ADTU-Denmark/Parts)
[Basic\_Part](https://2019.igem.org/Team%3ADTU-Denmark/Basic_Part)
[Composite\_Part](https://2019.igem.org/Team%3ADTU-Denmark/Composite_Part)
[Part\_Collection](https://2019.igem.org/Team%3ADTU-Denmark/Part_Collection)

Journal

[Notebook](https://2019.igem.org/Team%3ADTU-Denmark/Notebook)
[Experiments](https://2019.igem.org/Team%3ADTU-Denmark/Experiments)

People

[Team Members](https://2019.igem.org/Team%3ADTU-Denmark/Team)
[Collaborations](https://2019.igem.org/Team%3ADTU-Denmark/Collaborations)
[Attributions](https://2019.igem.org/Team%3ADTU-Denmark/Attributions)

[Safety](https://2019.igem.org/Team%3ADTU-Denmark/Safety)
Human Practices

[Integrated Human Practices](https://2019.igem.org/Team%3ADTU-Denmark/Integrated_Human_Practices)
[Human Practices](https://2019.igem.org/Team%3ADTU-Denmark/Human_Practices)
[Education & Engagement](https://2019.igem.org/Team%3ADTU-Denmark/Public_Engagement)
[BioBrick Tutorial](https://2019.igem.org/Team%3ADTU-Denmark/BioBrick_Tutorial)

![](https://static.igem.org/mediawiki/2019/e/ea/T--DTU-Denmark--background3.png)
![](https://static.igem.org/mediawiki/2019/4/4b/T--DTU-Denmark--background4.png)
![](https://static.igem.org/mediawiki/2019/e/ea/T--DTU-Denmark--background3.png)

![](https://static.igem.org/mediawiki/2019/d/d0/T--DTU-Denmark--experimentheaderscg.svg "Comming soon")

## Experiments

If you've ever participated in iGEM, then you know just how many hours we have spent in the lab. Even though we didn't quite install half a dozen beds in the break room like we wanted to, we still felt like sharing our experiences with all of you. Behold! Our protocols.

## Protocols

Lab friendly downloads available for all protocols

*Aspergillus niger* protoplast

This protocol is adapted from a protocol used by DTU Bioengineering.

### Materials

* Fungal plates (either streaked or 3-point)
* Drigalski spatula
* 500 mL shake flasks
* Counting chamber
* Solutions (APB, ATP, PCT, Milli-Q, TM)
* 30 °C incubator with shaking
* Sterile tea spoon
* Mira cloth in funnel (sterile)
* Glucanex
* Magnet stirrer
* Magnets
* 50 mL sterile falcon tubes
* 0.45 µm filters
* 50 mL syringe
* Centrifuge for falcon tubes

### Media

* Aspergillus transformation buffer (ATB)
* Aspergillus protoplastation buffer (APB)
* 500 mL shake flasks
* YPD media

### Procedure

#### Initiation

* Streak spore suspension of host strain on YPD plates supplemented with uridine (for this particular fungus) and let grow for a week (there should be black spores!)
* All solutions should be sterile.

#### Day 1 (inoculation)

* Add 95 mL of YPD media supplemented with uridine to a shake flask and transfer 5 mL of the YPD media to a plate with A. niger. Collect conidia and spores from plate by carefully scraping off the conidia using a drigalski (the spores are hydrophobic and would therefore rather just fly around than actually get into suspension so be careful not to make a mess here). This should give a concentration of around 108 spores/100 mL. It is a good idea to make more than one shake flask at a time.
* Incubate shake flasks at 30 °C, 150 RMP for 48 h.

#### Day 3 (mycelial harvest)

* Place the sterile funnel with a mira cloth in to a sterile blue cap bottle and transfer the contents of the shake flasks to the mira cloth (content should be brown and thick).
* Wash the mycelia using Aspergillus protoplastation buffer (APB) to remove residual glucose from the mycelia (this can inhibit protoplastation). You need to use quite a bit of APB. squeeze out remaining liquid using a sterile spoon. Then, transfer the mycelium to falcon tubes ( ≈2 g per tube => 1 shake flask ≈ 2 falcon tubes).

#### Protoplastation

* Add glucanex to APB to get a final concentration of 40 mg glucanex per mL APB and dissolve glucanex via gentle magnetic stirring and no heat.
* Sterile filter 20 mL of APB+glucanex to each falcon tube using a 0.45 µm filter and a 50 mL syringe (there is a bit of resistance in the filter but that's ok).
* Shake/incubate enzyme-mycelium mix at 30 °C, 150 rpm for 2-3 h.
* From now on, whenever you pipette anything with the cells in it, cut of the edge of the pipette tip and CAREFULLY(!) pipette the cells. If you don't do this, they break as they don't have a cell wall to keep them stable.
* Evaluate the number and quality of protoplasts in a microscope and discard a batch that is too diluted (i.e. < 105 protoplasts/mL). Approved protoplast solutions are then diluted by pouring APB up to 40 mL. and the tubess are balanced. Dilute Aspergillus transformation buffer (ATB) to 1/2x with sterile Milli-Q water and carefully place 5 mL of this on top of the APB, creating an overlay. Centrifuge samples on rotor settings rotor code Sla-600TC; time: 13 min; Speed: 3000g; Temperatire: 16 °C, Acc: 2, Brake: 2 (NB! due to slow acc and brake, this takes forever!)
* In the interphase between the two liquids, a halo of white slurry consisting of concentrated protoplasts should be observed. If there is cell wall debris mixed in with the protoplasts, that's ok. They can still be used. Withdraw the protoplasts with a pipette and wash them in a new falcon tube. Add ATB up to 40 mL and pellet the protoplasts at 3000g for 13 min (acc. 2, brake 2). Discard supernatant by decanting.
* Count protoplasts in microscope by diluting a small sample 1:100.
* Resuspend protoplasts in 4 mL ATB to obtain concentrated solution.

### Click below for a lab friendly version of this protocol

[Download File](https://static.igem.org/mediawiki/2019/3/3e/T--DTU-Denmark--Aspergillus_protoplast_protocol.pdf)

*Aspergillus* transformation

A quick overview of the two different transformation protocols

### Materials

* DNA
* Protoplasts
* Falcon tubes
* PCT
* TM molten agar
* TM plates
* ATB

### Procedure

#### Regular protocol

* Add at MOST 20 µL DNA ( or max 25% of protoplast volume) and 100 µL of protoplasts in a 50 mL falcon tube.
* Incubate in the falcon tube at RT for at least 30 min.
* Add 1mL of PCT.
* Gently mix by gently swirling the tube in a circular motion – careful they are fragile. Do not vortex or pipette mix.
* Incubate for 5 min at RT.
* Add 3 mL of ATB.
* Add 12 mL of molten (40-45 °C) TM agar.
* Immediately pour mixture directly onto TM plates, and swirl to spread mixture evenly.
* Let the plates settle for a few minutes and incubate at 37 °C for 4 days.

#### Quick protocol

* Add at MOST 25 µL (1500-5000 ng) DNA ( or max 25% of protoplast volume) and 100 µL of protoplasts in a 2 mL Eppendorf tube. 1 µL (100 ng) pac6 as positive control.
* Add 150 μL PCT with large-nozzle pipette tip.
* Gently mix by swirling – careful the protoplasts are fragile.
* Incubate 10-30 min at room temperature.
* Add 250 μL ATB.
* Distribute transformation mix on osmotic-stabilized selective media and let the agar absorb the mix before incubating

### Click below for a lab friendly version of this protocol

[Download File](https://static.igem.org/mediawiki/2019/3/3b/T--DTU-Denmark--Aspergillus_Transformation_protocols.pdf)

Bacterial glycerol stock

Protocol for producing a glycerol stock of *E. coli* adapted by Jacob Mejlsted from [Addgene's protocol](https://www.addgene.org/protocols/create-glycerol-stock/).

### Materials

#### Consumables

* 2 mL screw top tube or cryovial

#### Chemicals

* 50% glycerol
* Cell culture

### Procedure

#### Making the stock

* After having cultivated a culture overnight, add 500 µL culture to 500 µL 50% glycerol in the selected tube
* The stock can now be frozen at -80 °C
   If the stock is repeatedly thawed and frozen, it will reduce the shelf life of the culture

#### Using the stock

* To recover bacteria, take a innoculation loop, toothpick or pipete tip and scrape a small amount of bacteria off the top.
* Transfer this to a plate or a tube with liquid media for growth.
  Normally, media with antibiotics are used. Here Amp and Cam are among the most used.

### Click below for a lab friendly version of this protocol

[Download File](https://static.igem.org/mediawiki/2019/b/bf/T--DTU-Denmark--Glycerol_stock.pdf)

 Biolector

How to load and clean biolector plate.

### Materials

* Biolector
* Minimal media
* P1000 pipette and tips
* P10 pipette and tips
* Spore suspensions
* Biolector plate
* Lid for the biolector plate

### Procedure

* **NB! Make sure to do all work in a clean LAF-bench!**
* Clean plate with ethanol and a cotton swab and leave to dry
* Clean additionally using UV for >30 min.
* Add 1500 µl minimal media (+supplements if necessary) to each well.
* Add spores to the wells to achieve a final concentration of 106 or 107 spores per mL.
* Add a lid for the plate. These are sticky and should be breathable, i.e. the white, kind of fabric-y lids
* Run Biolector for 36 hours
* After run, fill all wells with ethanol and clean using "soft" things such as a pipette tip to get to the corners and a cue tip to clean the sides. Repeat this process until the plate is clean.

### Click below for a lab friendly version of this protocol

[Download File](https://static.igem.org/mediawiki/2019/1/1f/T--DTU-Denmark--Biolector.pdf)

Bioreactor cultivation

Protocol for preparation and execution of the 1 L bioreactor (Sartorius Stedim 1L) cultivations. Adapted from DTU course material by Helena Utzon.

