iGEM 2020 — Team DTU-Denmark

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# Team:DTU-Denmark/Notebook

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Project

[Description](https://2020.igem.org/Team%3ADTU-Denmark/Description)

---

[Contribution](https://2020.igem.org/Team%3ADTU-Denmark/Contribution)
[Engineering](https://2020.igem.org/Team%3ADTU-Denmark/Engineering)

---

[Implementation](https://2020.igem.org/Team%3ADTU-Denmark/Implementation)
[Proof of concept](https://2020.igem.org/Team%3ADTU-Denmark/Proof_Of_Concept)

Labwork

[Design](https://2020.igem.org/Team%3ADTU-Denmark/Design)
[Experiments](https://2020.igem.org/Team%3ADTU-Denmark/Experiments)
[Measurement](https://2020.igem.org/Team%3ADTU-Denmark/Measurement)
[Safety](https://2020.igem.org/Team%3ADTU-Denmark/Safety)

Modelling

[Model](https://2020.igem.org/Team%3ADTU-Denmark/Model)
[Software](https://2020.igem.org/Team%3ADTU-Denmark/Software)

[Results](https://2020.igem.org/Team%3ADTU-Denmark/Results)
[Human Practices](https://2020.igem.org/Team%3ADTU-Denmark/Human_Practices)

Parts

[Parts](https://2020.igem.org/Team%3ADTU-Denmark/Parts)
[Part Collection](https://2020.igem.org/Team%3ADTU-Denmark/Part_Collection)

People

[Team](https://2020.igem.org/Team%3ADTU-Denmark/Team)
[Collaboration](https://2020.igem.org/Team%3ADTU-Denmark/Collaborations)
[Science communication](https://2020.igem.org/Team%3ADTU-Denmark/Education)
[Notebook](https://2020.igem.org/Team%3ADTU-Denmark/Notebook)
[Attributions](https://2020.igem.org/Team%3ADTU-Denmark/Attributions)
[Inclusion](https://2020.igem.org/Team%3ADTU-Denmark/Inclusion)

---

[Sponsorship](https://2020.igem.org/Team%3ADTU-Denmark/Sponsorship)

[Awards](https://2020.igem.org/Team%3ADTU-Denmark/Awards)
![](https://static.igem.org/mediawiki/2020/a/ae/T--DTU-Denmark--frontpage_prize_new.svg)

![](https://static.igem.org/mediawiki/2020/a/ae/T--DTU-Denmark--banner-notebook.svg)

* Back to top

## Here is an overview of what we have done throughout our project.

* Feb
* Mar
* Apr
* May
* Jun
* Jul
* Aug
* Sep
* Oct

# February notebook

## February 10, 2020 - February 16, 2020

**General:**
We met each other for the first time, formed subgroups and decided having a meeting every Monday followed by dinner.

**Wetlab:**
We had a meeting for Introduction to lab Safety with the faculty members.

## February 17, 2020 - February 23, 2020

**General:**
All subgroups had their introductory meetings where they brainstormed their goals.

## February 24, 2020 - March 1, 2020

**General:**
We did research to get inspiration for project ideas.

**Outreach:**
We took over SoMe accounts and discussed logo ideas for our project.

# March notebook

## March 2, 2020 - March 8, 2020

**General:**
We brainstormed ideas for the project and read previous iGEM Judging forms. The team also submitted a Lab safety test.

**Outreach:**
We took over Instagram, Twitter and Facebook accounts for the DTU iGEM team.

**Economy:**
We took pictures of team members for funding applications and started contacting people for funding advice.

**Team Building:**
We had our first team building event, a fun PubQuiz at a local pub, and the second event was planned during this week.

**Admin:**
We prepared a team contract and got everyone’s signatures on it.

![](https://static.igem.org/mediawiki/2020/8/89/T--DTU-Denmark--1stteampic.png)

### First picture of the whole team including 3 of our 4 supervisors.

## March 9, 2020 - March 15, 2020

**General:**
All team members pitched different ideas and received feedback from the team and supervisors. A few examples are: water remediation - to remove pesticides, psychotropic drugs - fabricating novel synthetic psychotropic drugs to fight psychological disorders, and optimizing hyphae production in filamentous fungi.

**Outreach:**
We attended and presented at the Open House event at our university, where we talked about synthetic biology and iGEM.

**Economy:**
We got the first team picture taken.

**Admin:**
We registered as an iGEM Team and discussed the Nordic iGEM meet up in Linköping.

## March 16, 2020 - March 22, 2020

**General:**
We narrowed down our ideas range, and further discussed the brainstorming ideas. We set a deadline for deciding on an idea next week. We were all sent home and travel bans had come into place. DTU was closed due to Covid-19 Pandemic. We also discussed options for the BioBrick Tutorial.