### Materials

#### Materials

* 1 L Sartorius Stedim 1L bioreactor
* Sartorius Stedim BIOSTAT Q Plus control tower
* Off-gas
* Minimal media
* Sigma 204 Antifoam
* Sterile arginine solution
* Acid (2 M Sulfuric acid)
* Base (2 M Potassium hydroxide)
* Spore suspensions

#### Consumables

* 1 mL syringes
* Lab grease
* White strips

### Procedure

#### Assembly of the bioreactors

* Mount the inoculation, sampling, and temperature port from the bottom of the lid and secure them with the bolt from the top of the lid.
* Mount the impellers at the correct height.
* Attach the sparger and baffles, and secure them with the bolt at the top of the bioreactor.
* Mount the condenser, preferably as close to the control tower as possible.
* The mouth of the condenser is loosely packed with a bit of glass wool, and the condenser is closed off.
* The temperature and sampling pipe is mounted, and the sampling tubing is attached and sealed off with both a butterfly clamp and a regular clamp.
* The inoculation port (with membrane) is assembled and mounted.
* The 4-way connector is attached to the bioreactor, and the un-needed connections are closed off with a knotted piece of tubing.
* Marprene tube is attached to the air inlet, and an air filter is attached to the tubing in the right direction.
* Assemble the bioreactor, using a bit of lab grease on the rubber gaskets.
* Connect the empty 250 mL Blue Cap bottles (acid/base) to the 4-way connector, with tubing appropriate for the control tower pumps.
* Clamp tubing between the bioreactor and bottles, as close to the reactor as possible. Also, attach a clamp the tube leading to the air filter.
* Secure all tubing with white strips.
* Fill the reactor with 1 L of minimal medium ([see media recipe](#Minimal-media)).
* Calibrate the pH-electrode, and pressure test the reactor.
* Cover all open ends and filters with tin foil to protect them during autoclaving.
* Check all clamps prior to autoclavation, and loosen the main screw to prevent overpressure.
* Autoclave the bioreactor.
* After autoclavation, close the loose fitting and mount the air supply. Set the flow rate on the DCU to 0.75 vvm, and remove all clamps except for on the sampling tubing.
* Once the medium has cooled down, connect the condenser tubing, the temperature jacket tubing, pH-electrode, DO-electrode, and the stirrer.
* Set the stirrer to 800 rpm and check the accuracy of the pH-electrode. Calibrate if needed.
* Mount the tubing for the acid and base on their respective pumps, run the acid/base through the tubing and switch to “auto”.
* Calculate the inoculum volume to a final concentration of 106 spores/mL, and inoculate the bioreactors with the previously prepared spore suspensions ([See Here](#Sporesuspensions)).
* Add 50 µL of Sigma 204 Antifoam to the reactor.
* Due to species limitations, 0.7 g of arginine was added to the media of the positive control.
* Set the timer to zero at the DCU, and start the batch fermentation at the desired process values.
* Please find the ramps and process conditions for our bioreactor cultivations below.

#### Bioreactor conditions

| Process conditions | |
| --- | --- |
| Airflow inlet | |
| Time (min) | ValueVVM (L/L/M) |
| 0 | 0.1 |
| 300 | 0.16 |
| 420 | 0.4 |
| 600 | 1 |
| pH | |
| Time (min) | ValuepH |
| 0 | 3 |
| 600 | 5 |
| Stirrer speed | |
| Time (min) | ValueRPM |
| 0 | 100 |
| 300 | 200 |
| 420 | 300 |
| 600 | 500 |
| 720 | 800 |
| Temperature | |
| Time (min) | Value(°C) |
| Start-end | 30 |

* Sampling can then be performed as described in [the sampling protocol](#Samplingprocedures)

### Click below for a lab friendly version of this protocol

[Download File](https://static.igem.org/mediawiki/2019/e/eb/T--DTU-Denmark--Bioreactor.pdf)

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 assemblies, such as 3A, Gibson, or Golden Gate. 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
* Sterile water
* LB media with apropriate antibiotics

### Procedure

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

* Pick a number of transformants, typically 3-10, from each plate of interest and mark them on the back of the plate.
* Set up 2 eppendorf tubes for each colony and mark them accordingly:
* Fill the first one (1) with 15 µL MQ water.
* The other one (2) remains empty for now.
* Transfer each colony to the eppendorf containing 15 µL water using a sterile toothpick, inoculation loop or autoclaved pipette tip.
* 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.
* Boil the (1) tubes for 10 minutes at 98 °C. Prepare the PCR mastermix, while the colonies are boiling.

#### Setting up the PCR itself:

* 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 |

* Run the PCR in the thermocycler (use [this website](https://tmcalculator.neb.com/#!/main) to calculate temperatures used based upon the primers and polymerase used).
* Run the products on a gel to check for correct insertion.
* 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.

### Click below for a lab friendly version of this protocol

[Download File](https://static.igem.org/mediawiki/2019/4/41/T--DTU-Denmark--Colony_PCR.pdf)

DNS glucose assay

The procedure determine the glucose concentration of a sample using 3,5-dinitroalicylic acid (DNS).
Adapted by Philip Sørensen and Jacob Mejlsted from [D. Navarro et al.](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2919459/) and [Worthington biochem.](http://www.worthington-biochem.com/AA/assay.html)

### Materials

#### Materials

* Plate reader

#### DNS reagent

* 1 gram of 3,5-dinitrosalicylic acid
* Milli-Q water
* 30.0 grams sodium potassium tartrate tetrahydrate
* 20 mL 2 M NaOH

#### Consumables

* Microtiter plate

### Procedure

#### Preparation of DNS reagent

* Dissolve 1 g of 3,5-dinitrosalicylic acid in 50 mL Milli-Q water
* Add slowly 30.0 grams of sodium potassium tartrate tetrahydrate
* Add 20 mL of 2 M NaOH
* Dilute to a final volume of 100 mL with reagent grade water. Protect from carbon dioxide and store no longer than 2 weeks.

#### Running the assay

* 100 µL media/sample is added to the microtiter plate
* 100 µL DNS regeant is added to each sample
* Plate is heated to 98 °C for 10 min
* Absorbance at 540 nm is measured
* **Remember to include a standard to determine actual concentrations**

### Click below for a lab friendly version of this protocol

[Download File](https://static.igem.org/mediawiki/2019/0/04/T--DTU-Denmark--DNS_assay.pdf)

DpnI digestion of PCR products

Adapted by Jacob Mejlsted from [NEB's product](https://international.neb.com/products/r0176-dpni#Product%20Information) information and [Barrick Lab](http://barricklab.org/twiki/bin/view/Lab/ProtocolsDpnIDigestion)
This protocol describes digestion of DNA with DpnI. This can be used after a PCR reaction to remove the template DNA. It is recommended that the PCR product is purified before this digestion is conducted, but not necessary.

### Materials

#### Chemicals

* DpnI enzyme

### Procedure

#### DpnI digestion

* Add 1 µL of DpnI to finished 50 µL PCR reactions (or 0.5 µL to 25 µL reactions). Pipet or invert to mix.
* Incubate the mixture at 37 °C for 1-2 hrs.
   Alternatively, the solution can be left overnight at room temperature. Periodic mixing may aid digestion (but is unnecessary).
* PCR cleanup or gel-purify the reaction for downstream processes OR heat inactivate at 80 °C for 20 min.

### Click below for a lab friendly version of this protocol

[Download File](https://static.igem.org/mediawiki/2019/6/69/T--DTU-Denmark--DpnI_digest.pdf)

*E. coli* competent cell

Adapted by Jacob Mejlsted & Kyle Rothschild

### Materials

#### Equipment

* Autoclave
* Freezer
* Centrifuge
* Spectrophotometer

#### Consumables

* PCR tubes
* 50 mL falcon tubes

#### Chemicals

* MgCl2
* CaCl2
* SOC/SOB
* Glycerol

### Procedure

#### Day 1:

* Autoclave the following:

+ Minimum 200 mL 0.1 M MgCl2
+ Minimum 150 mL 0.1 M CaCl2
+ Minimum 100 mL 85 mM CaCl2 + 15% glycerol (v/v)
+ Minimum 700 mL SOC
+ 3 x shakeflasks (500 mL)

* Freeze at -20 °C (after autoclaving)

+ 0.1 M MgCl2
+ 0.1 M CaCl2
+ 85 mM CaCl2 + 15% glycerol (v/v)
+ Minimum 10 x 50 mL falcon tubes
+ PCR tubes

#### Day 2:

* Culture growth
* Early in the morning, start cooling the Sorval centrifuge to 4 °C
* Pour 200 mL SOB media into each of the shakeflask (one shakeflask per starter culture).
* Mark the shakeflasks to match the startercultures
* Measure the OD 600 of each starterculture, and inoculate the shakeflask with a volume so the final OD 600 value in the shakeflask culture becomes 0.01
* Grow the shakeflask culture at 37 °C with shaking. Measure OD values of the sample every 20 minutes once the OD 600 value is above 0.2.
* When OD 600 is between 0.3 and 0.4, put the cultures into an ice bath immediately, and swirl the shake flash around in the cold water to cool culture. Chill the culture in the ice water for 20-30 minutes, occasionally swirling the cultures.
* FROM THIS STAGE ON, KEEP CELLS AT ICE/4 °C AT ALL TIMES
* For each shakeflask culture, pour the culture into 3 x 50 mL frosted falcon tubes from the freezer.
* Keep the tubes on ice
* Centrifuge falcon tubes at 3000 x g for 15 min at 4 °C (Spin #1 of 4)
* Discard supernatant, and resuspend cells in 15 mL icecold 0.1 M MgCl2
* Keep tubes with cells on ice
* Pool the resuspended cells into one of their matching falcon tubes, so you now have 3 different 50 mL falcon tubes, one with cells corresponding to each of the starter cultures you had.
* Keep tubes on ice
* Centrifuge falcon tubes at 2000 x g for 15 min at 4 °C (Spin #2 of 4)
* Discard the supernatant, and resuspend pellet in 40 mL icecold 0.1 M CaCl2
* Keep tubes on ice
* Let cell suspensions stand in ice for 20-30 minutes
* Centrifuge falcon tubes at 2000 x g for 15 min at 4 °C (Spin #3 of 4)
* Discard supernatant, and resuspend pellet in 10 mL icecold 85 mM CaCl2 + 15 % glycerol
* Keep tubes on ice
* Centrifuge falcon tubes at 1000 x g for 15 minutes at 4 °C (Spin #4 of 4)
* Pellet might look small and will be a bit fragile. Handle tubes with care when taking them out of centrifuge
* Attention: The next few steps are best done on ice inside a LAF bench
* Resuspend pellet in 800 µL ice-cold 85 mM CaCl2 + 15 % glycerol
* Put falcon tubes on ice
* Immediately after cells are confirmed resuspended, aliquot 30 µL of the competent cell culture into the chilled PCR tubes
* Put tubes into -80 °C freezer as fast as possible

### Click below for a lab friendly version of this protocol

[Download File](https://static.igem.org/mediawiki/2019/c/ce/T--DTU-Denmark--E_coli_compentent_cells.pdf)

Fluorescence measurements

Here, a description of the instruments, settings, and general protocol for the fluorescence measurements can be found.

### Materials

#### Materials

* SpectraMax iD3 or equivalent plate reader

#### Consumables

* Corning® Clear Polystyrene 96-Well Microplate

### Procedure

#### Fluorescence measurements

* If not done already, 100 µL of sample was loaded into the well of a spectophotometry plate.
* The RFP was measured using the following settings: (Ex/Em)

+ mCherry: 580/625
+ GFP: 488/530

### Click below for a lab friendly version of this protocol

[Download File](https://static.igem.org/mediawiki/2019/7/73/T--DTU-Denmark--Fluorescens_measurements.pdf)

Gibson assembly (combined)

Protocol for PCR & Gibson adapted from the Gibson Assembly Cloning Guide 2nd edition
Protocol for template removal adapted from [NEBcloner](http://nebcloner.neb.com/#!/protocol/re/single/DpnI)
Protocol for PCR Purification adapted from Qiagen [QIAquick® PCR Purification Kit](https://www.qiagen.com/dk/resources/resourcedetail?id=e0fab087-ea52-4c16-b79f-c224bf760c39&lang=en)
Protocol for MiniPrep adapted from Qiagen's [QIAprep® Spin Miniprep Kit](https://www.qiagen.com/us/resources/download.aspx?id=56b0162c-23b0-473c-9229-12e8b5c8d590&lang=en)

### Procedure

#### PCR Amplfication of DNA Fragments

* Prepare PCR reaction (see table)

| Component | Volume |
| --- | --- |
| Insert or vector DNA (100 pg/µL - 1 ng/µL in TE) | 0.5 µL |
| 10 µM Forward Primer | 2.5 µL |
| 10 µM Reverse Primer | 2.5 µL |
| 10 mM dNTPs | 1 µL |
| 5X Phusion HF Buffer | 10 µL |
| Phusion DNA Polymerase (2 U/µL) | 0.5 µL |
| Nuclease-free Water | 33 µL |
| Total | 50 µL |

* Run reaction in a thermocycler

| Step | Temperature | Duration | Number of Cycles |
| --- | --- | --- | --- |
| Initial denaturation | 98 °C | 30 seconds | 1 cycle |
| Amplification | 98 °C | 10 seconds | 25-30 cycles |
| Primer Tm | 20 seconds |
| 72 °C | 30 seconds/kb |
| Final extension | 72 °C | 5 minutes | 1 cycle |
| Hold | 4 °C | - | 1 cycle |

#### Template Removal

* Set up the reaction as follows:

| Component | 50 µL Reaction |
| --- | --- |
| Component | 50 µL Reaction |
| DNA | 1 µg |
| 10X CutSmart Buffer | 5 µL (1X) |
| DpnI | 1.0 µL (or 10 units) |
| Nuclease-free Water | to 50 µL |

* Incubate at 37 °C for 5–15 minutes (DpnI is Time-Saver qualified).
* Optional: Heat inactivate at 80 °C for 20 min if not doing a DNA purification step

#### PCR Purification

* Add 5 volumes Buffer PB to 1 volume of the PCR reaction and mix. If the color of the mixture is orange or violet, add 10 µL 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn yellow
* Place a QIAquick column in a provided 2 mL collection tube
* To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 s. Discard flow-through and place the QIAquick column back in the same tube.
* To wash, add 750 µL Buffer PE to the QIAquick column centrifuge for 30–60 s. Discard flow-through and place the QIAquick column back into the same tube.
* Centrifuge the QIAquick column once more in the provided 2 mL collection tube for 1 min to remove residual wash buffer.
* Place each QIAquick column in a clean 1.5 mL microcentrifuge tube.
* To elute DNA, add 50 µL Buffer EB (10 mM Tris·Cl, pH 8.5) or water (pH 7.0–8.5) to the center of the QIAquick membrane and centrifuge the column for 1 min. 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.

#### Mid-way analysis

* Add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.
* Run the gel and evaluate the results
* NanoDrop the purified DNA to determine the concentration

#### Gibson Assembly using a HiFi 1-Step Kit

* Thaw Gibson Assembly HiFi 1-step Master Mix on ice
* Combine insert and vector DNA to a total volume of:
  5 µL for the 2X kit or
  7.5 µL for the HC 4X kit
* Vortex the master mix
* On ice, combine
  5 µL DNA and 5 µL Master Mix for the 2X kit or
  7.5 µL DNA and 2.5 µL Master Mix for the HC 4X kit
  Mix well andbriefly centrifuge
* Incubate at 50 °C for 1 hour
* Store reactions at -20 °C or use in 1-2 µL per reaction for transformation

#### Transformation protocol

* Thaw chemically-competent cells on ice
* 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
* Place the mixture on ice for 30 minutes. Do not mix
* Heat shock at 42 °C for 30 seconds. Do not mix
* Transfer tubes to ice for 2 minutes
* Add 950 µL of room temperature SOC media to the tubes
* Incubate the tube for 37 °C for 60 minutes. shake vigerously (250 rpm) or rotate
* Warm selection plates to 37 °C
* Spread 100 µL of the cells onto the selection plates.
* Note: Use Amp plates for the positive control
* Incubate overnight at 37 °C.

#### Day 2:

* Plate counting: Count CFU and control plates
* Innoculate LB + antibiotic media for O/N culture

#### Day 3: Miniprep

* Use a miniprep kit to purify the assembled plasmids.

### Click below for a lab friendly version of this protocol

[Download File](https://static.igem.org/mediawiki/2019/6/6e/T--DTU-Denmark--Gibson_Assembly.pdf)

Golden Gate assembly with BsaI (Type IIs assembly)

This protocol is made to assembly parts with TypeIIs enzyme BsaI HF v2. These parts will have the standard MoClo overhangs. Protocol adapted by Philip Sørensen from a [Evry Paris-Saclay's protocol](https://static.igem.org/mediawiki/2017/e/e8/T--Evry_Paris-Saclay--protocol--pdf--gate.pdf).

### Materials

#### Reagens

* T4 DNA ligase
* 10X T4 DNA ligase buffer
* BsaI HF v2 enzyme (NEB)
* Reciever plasmid
* DNA fragments with BsaI overhangs
* Milli-Q water

#### Materials

* PCR tubes
* 10 µL pipette tips
* Thermocycler

### Procedure

#### Setting up the reaction

* In a PCR tube, mix the following:
  - 0.5 µL of T4 DNA Ligase
  - 2 µL of 10X T4 DNA Ligase Buffer
  - 0.5 µL of BsaI HF v2 restriction enzyme
  - 100 ng of receiver plasmid
  - Equimolar amounts of inserts
  - Milli-Q for a total volume of 20 µL.
* Mix gently
* Place the tube on a thermocycler

#### Thermocycler program

| Step | Temp | Time |
| --- | --- | --- |
| Activation of BsaI HF v2 | 37 °C | 5 min |
| Activation of T4 ligase | 16 °C | 5 min |
| Repeat step 1 & 2 for 25 cycles |  |  |
| Inactivation BsaI HF v2 | 65 °C | 20 min |
| Inactivation T4 ligase | 85 °C | 10 min |
| Hold | 4 °C | Hold |

* The cloned plasmids can then be transformed as described in the [Hifi assembly and transformation protocol.](#HifiDNAass)

### Click below for a lab friendly version of this protocol

[Download File](https://static.igem.org/mediawiki/2019/f/fa/T--DTU-Denmark--Golden_Gate_BsaI.pdf)

Golden Gate assembly with SapI (Type IIs assembly)

This protocol is made to assembly parts with TypeIIs enzyme SapI. Protocol adapted by Philip Sørensen from a [Evry Paris-Saclay's protocol](https://static.igem.org/mediawiki/2017/e/e8/T--Evry_Paris-Saclay--protocol--pdf--gate.pdf).

### Materials

#### Reagens

* T4 DNA ligase
* 10X T4 DNA ligase buffer
* SapI enzyme (NEB)
* Reciever plasmid
* DNA fragments with SapI overhangs
* Milli-Q water

#### Reagens

* PCR tubes
* 10 µL pipette tips
* Thermocycler

### Procedure

#### Setting up the reaction

* In a PCR tube, mix the following:
  - 0.5 µL of T4 DNA Ligase
  - 2 µL of 10X T4 DNA Ligase Buffer
  - 0.5 µL of Type IIS restriction enzyme
  - 100 ng of receiver plasmid
  - Equimolar amounts of inserts
  - Milli-Q for a total volume of 20 µL.
* Mix gently
* Place the tube on a thermocycler

#### Thermocycler program

| Step | Temp | Time |
| --- | --- | --- |
| Activation of SapI | 37 °C | 5 min |
| Activation of T4 ligase | 16 °C | 5 min |
| Repeat step 1 & 2 for 25 cycles |  |  |
| Inactivation SapI | 65  °C | 20 min |
| Inactivation T4 ligase | 85  °C | 10 min |
| Hold | 4  °C | Hold |

* The cloned plasmids can then be transformed as described in the [Hifi assembly and transformation protocol.](#HifiDNAass)

### Click below for a lab friendly version of this protocol

[Download File](https://static.igem.org/mediawiki/2019/c/c4/T--DTU-Denmark--Golden_Gate_SapI.pdf)

Hifi DNA assembly and transformation

Adapted by Jacob Mejlsted & Joen Haahr Jensen from NEB's [HiFi assembly protocol](https://international.neb.com/protocols/2014/11/26/nebuilder-hifi-dna-assembly-reaction-protocol).