**Outreach:**
We discussed possible future events.

**Economy:**
We formed a list of possible funding to apply for, taking inspiration from last year's DTU iGEM team.

**Team Building:**
We brainstormed ideas for future meet ups if restrictions allow it - Escape room might be a possibility.

![](https://static.igem.org/mediawiki/2020/3/3d/T--DTU-Denmark--DTUopenhouse.png)

### Happy BioBuilders at DTU open house.

## March 23, 2020 - March 29, 2020

**General:**
We decided to work on improving the morphology of filamentous fungi and creating a signal peptide library. BioBrick Tutorial was postponed due to Covid-19 Pandemic, making a virtual event may not be feasible. We made a new subgroup for WIki.

**Outreach:**
We worked on designing a logo.

**Drylab:**
We started looking into previous models of morphology of filamentous fungi. We sought to find out what initiatives have been taken, what challenges have been discovered and how these have been met.

**Wetlab:**

* ***Morphology*:** We discussed different strategies for engineering morphology.
* ***Protein secretion*:** We did research on signal peptides.

**Human Practice:**
We brainstormed a list of people to whom we would reach out.

**Economy:**
We requested reference letters from supervisors and faculty personnel for funding applications. We prepared our first draft for a funding application and got our budget approved from the supervisors.

**Team building:**
We had an Online movie night (Netflix party).

**Admin:**
We started preparing for our lab work by coordinating planning and protocols, in case the restrictions are lifted later.

# April notebook

## March 30, 2020 - April 5, 2020

**General:**
We did further research on ideas and presented our research slides to the team. We continue to have meetings on Zoom in order to comply with the Danish lockdown. Our iGEM ambassador had a meeting with other iGEM ambassadors to discuss lockdown restrictions and their influence on iGEM 2020. We cancelled the BioBrick Tutorial due to Covid-19 Pandemic. We agreed on making an offline version of our wiki.

**Outreach:**
We prepared and posted a teaser for our project revealed on the social media platforms. We decided to do a crochet pattern of a fungus as a kind of team mascot.

**Drylab:**
We brainstormed different ideas for growth modelling of filamentous fungi based on the literature that we had read and furthermore, we started gathering relevant data sets of microscopic images from different papers.
**Wetlab:**

* ***Morphology*:** We did research on different morphologies of filamentous fungi - pelleted, dispersed and so on. We discussed different filamentous fungi as chassis organisms.
* ***Protein secretion*:** We did research on different proteins and enzymes of interest for increased protein secretion.

**Human Practice:**
We reviewed the list of people to contact, and took some inspiration from the list of people from last year's team.
**Economy:**
We got some feedback on our funding application cover letter.

![](https://static.igem.org/mediawiki/2020/d/df/T--DTU-Denmark--zoompic.png)

### Even though we were restricted to being home due to Covid-19, our teamspirit and moral was sky high!

## April 6, 2020 - April 12, 2020

**General:**
We formulated a hypothesis for our project and decided to work with A. niger. We had presentations of different genes of interest for morphology and signal peptides.

**Outreach:**
We made our first draft of the logo/mascot and worked on creating crochet mascots.

**Drylab:**
We started our discussion of what features would be relevant to include in our growth model, we were also granted access to the growth model developed by the DTU biobuilders 2018, which could then take part in our discussions.

**Wetlab:**

* ***Morphology*:** We made a first draft with a list of genes of interest to engineer morphology.
* ***Protein secretion*:** We started testing the *in silico* tool SignalP 5.0.

## April 13, 2020 - April 19, 2020

**General:**
We had Easter holiday in Denmark. Therefore, no iGEM meetings were held this week.

## April 20, 2020 - April 26, 2020

**General:**
We discussed the goal of the project: a better fungal cell factory?

**Outreach:**
We contacted photographers for headshots of each team member.

**Drylab:**
We worked on converting the model from the DTU BioBuilders 2018 from R to Python to use it as a starting template. We continued the discussion of potential parameters of interest for modelling the morphology of A. niger, in particular we discussed branching frequency, hyphal length, and hyphal densities.

**Wetlab:**

* ***Morphology*:** We made a list of potential target genes. We made presentations on pros/cons of working with each gene.
* ***Protein secretion*:** We held a discussion on whether a library of signal peptides is feasible to make. We did further research on signal peptides. Glucoamylase was identified as a protein of interest.

**Human Practice:**
We sent emails to bioindustry personnel. We also discovered that we needed to contact people with a bioinformatics viewpoint.

## April 27, 2020 - May 3, 2020

**General:**
We found out that this year’s Jamboree will be online.

**Outreach:**
We were contacted by IIT Madras regarding a collaboration. We discussed a Wetlab-based collaboration idea with a Norwegian team.

**Drylab:**
We discussed how to improve the growth models that had already been developed, such as graph based models, using 2D/3D images and mixing. We continued the discussion of what other parameters to consider, and we finished the conversion of the 2018 growth model from R to python.

**Wetlab:**

* ***Morphology*:** We discussed how to obtain quantitative data and ideas were exchanged on possible types of microscopes that we could consider for this.
* ***Protein secretion*:** We discussed different strategies for quantitative measurements: fluorescence assays, spectrophotometers, etc. We worked on identifying ten native signal peptides.

**Human Practice:**
We had meetings with Mhairi McIntyre Workman (Associate Manager - Novo Nordisk), Kim Hansen (Senior science manager - Novozymes), Jens Frisvad (Professor - DTU Bioengineering), Dr. Edyta Szewczyk (Senior Scientist in Biology - Bolt Threads), and Kenneth Jensen (Science Manager and Project Leader - Novozymes).

**Economy:**
We discussed different costs related to the project. More recommendation letters were received during this week.

# May notebook

## May 4, 2020 - May 10, 2020

**General:**
We discussed different options for filming for the promotional and presentation videos.
**Outreach:**
We had a meeting with IIT Madras, they had to withdraw due to Covid-19 Pandemic.

**Drylab:**
We wanted to base our growth model on experimental data, so we started looking into methods for doing good microscopy of fungi and we also looked at different image analysis tools that could help us extract parameters from the data sets that we had received.
**Wetlab:**

* ***Morphology*:** We held a presentation on genes, all seven genes were decided for engineering.
* ***Protein secretion*:** We worked on researching potential signal peptides for our project.

**Human Practice:**
We had meetings with Morten Skov Jacobsen (Head of fermentation pilot plant - Novozymes) and José Luis Martinez Ruiz (Associate Professor - DTU Bioengineering).

## May 11, 2020 - May 17, 2020

**General:**
We had no meetings or work on iGEM during this week due to exams at DTU.

## May 18, 2020 - May 24, 2020

**General:**
We had no meetings or work on iGEM during this week due to exams at DTU.

## May 25, 2020 - May 31, 2020

**General:**
We discussed our prospects for lab work with our supervisors. They promised to find some spots for us. We made a new subgroup for making project videos.

**Outreach:**
We held a discussion as a follow up on our collaboration with IIT-Madras.

**Drylab:**
We continued our work on investigating microscopy protocols and started testing different image analysis tools on the data sets that we had received.

**Wetlab:**

* ***Morphology*:** We started planning different strategies for cloning of our chosen genes. We made flowsheets of our experimental strategy.
* ***Protein secretion*:** We retrieved protein secretion data to find the most secreted proteins. We found some DNA sequences of signal peptides and analyzed them using SignalP-5.0.

**Human Practice:**
We held a meeting with Drylab to discuss model ideas with different bioindustry personnel.

**Economy:**
We got a sponsorship from Amplicon.

**Admin:**
We also discussed different medal criteria with the team.

# June notebook

## June 1, 2020 - June 7, 2020

**General:**
We all signed up for Benchling and Kyle gave a Benchling tutorial.

**Outreach:**
We published two crochet patterns for our team mascots on Ravelry. We are still working on the IIT-Madras collaboration. We set a date for team photography.

**Drylab:**
We worked on modeling.

**Wetlab:**

* ***Morphology*:** We made a GMO risk assessment of our project. We made a decision to use Benchling as a lab book. We started working on designing primers and decided on using CRISPR Cas9 together with USER cloning to create knockouts.
* ***Protein secretion*:** We made a Flowsheet of our experimental strategy and started designing primers for knockout and insertion of *glaA*.

**Human Practice:**
We worked on documenting talks from bioindustry personnel.

![](https://static.igem.org/mediawiki/2020/1/19/T--DTU-Denmark--Aspergillus-conid-picture.png)
![](https://static.igem.org/mediawiki/2020/2/27/T--DTU-Denmark--Mushroom-picture.png)

### The two crochet patterns that were published on Ravelry.

## June 8, 2020 - June 14, 2020

**General:**
We went to the lab for the first time! With initial restrictions only allowing three people to be in our lab at any one time

**Outreach:**
A collaboration agreement document was prepared with IIT-Madras. We participated in the COPI (Corona Outreach Program Initiative) started by Uppsala iGEM team.