### Materials

(X is the number of reactions)

#### Consumables

* X PCR tubes for each reaction + 1 for positive control
* X eppendorf tupe for each reaction + 1 for postive control
* X selection plate for each reaction
* 1 Amp plate for positive control

#### Chemical

* Hifi DNA assembly Master mix
* Sterile Milli-Q water
* Competent *E. coli* cells
* Prepared DNA fragments for assembly

### Procedure

#### Assembly protocol

* Set up the following reaction on ice:
  \* 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

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

* 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

* Thaw chemically-competent cells on ice
* 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
* Place the mixture on ice for 30 minutes. Do not mix
* Heat shock at 42 °C for 30 seconds. Do not mix
* Transfer tubes to ice for 2 minutes
* Add 950 µL of room temperature SOC media to the tubes
* Incubate the tube for 37 °C for 60 minutes. shake vigerously (250 rpm) or rotate
* Warm selection plates to 37 °C
* Spread 100µL of the cells onto the selection plates.
* Note: Use Amp plates for the positive control
* Incubate overnight at 37 °C.

### Click below for a lab friendly version of this protocol

[Download File](https://static.igem.org/mediawiki/2019/7/7f/T--DTU-Denmark--Hifi_DNA_assembly.pdf)

Minimal media

Protocol for production of 1 liter of minimal media

### Materials

#### Materials

* 50 mL of 20% w/V D-glucose stock
* 50 mL of 20x nitrate salts stock
* 1 mL 1000x Trace elements stock
* 1 mL of 1% thiamine stock
* 20 g agar

### Procedure

#### Media preparation

* Mix ingredients in flask and add MiliQ water to 1 litre
* Mix with magnet stirrer until the sugar is dissolved
* Autoclave at 121 °C for 20 min

### Click below for a lab friendly version of this protocol

[Download File](https://static.igem.org/mediawiki/2019/f/f0/T--DTU-Denmark--Minimal_media.pdf)

Miscellaneous protocols

Here is a list of miscellaneous protocols, all of which were performed according to the manufacturer's and/or the referenced specifications. .

### Protocols

#### Gel electrophoresis

* Gels contained 1% agarose and were stained with 1000X SYBR Safe (Invitrogen)
* DNA fragment solutions were combined with 6X Purple Loading Dye (New England Biolabs).
* Samples were run in 1X TAE running buffer at 100 V with a 1kb DNA ladder (New England Biolabs).
* Electrophoresis was run on the RunOne Electrophoresis System (Embi Tec).
* Pictures of the resulting gels were taken with the ChemiDoc™ XRS+ Imaging System or equivalent.

#### Gel purifications

* Isolated DNA bands were purified via the QIAquick Gel Extraction kit (QIAGEN).

#### PCR spin purifications

* When deemed necessary, plasmid DNA was spin purified, rather than gel purified, via the QIAquick PCR Purification Kit (QIAGEN).

#### Plasmid miniprep purification

* Plasmids were purified using the QIAprep Spin Miniprep Kit (QIAGEN).

#### DNA sequencing

* DNA was verified by Sanger sequencing, using the Eurofins’ overnight sanger sequencing service.

#### Primer/DNA resuspension

* Primers were diluted to a stock concentration of 100 µM and a working concentration of 10 µM.
* Synthetic DNA (e.g. synthetic promoters) were diluted to 25 ng/µL or 40 ng/µL, depending on downstream uses.

PacI+Nt.BbvCI digest for USER linarisation

This protocol is for linearising backbones with the PacI+Nt.BbvCI USER casette. This will allow assembly of PCR product with USER tails to be assembled into this linearised plasmid.

### Materials

#### For digest I

* Plasmid: 40 µL
* PacI: 2 µL
* Cutsmart buffer (NEB): 10 µL
* MQ water: 48 µL

#### For digest II

* Finished digest I
* Nt.BbvCI: 2 µL

### Procedure

#### Digest I

* MIx all the reagents as mentioned above, and incubate at 37 °C overnight

#### Digest II

* Take the digest I reaction from the 37 °C incubator, add in the 2 µL Nt.BbvCI to the reaction mixture, and incubate for 2 hours at 37 °C
* Heat inactivate the samples at 80 °C for 30 min

### Click below for a lab friendly version of this protocol

[Download File](https://static.igem.org/mediawiki/2019/7/7a/T--DTU-Denmark--USER_digestion.pdf)

PCR protocol for Phusion® high-fidelity DNA polymerase

Protocol for PCR using the Phusion polymerase adapted by Jacob Mejlsted from the [NEB protocol for the same product](https://international.neb.com/Protocols/0001/01/01/pcr-protocol-m0530).

### Materials

#### DNA

#### Consumables

* PCR tubes (1 per reaction + 1 for positive control)

#### Chemicals

* Forward primers
* Reverse primers
* Nuclease-free water
* Phusion DNA Polymerase
* 5X Phusion HF or GC Buffer
* 10 mM dNTPs
* DMSO (optional)

### Procedure

#### PCR Amplfication of DNA Fragments

* Prepare PCR reaction (see table)

| Component | 20 µL Reaction | 50 µL Reaction | Final Concentration |
| --- | --- | --- | --- |
| Nuclease-free water | to 20 µL | to 50 µL |  |
| 5X Phusion HF or GC Buffer | 4 µL | 10 µL | 1X |
| 10 mM dNTPs | 0.4 µL | 1 µL | 200 µM |
| 10 µM Forward Primer | 1 µL | 2.5 µL | 0.5 µM |
| 10 µM Reverse Primer | 1 µL | 2.5 µL | 0.5 µM |
| Template DNA | variable | variable | < 250 ng |
| DMSO (optional) | (0.6 µL) | (1.5 µL) | 3% |
| Phusion DNA Polymerase | 0.2 µL | 0.5 µL | 1.0 units/50 µL PCR |

* Alternatively, a master mix can be prepared

| Reactant | Per reaction (50µL) [µL] | Mastermix [µL] |
| --- | --- | --- |
| Number of reactions | 1 | 10 |
| 5X Phusion HF or GC Buffer | 10 | 100 |
| 10 mM dNTPs | 1 | 10 |
| 10 µM Forward Primer | 2.5 | Added individually |
| 10 µM Reverse Primer | 2.5 | Added individually |
| Template DNA | variable | Added individually |
| Phusion DNA Polymerase | 0.5 | 5 |
| DMSO (optional) | 0 | 0 |
| Milli-Q | 33.5 | 335 |

* Run reaction in a thermocycler
  \*15 seconds/kb works for most reactions. 30 seconds/kb can be used for more complex reaction, such as cDNA.

| Step | Temperature | Duration | Number of Cycles |
| --- | --- | --- | --- |
| Initial denaturation | 98 °C | 30 seconds | 1 cycle |
| Amplification | 98 °C | 10 seconds | 25-30 cycles |
| Primer Tm | 20 seconds |
| 72 °C | 15-30 seconds/kb\* |
| Final extension | 72 °C | 5-10 minutes | 1 cycle |
| Hold | 4 °C | - | 1 cycle |

### Click below for a lab friendly version of this protocol

[Download File](https://static.igem.org/mediawiki/2019/5/51/T--DTU-Denmark--Phusion_protocol.pdf)

PCR using OneTaq

Adapted by Jacob Mejlsted from the [NEB protocol for the same product](https://international.neb.com/protocols/2012/10/11/onetaqdnapolymerasem0480).

### Materials

* 1 PCR tube per reaction

#### Chemicals

* OneTaq standard reaction buffer
* dNTPs
* Primers
* DNA template
* OneTaq DNA Polymease

### Procedure

#### Method

* To a tube add the following

| Component | 25 μL reaction\*\*\* | 50 μL reaction\*\*\* | Final Concentration |
| --- | --- | --- | --- |
| 5X OneTaq Standard Reaction Buffer\* | 5 µL | 10 μL | 1X |
| 10 mM dNTPs (#N0447) | 0.5 µL | 1 μL | 200 µM |
| 10 µM Forward Primer | 0.5 µL | 1 μL | 0.2 µM |
| 10 µM Reverse Primer | 0.5 µL | 1 μL | 0.2 µM |
| OneTaq DNA Polymerase | 0.125 µL | 0.25 µL | 1.25 units/50 µL PCR\*\* |
| Template DNA\*\* | variable | variable | < 1,000 ng |
| Nuclease-free water | to 25 µL | to 50 µL |  |

\*OneTaq GC Reaction Buffer and High GC Enhancer can be used for difficult amplicons
\*\*For plasmids or viral DNA, use 1 pg–10 ng DNA for a 50 µL reaction.

\*\*\*It can be advantagous to pool some of the parts into a master mix, as some labs cannot dispense 0.125 µL accurately.

* Alternatively, a master mix can be prepared

| Reactant | Per reaction (50µL) [µL] | Mastermix [µL] |
| --- | --- | --- |
| Number of reactions | 1 | 10 |
| 5X OneTaq Standard Reaction Buffer\* | 10 | 100 |
| 10 mM dNTPs (#N0447) | 1 | 10 |
| 10 µM Forward Primer | 1 | Added individually |
| 10 µM Reverse Primer | 1 | Added individually |
| OneTaq DNA Polymerase | 0.25 | 2.5 |
| Template DNA | 1 | Added individually |

* Gently mix the reaction. Collect all liquid to the bottom of the tube by a quick spin if necessary.

| STEP | TEMP | TIME |
| --- | --- | --- |
| Initial Denaturation | 94 °C | 30 seconds |
| 30 Cycles | 94 °C | 15-30 seconds |
| 45-68 °C | 15-60 seconds |
| 68 °C | 1 minute per kb |
| Final Extension | 68 °C | 5 minutes |
| Hold | 4-10 °C |  |

* PCR products can then by digested by DpnI, purified, stored at -20 C, or a combination.

### Click below for a lab friendly version of this protocol

[Download File](https://static.igem.org/mediawiki/2019/a/a3/T--DTU-Denmark--OneTaq_protocol.pdf)

PCR using Q5® high-fidelity 2X master mix

Protocol for PCR using the Q5 2X master mix adapted by Jacob Mejlsted from the [NEB protocol for the same product](https://international.neb.com/protocols/2012/12/07/protocol-for-q5-high-fidelity-2x-master-mix-m0492).

### Materials

#### DNA

#### Consumables

* PCR tubes (1 per reaction + 1 for positive control)

#### Chemicals

* Forward primers
* Reverse primers
* Nuclease-free water
* Q5 2X Master Mix

### Procedure

#### PCR Amplfication of DNA Fragments

* Prepare PCR reaction (see table)

| Component | 25 µL reaction | 50 µL reaction |
| --- | --- | --- |
| Q5 High-Fidelity 2X Master Mix | 12.5 µL | 25 µL |
| 10 µM Forward Primer | 1.25 µL | 2.5 µL |
| 10 µM Reverse Primer | 1.25 µL | 2.5 µL |
| Template DNA\* | variable (0ften 0.5 µL) | variable (0ften 1 µL) |
| Nuclease-Free Water | to 25 µL | to 50 µL |
| Total | 25 µL | 50 µL |

\*Template DNA:
For a 50 µL reaction, 1 ng-1 µg is recommended for genomic DNA and 1 pg-10 ng is recommended for plasmid or viral DNA.

* Run reaction in a thermocycler

| Step | Temperature | Duration | Number of Cycles |
| --- | --- | --- | --- |
| Initial denaturation | 98 °C | 30 seconds | 1 cycle |
| Amplification | 98 °C | 10 seconds | 25-30 cycles |
| Primer Tm | 20 seconds |
| 72 °C | 30 seconds/kb |
| Final extension | 72 °C | 2 minutes | 1 cycle |
| Hold | 4 °C | - | 1 cycle |

* The PCR products can then be stored at -20 °C, used directly, or purified using PCR purification or gel extraction

### Click below for a lab friendly version of this protocol

[Download File](https://static.igem.org/mediawiki/2019/f/f6/T--DTU-Denmark--Q5_master_mix.pdf)

PCR using X7 polymerase

This protocol describes the required components and method for a X7 polymerase mediated PCR reaction. .

### Materials

#### Consumables

* PCR strip/tubes

### Procedure

#### PCR Amplfication of DNA Fragments

* Prepare PCR reaction (see table below)

| COMPONENT | 50 µL REACTION |
| --- | --- |
| CXL buffer | 10 µL |
| 10 mM dNTPs | 1 µL |
| 10 µM Forward Primer | 2.5 µL |
| 10 µM Reverse Primer | 2.5 µL |
| Template DNA | Variable (often 1 µL) |
| Pfu X7 Polymerase | 0.5 µL |
| DMSO | 1.5 µL |
| Nuclease-Free Water | to 50 µL |

* Run reaction in a thermocycler

#### Thermocycler PCR program

| Step | Temperature | Duration | Number of Cycles |
| --- | --- | --- | --- |
| Initial denaturation | 95 °C | 2 minutes | 1 cycle |
| Amplification | 95 °C | 30 seconds | 25-30 cycles |
| Primer Tm | 30 seconds |
| 72 °C | around 1 min/kb\* |
| Final extension | 72 °C | 5 minutes | 1 cycle |
| Hold | 4 °C | - | 1 cycle |

* The PCR products can then be stored at -20 °C, used directly, or purified using PCR purification or gel extraction.

### Click below for a lab friendly version of this protocol

[Download File](https://static.igem.org/mediawiki/2019/6/66/T--DTU-Denmark--X7_polymerase_protocol.pdf)

Protein extraction

This protocol is an adaptation of a GUS (β-glucuranidase) assay provided by Zofia Dorota Jarczynska and adapted by Jacob Mejlsted.

### Materials

#### Extraction buffer (1L)

* 50 mM Na3PO4, pH 7 (50 mL of 1 M stock solution)
* 10 mM β-mercaptoethanol (0.7 mL of 14.4 M stock solution)
* 10 mM Na2EDTA (20 mL of 0.5 M Na2EDTA stock solution)
* 0.1% Sodium Lauryl Saccosine (10 mL of 10% Sarcosyl)
* 0.1% Triton X-100 (10 mL f 10% Triton)
* Milli-Q water up to 1L

#### Equipment

* Centrifuge with cooling
* Plate reader
* Homogenizer
* Beads (big, metal)
* Sterile funnels with mira cloth

#### Chemicals

* Bradford reagent (Like SigmaAldrich B6916)
* BSA standard (Bovine Serum Albumin 2 mg/mL)

### Procedure

#### Protein Extraction

* **CRITICAL** Set the centrifuge at 4 °C
* Seperate biomass from supernantant using mira cloth or other methods
* Take ~100 mg biomass and place in a FastPrep tube with one big metal bead
* Place the tubes in liquid nitrogen
* Homogenize for 1 min at 45 Hz
* Add 500 µL extraction buffer
* Homogenize for 2 min at 45 Hz
* Centrifuge for 2 min at 10,000 x g at 4 °C
* Collect the liquid phase and place in a new tube
* Centrifuge for 15 min at 10,000 x g at 4 °C
* Aliquote the supernantant into new tubes
* Store at -80 °C

#### Bradford Assay

* Transfer 100 µL protein sample into a microtiter place
* Measure fluorescense at the appripriate wavelenghts

#### Fluorescence Assay (optional)

* Mix 20 µL protein sample with 230 µL Bradfor reagent
* Transfer to a new microtiter plate
* Measure the blue color at 595 nm
* **Remember to include a standard curve and Bradford reagent**
* Determine protein concentration

### Click below for a lab friendly version of this protocol

[Download File](https://static.igem.org/mediawiki/2019/a/a9/T--DTU-Denmark--Protein_extraction_protocol.pdf)

Retrieving DNA from the iGEM distribution kit

Adapted by Jacob Mejlsted from [iGEM's protocol](http://parts.igem.org/Help%3AProtocols/Transformation)

### Materials

#### Materials

* Resuspended DNA to be transformed
* 10 pg/µL Positive transformation control DNA (e.g. pSB1C3 w/ BBa\_J04450, RFP on high-copy chloramphenicol resistant plasmid. Located in the Competent Cell Test Kit.)
* Competent Cells (50 µL per sample)
* 1.5 mL Microtubes
* SOC Media (950 µL per sample)
* Petri plates w/ LB agar and antibiotic (2 per sample)

#### Equipment

* Floating Foam Tube Rack
* Ice & ice bucket
* Lab Timer
* 42 °C water bath
* 37 °C incubator
* Sterile spreader or glass beads
* Microcentrifuge

### Procedure

#### Method: Day 1

* Resuspend DNA in selected wells in the Distribution Kit with 10 µL dH2O. Pipet up and down several times, let sit for a few minutes. Resuspension will be red from cresol red dye.
* Label 1.5 mL tubes with part name or well location. Fill lab ice bucket with ice, and pre-chill 1.5 mL tubes (one tube for each transformation, including your control) in a floating foam tube rack.
* Thaw competent cells on ice: This may take 10-15 min for a 260 µL stock. Dispose of unused competent cells. Do not refreeze unused thawed cells, as it will drastically reduce transformation efficiency.
* Pipette 50 µL of competent cells into 1.5 mL tube: 50 µL in a 1.5 mL tube per transformation. Tubes should be labeled, pre-chilled, and in a floating tube rack for support. Keep all tubes on ice. Don’t forget a 1.5 mL tube for your control.
* Pipette 1 µL of resuspended DNA into 1.5 mL tube: Pipette from well into appropriately labeled tube. Gently pipette up and down a few times. Keep all tubes on ice.
* Pipette 1 µL of control DNA into 2 mL tube: Pipette 1 µL of 10 pg/µL control into your control transformation. Gently pipette up and down a few times. Keep all tubes on ice.
* Close 1.5 mL tubes, incubate on ice for 30 min: Tubes may be gently agitated/flicked to mix solution, but return to ice immediately.
* Heat shock tubes at 42 °C for 45 sec: 1.5 mL tubes should be in a floating foam tube rack. Place in water bath to ensure the bottoms of the tubes are submerged. Timing is critical.
* Incubate on ice for 5 min: Return transformation tubes to ice bucket.
* Pipette 950 µL SOC media to each transformation: SOC should be stored at 4 °C, but can be warmed to room temperature before use. Check for contamination.
* Incubate at 37 °C for 1 hours, shaking at 200-300 rpm
* Pipette 100 µL of each transformation onto petri plates. Spread with sterilized spreader or glass beads immediately. This helps ensure that you will be able to pick out a single colony.
* Incubate transformations overnight (14-18 hours) at 37 °C: Incubate the plates upside down (agar side up). If incubated for too long, colonies may overgrow and the antibiotics may start to break down; un-transformed cells will begin to grow.

#### Method: Day 2

* Pick single colonies: Pick single colonies from transformations: do a colony PCR to verify part size, make [glycerol stocks](#proto3aassembly), grow up cell cultures and miniprep.
* Count colonies for control transformation: Count colonies on the 100 μL control plate and [calculate your competent cell efficiency](http://parts.igem.org/Help%3ACompetent_Cell_Test_Kit). Competent cells should have an efficiency of 1.5x108 to 6x108 CFU/µg DNA.

### Click below for a lab friendly version of this protocol

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Sampling procedures

Here, the procedure for sampling of the shake flask and bioreactor cultivations is described.

### Materials

#### Consumables

* 10 mL syringe (bioreactor)
* 15 mL falcon tube
* 1.5 mL eppendorf tube
* Corning® Clear Polystyrene 96-Well Microplate

### Procedure

#### Shake flask cultivation

* In a clean bench, approximately 1 mL of sample containing biomass was sampled via pipetting.
* 100 µL sample containing biomass was transferred to a spectrophotometry plate.
* 200 µL of sample was spun down at 10.000 g for 3 min and 100 µL transferred to a spectrophotometry plate.
* Immediately after sampling, samples in the spectrophotometry plate was measured for fluorescence according to the [fluorescence measurement protocol](#FluorescenceMeasurements), and the remainder of the sample in the eppendorf tubes stored at -20 °C.

#### Bioreactor cultivation

* For sampling of the bioreactor, extra care was taken to prevent contamination.
* The sterile 10 mL syringe was sprayed with 70% ethanol and inserted in the sampling tube.
* The clamps on the sampling tube were loosened and 5 mL sample was drawn and discarded.
* The syringe was again sprayed with 70% ethanol, and reinserted in the sampling tube.
* Approximately 10 mL sample containing biomass was drawn and transferred to a 15 mL falcon tube.
* The clamps on the sampling tube were tightened, and the sampling tube sprayed with 70% ethanol.
* The samples were stored at -20 C for later protein purification.

### Click below for a lab friendly version of this protocol

[Download File](https://static.igem.org/mediawiki/2019/3/39/T--DTU-Denmark--Sampling_procedures.pdf)

Shake flask cultivation

Here, we describe the protocol for the set-up and execution of the shake flask cultivations. Please, [click here](#Minimal-media) for the media recipe, [here](#Samplingprocedures) for sampling procedures for the specifics regarding sampling, and [here](#FluorescenceMeasurements) for details regarding the fluorescence measurements.

### Materials

#### Materials

* Pyrex shake flasks (with baffles)
* Minimal media
* Spore suspension

#### Consumables

* Cotton stopper
* Tin foil

### Procedure

#### Preparations and inoculation

* A breathable cotton ball was added to the mouth of a glass shake flask (with baffles), covered with tin foil, and then autoclaved.
* 150 mL minimal media was added to each shake flask.
* The shake flasks were inoculated with their respective spore solutions to a final concentration of 106 spores/mL.
* Shake flasks were incubated for 5 days as described below.

#### Conditions

* Shake flasks were incubated at 30 °C at 150 rpm for 5 days.
* Samples were taken as regularly as described in sampling procedures.

### Click below for a lab friendly version of this protocol

[Download File](https://static.igem.org/mediawiki/2019/2/25/T--DTU-Denmark--Shake_flask_cultivation.pdf)

Spore suspensions

The following is a quick and easy protocol for creating the spore suspensions used for storage and the inoculation of the shake flask, biolector, and bioreactor cultivations. .

### Materials

#### Materials

* Fully grown fungi plates (with spores)
* Drigalski spatula
* 96% ethanol
* FireBoy or other flame-sterilizing equipment
* Sterile Milli-Q water
* Sterile mira cloth
* Sterile falcon tubes
* P1000 pipette and tips
* Scissors
* Microscope
* Counting chamber

### Procedure

#### Creating the spore suspensions

* Add mira cloth to a falcon tube.
* Pour 2-3 mL of Milli-Q water onto a plate
* Clean and sterilize a drigalski spatula using ethanol and fire.
* Let the drigalski cool off in the water before commencing the scraping. Then, carefully scrape off the spores from the plate. This should be done with the lid still on and VERY careully as the spores are hydrophobic and would much rather just fly around than being suspended into the water.
* When all of the spores have been suspended into the water, transfer the liquid to the mira cloth using a pipette with the tip cut off.
* When this is done, count the spores in the microscope. Usually, a 1000x dilution is necessary.
* The spore suspension can be kept for up to 3 months at 4 °C.

### Click below for a lab friendly version of this protocol

[Download File](https://static.igem.org/mediawiki/2019/8/85/T--DTU-Denmark--Spore_suspensions.pdf)

Transformation media

Protocol for production of 1 liter of fungal transformation media

### Materials

* 342.30 g sucrose
* 50.0 mL Nitrate salts
* 1 mL Trace metal elements
* 1 mL Thiamine
* 21 g Bacto agar

### Procedure

* Mix ingredients in flask and add MiliQ water to 1 litre.
* Mix with magnet stirrer until the sugar is dissolved.
* Autoclave at 121 °C for 20 min.

### Click below for a lab friendly version of this protocol

[Download File](https://static.igem.org/mediawiki/2019/8/8e/T--DTU-Denmark--Transformation_media.pdf)

USER-cloning

The following describes the USER-cloning protocol used during our project.

### Materials

#### Reagents

* USER-linearized insert(s)
* USER-linearized backbone
* USER enzyme
* CutSmart buffer
* Milli-Q water

#### Consumables

* PCR-strip/tubes

### Procedure

#### Preparing USER-reaction

* In a PCR-tube, combine;
  + 200 ng USER-linearized insert(s)
  + 100 ng USER-linearized vector backbone
  + 1 µL 10X CutSmart buffer
  + 1 µL USER enzyme
  + H2O to 10 µL.* Incubate reaction mix as described below.

#### Incubating reaction mix

* In a PCR-machine, incubate as follows.

+ 37 °C for 25 min
+ 25 °C for 10 min
+ 20 °C for 10 min
+ 15 °CC for 10 min
+ 10 °C until further use

* Transform reaction mixture in *E. coli* as described in the [Hifi assembly protocol](#HifiDNAass)

### Click below for a lab friendly version of this protocol

[Download File](https://static.igem.org/mediawiki/2019/4/40/T--DTU-Denmark--USER_cloning.pdf)

[![](https://static.igem.org/mediawiki/2019/f/fe/T--DTU-Denmark--happylogotemp.png)](https://2019.igem.org/Team%3ADTU-Denmark)

##### Useful links

[Home](https://2019.igem.org/Team%3ADTU-Denmark)
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[Project description](https://2019.igem.org/Team%3ADTU-Denmark/Description)
·
[Modelling](https://2019.igem.org/Team%3ADTU-Denmark/Model)
·
[Parts overview](https://2019.igem.org/Team%3ADTU-Denmark/Parts)
·
[Notebook](https://2019.igem.org/Team%3ADTU-Denmark/Notebook)
·
[Team](https://2019.igem.org/Team%3ADTU-Denmark/Team)
·
[Safety](https://2019.igem.org/Team%3ADTU-Denmark/Safety)
·
[Human practices](https://2019.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

![The logos of our three biggest supporters, DTU Blue Dot, Novo Nordisk fonden and Otto Mønsted fonden](https://static.igem.org/mediawiki/2019/9/91/T--DTU-Denmark--biggrants.svg "The logos of our three biggest supporters, DTU Blue Dot, Novo Nordisk fonden and Otto Mønsted fonden")
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# Team:DTU-Denmark/Results

[![logo](https://static.igem.org/mediawiki/2019/f/fe/T--DTU-Denmark--happylogotemp.png)](https://2019.igem.org/Team%3ADTU-Denmark)

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* [Project](https://2019.igem.org/Team%3ADTU-Denmark/Description)

  [Description](https://2019.igem.org/Team%3ADTU-Denmark/Description)
  [Design](https://2019.igem.org/Team%3ADTU-Denmark/Design)
  [Promoter Design](https://2019.igem.org/Team%3ADTU-Denmark/Design_Promoter)
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  [Results](https://2019.igem.org/Team%3ADTU-Denmark/Results)
  [Demonstrate](https://2019.igem.org/Team%3ADTU-Denmark/Demonstrate)
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* [Modelling](https://2019.igem.org/Team%3ADTU-Denmark/Model)

  [Model](https://2019.igem.org/Team%3ADTU-Denmark/Model)
  [Software](https://2019.igem.org/Team%3ADTU-Denmark/Software)
* [Parts](https://2019.igem.org/Team%3ADTU-Denmark/Parts)

  [Parts Overview](https://2019.igem.org/Team%3ADTU-Denmark/Parts)
  [Basic Parts](https://2019.igem.org/Team%3ADTU-Denmark/Basic_Part)
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  [Part Collection](https://2019.igem.org/Team%3ADTU-Denmark/Part_Collection)
* [Journal](https://2019.igem.org/Team%3ADTU-Denmark/Notebook)

  [Notebook](https://2019.igem.org/Team%3ADTU-Denmark/Notebook)
  [Experiments](https://2019.igem.org/Team%3ADTU-Denmark/Experiments)
* [People](https://2019.igem.org/Team%3ADTU-Denmark/Team)

  [Team Members](https://2019.igem.org/Team%3ADTU-Denmark/Team)
  [Collaboration](https://2019.igem.org/Team%3ADTU-Denmark/Collaborations)
  [Attributions](https://2019.igem.org/Team%3ADTU-Denmark/Attributions)
* [Human Practices](https://2019.igem.org/Team%3ADTU-Denmark/Integrated_Human_Practices)

  [Integrated Human Practices](https://2019.igem.org/Team%3ADTU-Denmark/Integrated_Human_Practices)
  [Human Practices](https://2019.igem.org/Team%3ADTU-Denmark/Human_Practices)
  [Education and Engagement](https://2019.igem.org/Team%3ADTU-Denmark/Public_Engagement)
  [Biobrick Tutorial](https://2019.igem.org/Team%3ADTU-Denmark/BioBrick_Tutorial)

[LEAP](https://2019.igem.org/Team%3ADTU-Denmark)

Project

[Description](https://2019.igem.org/Team%3ADTU-Denmark/Description)
[Design](https://2019.igem.org/Team%3ADTU-Denmark/Design)
[Promoter Design](https://2019.igem.org/Team%3ADTU-Denmark/Design_Promoter)
[Vector Design](https://2019.igem.org/Team%3ADTU-Denmark/Design_Plasmid)
[Measurement Design](https://2019.igem.org/Team%3ADTU-Denmark/Design_Measurement)
[Results](https://2019.igem.org/Team%3ADTU-Denmark/Results)
[Demonstrate](https://2019.igem.org/Team%3ADTU-Denmark/Demonstrate)
[Characterization](https://2019.igem.org/Team%3ADTU-Denmark/Contribution)

Modelling

[Model](https://2019.igem.org/Team%3ADTU-Denmark/Model)
[Software](https://2019.igem.org/Team%3ADTU-Denmark/Software)

Parts

[Parts](https://2019.igem.org/Team%3ADTU-Denmark/Parts)
[Basic\_Part](https://2019.igem.org/Team%3ADTU-Denmark/Basic_Part)
[Composite\_Part](https://2019.igem.org/Team%3ADTU-Denmark/Composite_Part)
[Part\_Collection](https://2019.igem.org/Team%3ADTU-Denmark/Part_Collection)

Journal

[Notebook](https://2019.igem.org/Team%3ADTU-Denmark/Notebook)
[Experiments](https://2019.igem.org/Team%3ADTU-Denmark/Experiments)

People

[Team Members](https://2019.igem.org/Team%3ADTU-Denmark/Team)
[Collaborations](https://2019.igem.org/Team%3ADTU-Denmark/Collaborations)
[Attributions](https://2019.igem.org/Team%3ADTU-Denmark/Attributions)

[Safety](https://2019.igem.org/Team%3ADTU-Denmark/Safety)
Human Practices

[Integrated Human Practices](https://2019.igem.org/Team%3ADTU-Denmark/Integrated_Human_Practices)
[Human Practices](https://2019.igem.org/Team%3ADTU-Denmark/Human_Practices)
[Education & Engagement](https://2019.igem.org/Team%3ADTU-Denmark/Public_Engagement)
[BioBrick Tutorial](https://2019.igem.org/Team%3ADTU-Denmark/BioBrick_Tutorial)

![](https://static.igem.org/mediawiki/2019/e/ea/T--DTU-Denmark--background3.png)
![](https://static.igem.org/mediawiki/2019/4/4b/T--DTU-Denmark--background4.png)

![](https://static.igem.org/mediawiki/2019/7/78/T--DTU-Denmark--resultsheader.svg)

## Results

All iGEM projects generate a lot of awesome results, and we are not an exception!

We set out to design a piece of software that could be used to create synthetic constitutive promoters, and we wanted to build a promoter library in *Aspergillus niger* as a proof of concept to demonstrate that the software worked on a fundamental level. By performing different experiments during the summer, we have been able to demonstrate the following:

* From our software “proHMMoter”, we are able to design promoters that are functional, while also being domesticated to be in compliance with common cloning standards.
* We have demonstrated that the promoters can work when cultivating in several different scales.
* The proHMMoter is able to produce promoters that have different dynamics, in a manner that can be predicted by modeling based on RNA-seq data
* proHMMoter can additionally be used to create promoters of varied strength

## Pick your Promoter

PLEAPglaA\_2
PLEAPsonB\_1
PLEAPgpdA\_1
PLEAPgpdA\_2
PLEAPmstA\_1
PLEAPunk\_1
PLEAPgfaA\_1
PLEAPhfbD\_1

## An03g06550 (BBa\_K3046001)

**Alias:** PLEAPglaA\_2

glaA encodes a secreted glucoamylase (1,4-alpha-D-glucan glucohydrolase), and is mostly expressed at the edge of the colonies. Furthermore, it is repressed by xylose and induced by maltose.

**Versions:** 1 naturalistic & 1 synthetically enhanced

![](https://static.igem.org/mediawiki/2019/thumb/5/50/T--DTU-Denmark--RNAseq_GlaA.png/650px-T--DTU-Denmark--RNAseq_GlaA.png)

RNA seq data derived from [1]

![](https://static.igem.org/mediawiki/parts/a/aa/T--DTU-Denmark--glaA_2promodynamics.png)

When analyzed in the microtiter scale on a biolector, the red fluorescence and biomass have been measured and the Dynamic Promoter Activity has been calculated. On the graph Dynamic Promoter Activity (DPA) is green, the biomass is measured in Light Scattering Units (LSU) is in blue, and red fluorescence (RFP) is shown in red. The graphs have been normalized for LSU

## An04g05630 (BBa\_K3046002)

**Alias:** PLEAPsonB\_1

The specific function of sonB\_1 in unknown, but it’s orthologs are associated with cellular response to DNA damage and regulation of the mitotic cell cycle.

**Versions:** 1 naturalistic & 1 synthetically enhanced

![](https://static.igem.org/mediawiki/2019/thumb/c/cd/T--DTU-Denmark--RNA_SonB.png/650px-T--DTU-Denmark--RNA_SonB.png)

RNA seq data derived from [1]

![](https://static.igem.org/mediawiki/parts/a/ac/T--DTU-Denmark--sonB_1promodynamics.png)

When analyzed in the microtiter scale on a biolector, the red fluorescence and biomass has been measured and the Dynamic Promoter Activity has been calculated. On the graph Dynamic Promoter Activity (DPA) is green, the biomass is measured in Light Scattering Units (LSU) is in blue, and red fluorescence (RFP) is shown in red. The graphs have been normalized for LSU

## An16g01830 (BBa\_K3046003)

**Alias:** PLEAPgpdA\_1

gpdA encodes a Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is an enzyme that is involved in glycolysis, and, according to new research, it may also be involved with initiation of apoptosis and vesicle shuttling.

**Versions:** 1 naturalistic & 1 synthetically enhanced

![](https://static.igem.org/mediawiki/2019/thumb/b/b6/T--DTU-Denmark--RNA_gdpA.png/650px-T--DTU-Denmark--RNA_gdpA.png)

RNA seq data derived from [1]

![](https://static.igem.org/mediawiki/parts/5/56/T--DTU-Denmark--gpdA_1promodynamics.png)

When analyzed in the microtiter scale on a biolector, the red fluorescence and biomass has been measured and the Dynamic Promoter Activity has been calculated. On the graph Dynamic Promoter Activity (DPA) is green, the biomass is measured in Light Scattering Units (LSU) is in blue, and red fluorescence (RFP) is shown in red. The graphs have been normalized for LSU

## An16g01830 (BBa\_K3046004)

**Alias:** PLEAPgpdA\_2

gpdA encodes a Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is an enzyme that is involved in glycolysis, and, according to new research, it may also be involved with initiation of apoptosis and vesicle shuttling.

**Versions:** 1 naturalistic & 1 synthetically enhanced

![](https://static.igem.org/mediawiki/2019/thumb/b/b6/T--DTU-Denmark--RNA_gdpA.png/650px-T--DTU-Denmark--RNA_gdpA.png)

RNA seq data derived from [1]

![](https://static.igem.org/mediawiki/parts/b/bd/T--DTU-Denmark--gpdA_2promodynamics.png)

When analyzed in the microtiter scale on a biolector, the red fluorescence and biomass has been measured and the Dynamic Promoter Activity has been calculated. On the graph Dynamic Promoter Activity (DPA) is green, the biomass is measured in Light Scattering Units (LSU) is in blue, and red fluorescence (RFP) is shown in red. The graphs have been normalized for LSU

## An12g07450 (BBa\_K3046005

**Alias:** PLEAPmstA\_1

mstA encodes a high-affinity sugar transporter and has been shown to be expressed when A. niger starts to run out of sugar. It doesn't care about the age/phase of the culture, only about sugar

**Special Ability:** sensitive sugar sensor, turns on when sugar is limited

**Versions:** 1 naturalistic & 1 synthetically enhanced

![](https://static.igem.org/mediawiki/2019/thumb/2/25/T--DTU-Denmark--RNA_MstA.png/650px-T--DTU-Denmark--RNA_MstA.png)

RNA seq data derived from [1]

![](https://static.igem.org/mediawiki/parts/c/c8/T--DTU-Denmark--mstA_1promodynamics.png)

When analyzed in the microtiter scale on a biolector, the red fluorescence and biomass has been measured and the Dynamic Promoter Activity has been calculated. On the graph Dynamic Promoter Activity (DPA) is green, the biomass is measured in Light Scattering Units (LSU) is in blue, and red fluorescence (RFP) is shown in red. The graphs have been normalized for LSU

## An07g08210 (BBa\_K3046006)

**Alias:** PLEAPunk\_1

This gene has not been characterized in Aspergillus niger yet, but in other aspergilli, it seems to be involved in signal transduction along actin filaments by activating kinases.

**Versions:** 1 naturalistic & 1 synthetically enhanced

![](https://static.igem.org/mediawiki/2019/thumb/3/37/T--DTU-Denmark--AN07pngRNA.png/650px-T--DTU-Denmark--AN07pngRNA.png)

RNA seq data derived from [1]

![](https://static.igem.org/mediawiki/parts/3/3e/T--DTU-Denmark--unk1promodynamics.png)

When analyzed in the microtiter scale on a biolector, the red fluorescence and biomass has been measured and the Dynamic Promoter Activity has been calculated. On the graph Dynamic Promoter Activity (DPA) is green, the biomass is measured in Light Scattering Units (LSU) is in blue, and red fluorescence (RFP) is shown in red. The graphs have been normalized for LSU

## An18g06820 (BBa\_K3046007)

**Alias:** PLEAPgfaA\_1

This gene encodes a gene called putative glutamine:fructose-6-phosphate amidotransferase (which is a mouthful) and is involved with the synthesis of the chitin that makes up the cell wall of fungi.

**Versions:** 1 naturalistic & 1 synthetically enhanced

![](https://static.igem.org/mediawiki/2019/thumb/a/a7/T--DTU-Denmark--RNA_gfaA.png/650px-T--DTU-Denmark--RNA_gfaA.png)

RNA seq data derived from [1]

![](https://static.igem.org/mediawiki/parts/0/0a/T--DTU-Denmark--gfa1promodynamics.png)

When analyzed in the microtiter scale on a biolector, the red fluorescence and biomass has been measured and the Dynamic Promoter Activity has been calculated. On the graph Dynamic Promoter Activity (DPA) is green, the biomass is measured in Light Scattering Units (LSU) is in blue, and red fluorescence (RFP) is shown in red. The graphs have been normalized for LSU

## An08g09880 (BBa\_K3046008)

**Alias:** PLEAPhfbD\_1

The hfbD gene supposedly codes for a hydrophobin gene that is active in the dormant conidia of Aspergillus niger. This gene has a special place in our hearts as it’s expression pattern is wonderfully specific.

**Versions:** 1 naturalistic & 1 synthetically enhanced

![](https://static.igem.org/mediawiki/2019/thumb/1/13/T--DTU-Denmark--hfbDwikiRNA.png/650px-T--DTU-Denmark--hfbDwikiRNA.png)

RNA seq data derived from [1]

![](https://static.igem.org/mediawiki/parts/7/77/T--DTU-Denmark--hfbD_1promodynamics.png)

When analyzed in the microtiter scale on a biolector, the red fluorescence and biomass has been measured and the Dynamic Promoter Activity has been calculated. On the graph Dynamic Promoter Activity (DPA) is green, the biomass is measured in Light Scattering Units (LSU) is in blue, and red fluorescence (RFP) is shown in red. The graphs have been normalized for LSU

## Library construction

We were able to integrate all our tested promoters into our promoter MoClo test device. As mentioned on our vector design page, the test device is designed with a screening mechanism for Golden Gate cloning that should result in red colonies if the plasmid has been re-ligated, and white colonies if the placeholder promoter has been replaced in favor of an insert promoter. As seen in figure 1, the system appears to work, as we can see mCherry being expressed by the vast majority of cells when the placeholder promoter has not been removed. When inserting the promoters, we can conversely see that the vast majority of colonies turn white, while a subset of the transformants keeps expressing mCherry. Sanger sequencing confirmed that the white colonies contained the inserted synthetic promoters as expected.

![Two agar plates can be seen: one with many red E. coli colonies and onw with white.](https://static.igem.org/mediawiki/2019/c/c8/T--DTU-Denmark--Resultsfig1.png)

Fig. 1: shows the transformants before and after the insertion of the MoClo promoter test plasmid. The left part of the figure shows that there is a production of mCherry if the MoClo promoter is not inserted, whereas to the right, it can be seen that the transformants with the inserted MoClo promoters stop producing mCherry.

## Qualitative results to demonstrate working promoters

After confirming that our test device worked as expected, we wanted to verify that we were able to express mCherry from our promoters. To do this, we inserted our promoter test device into the autonomously replicating AMA1-zing\_pyrG plasmid and used the plasmid to transform our *A. niger* ATCC1015 strain. From the fungal strains containing our test plasmids, we did a few short experiments to get some qualitative data on whether the promoters worked or not. We inoculated 10 mL cultures of minimal media in 50 mL ventilated falcon tubes and incubated them for 80 hours at 37 ℃ at 150 rpm. As seen in figure 2, the promoters were able to produce mCherry in sufficient quantities for it to be visible in the culture compared to the negative control.

![Six falcon tubes, each containing a fungal sample with different promoters expressing mCherry](https://static.igem.org/mediawiki/2019/e/ef/T--DTU-Denmark--result_pHS.png)

Fig. 2: Qualitative expression of mCherry from the LEAP promoters. To the left is the negative control, consisting of *A. niger* ATCC1015 with no test device transformed. The five tubes on the right all transformants containing the fungal promoter test device, each with a different synthetic promoter inserted into the test device.

![This figure shows results from a confocal microscope and shows a vast net of mycelia that is expressing mCherry and fluorescing brightly red](https://static.igem.org/mediawiki/2019/c/ca/T--DTU-Denmark--Redcell2.png)

Fig. 3: Confocal microscopy with excitation at 580 nm. As can be seen, mCherry is produced from both LEAP promoters, with no red colour from the negative control. The PLEAPglaA\_2 promoter appears to express mCherry to the same degree or higher than the positive control.

When looking at the culture samples through a confocal microscope, we are similarly able to see a clear difference in fluorescence between the sample and the negative control. The fluorescent images in figure 3 of the mCherry expressed by our fungal strains were taken by a confocal microscope. There's a clear fluorescence from synthetic promoter constructs, when compared to the negative control.

![This figure was taken on a confocal microscope and shows a vast net of mycelia that is expressing mCherry and fluorescing brightly red](https://static.igem.org/mediawiki/2019/c/ca/T--DTU-Denmark--Redcell2.png)

Fig. 3: Confocal microscopy with excitation at 580 nm. As can be seen, mCherry is produced from both LEAP promoters, with no red colour from the negative control. The PLEAPglaA\_2 promoter appears to express mCherry to the same degree or higher than the positive control.

## BioLector data

Microplate scale experiments was carried out by using a BioLector as mentioned. Each well was inoculated with 107 spores in 1,5 mL minimal media. For positive control, we used a PH4H3 histone promoter expressing mCherry in a reporter device that was genome integrated in *A. nidulans*. For negative control, *A. niger* transformed with an AMA1 plasmid with no mCherry device was used.

![This figure shows fluorescence measurements from the biolector where multiple graphs are plotted at once. Each graph shows the fluorescence intensity from the expression of one promoter, adjusted for biomass and normalised for background noise (based on the negative control). When adjusting for biomass, many of the promoters are producing more mCherry than the negative control, meaning an expression above 0 on this graph.](https://static.igem.org/mediawiki/2019/thumb/9/90/T--DTU-Denmark--RFU_ladder.png/654px-T--DTU-Denmark--RFU_ladder.png)

Fig. 4: When plotting the fluorescence at Emm/Exi = 580nm/625 nm, it can be observed that at least 7 of the synthetic promoters tested in this BioLector run was expressing mCherry at a level between the negative and positive control.

The graphs above display data collected from BioLector cultivations of a range of our synthetic promoters. The black lines represents the positive control, and all the other lines represent LEAP promoters of varying strengths. The results from figure 4 demonstrates our software’s ability to predict and create a synthetic promoter ladder, i.e. promoters of varying strengths, for a given organism. The biomass used for the graph is estimated by measuring light scattering units (LSU) of the different strains. Additional data is available both on our Demonstrate page, our parts pages on the Registry, and in our “pick-your-promoter” widget.

## Shakeflask data

We tested three of the LEAP promoters in 200 mL cultures in Erlenmeyer flasks, to see how the promoters would behave when scaling up from BioLector wells. Normalized fluorescence data from all the shake flasks can be seen in figure 6

![This figure shows fluorescence measurements from the shake flask experiments where five promoter were tested in duplicated. ](https://static.igem.org/mediawiki/parts/0/05/T--DTU-Denmark--shakeflasksunk1.png)

Fig. 6: Expressed fluorescent protein per total protein over time. X-axis is time in hours after inoculation. Y-axis is fluorescence given as an equivalent of the red fluorescent compound TexasRed.

From the figure above it can be observed that the PLEAPglaA\_1 and PLEAPsonB\_1 promoters express a relatively low amount of protein, whereas the PLEAPglaA\_2 and PLEAPunk\_1 promoters express a higher amount when adjusting for total protein expression. Our data from figure 6 suggest that PLEAPglaA\_2 has the highest promoter activity.

Based on these results, we believe that we have succeeded in creating a promoter library. For more details, check our in-depth measurements on the [demonstrate page](https://2019.igem.org/Team%3ADTU-Denmark/Demonstrate) page.

Further results from side projects

Besides our main mission, which has been to demonstrate that our proHMMoter software works by building a promoter library, we have also worked on a lot of smaller projects that are all related to the goal of making synthetic biology in filamentous fungi easier and more accessible in the future.
First of all, we have collected some qualitative data for PtrpC - an *Aspergillus* spp. promoter that is already present in the Registry of Standard Biological Parts, but a part that did not have any quantitative data. For more info on the characterisation we did, look at our [characterization](https://2019.igem.org/Team%3ADTU-Denmark/Contribution) page.
Furthermore, we have succeeded in creating a prototype design for genome integrating promoters into the *Aspergillus niger* genome. pPEA2P1 (BBa\_K3046031) was shown to express eGFP as expected, and the successful transformants were subsequently used for the PtrpC characterization mentioned above.

[![](https://static.igem.org/mediawiki/2019/f/fe/T--DTU-Denmark--happylogotemp.png)](https://2019.igem.org/Team%3ADTU-Denmark)

##### Useful links

[Home](https://2019.igem.org/Team%3ADTU-Denmark)
·
[Project description](https://2019.igem.org/Team%3ADTU-Denmark/Description)
·
[Modelling](https://2019.igem.org/Team%3ADTU-Denmark/Model)
·
[Parts overview](https://2019.igem.org/Team%3ADTU-Denmark/Parts)
·
[Notebook](https://2019.igem.org/Team%3ADTU-Denmark/Notebook)
·
[Team](https://2019.igem.org/Team%3ADTU-Denmark/Team)
·
[Safety](https://2019.igem.org/Team%3ADTU-Denmark/Safety)
·
[Human practices](https://2019.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

![The logos of our three biggest supporters, DTU Blue Dot, Novo Nordisk fonden and Otto Mønsted fonden](https://static.igem.org/mediawiki/2019/9/91/T--DTU-Denmark--biggrants.svg "The logos of our three biggest supporters, DTU Blue Dot, Novo Nordisk fonden and Otto Mønsted fonden")
![The logos of all of our sponsors, DTU, BioNordica, Eurofins Genomics, Qiagen, NEB New England biolabs, IDT Integrated DNA technologies and Twist bioscience](https://static.igem.org/mediawiki/2019/d/d9/T--DTU-Denmark--sponsorsfooter.svg "The logos of all of our sponsors, DTU, BioNordica, Eurofins Genomics, Qiagen, NEB New England biolabs, IDT Integrated DNA technologies and Twist bioscience")