**Drylab:**
We started a journal club.

**Wetlab:**

* ***Morphology*:** We had a successful amplification of BioBricks through PCR reactions for gene knockouts. We also did digestion of backbone plasmid. We made a mistake in gel purification, so we had to re-do PCR reactions.
* ***Protein secretion*:** We had a successful amplification of BioBricks through PCR reactions for gene knockouts.

**Team building:**
We had an outdoor meetup in a park for games and as we say in Denmark “hygge”.

**Admin:**
We reached out to the Dusseldorf iGEM team. We met up with SDU and KU online to plan a Danish iGEM meetup.

![](https://static.igem.org/mediawiki/2020/6/60/T--DTU-Denmark--Upp_insta.png)

### An Instagram post regarding our situation during Covid-19 by iGEM Uppsala as a part of COPI.

## June 15, 2020 - June 21, 2020

**Outreach:**
We are in discussions regarding collaborations with Mexico and Dusseldorf. We received the team headshots.

**Drylab:**
We asked paper authors for image data and received some more. We continued testing different image analysis packages for microscopic image analysis and we started discussing how to do good microscopy, considering what we learned from literature.
**Wetlab:**

* ***Morphology*:** We purified our plasmid and did PCR of Biobricks. We had a slight setback because of failed gel purification. We worked on some missing protocols.
* ***Protein secretion*:** We worked on creating a vector for K/O of *glaA*. We created a vector for reinsertion of *glaA* with different signal peptides into the *albA* region. We had found 10 different signal peptides of interest from homologous proteins.

**Economy:**
We submitted our abstract to ASBE V.

![](https://static.igem.org/mediawiki/2020/2/28/T--DTU-Denmark--grill%26chill.jpg)

### Grill&Chill - it was nice to be gathered as a team again!

## June 22, 2020 - June 28, 2020

**Outreach:**
We planned a possible collaboration and meeting with FCB-UANL from Mexico. We are doing Instagram presentations of team members.
**Drylab:**
We discussed pros and cons of the different image analysis tools that we had tested. We also discussed the microscopy protocols that we had seen and we met with Claus Sternberg to test the confocal microscope.

**Wetlab:**

* ***Morphology*:** We did Plasmid purification (of pFC330 and pFC902) and PCR of Bioblocks. We assembled our plasmid with BB (USER cloning and E.coli transformation).We created protoplasts of A. niger and did our first colony PCR.
* ***Protein secretion*:** We did a colony PCR of transformants with pGIA2PI vector for reinsertion of glaA, it showed a strange band, about 1000 bp too short. For the transformants with pPFC330 vector for knockout of glaA - colony PCR showed no clear bands.

**Human Practice:**
We had an online meeting with IIT-Madras.

**Team building:**
We had a Grill & Chill with all the supervisors this week and are planning further events.

# July notebook

## June 29, 2020 - July 5, 2020

**Outreach:**
We had meetings with IIT-Madras, FCB-UANL, and Korea-HS about collaborations.

**Drylab:**
We met with Claus Sternberg regarding microscopy, and agreed on doing microscope training on July 8th. We received better image data from paper authors to test out the imaging tools that we found.

**Wetlab:**

* ***Morphology*:** We sent our plasmids (purified from *E. coli* colonies) for sequencing. We started brainstorming ideas on a new proposed system for gene insertion using the Tet system. We worked on designing primers for reinsertion. We held a meeting with Claus on June 23rd to test a microscope.
* ***Protein secretion*:** We re-did USER cloning and lots of colony PCRs for the transformants.

**Admin:**
We submitted our preliminary safety form.

## July 6, 2020 - July 12, 2020

**Outreach:**
We are looking at a collaboration with Maastricht about an article for an iGEM journal. We will participate in DTU intro week with a talk on iGEM and synthetic Biology.

**Drylab:**
We continued discussing
**Wetlab:**

* ***Morphology*:** The vector constructs were determined to be correct through sequencing data, we went further with knockouts. We worked on insertion strategy and primers for the Tet system. We had a meeting with Katarina to test the Tet system in *A. niger*.
* ***Protein secretion*:** Our experiments were delayed by water damage. So we had to restart USER cloning, and make new PCR fragments. The sequencing data showed problems with *glaA* insert. We did Colony PCR troubleshooting by restriction enzyme digest but it didn’t work out.

**Team building:**
We went on a sailing trip around Copenhagen harbour.

![](https://static.igem.org/mediawiki/2020/9/9d/T--DTU-Denmark--sailing.png)

### Sailing in the canals of Copenhagen.

## July 13, 2020 - July 19, 2020

**Drylab:**
We will be working on image analysis because we want to improve estimates of parameters for branching angles, growth rate, and branching rate compared to the existing model. We are testing out tools for segmentation, binarization and vectorisation. We will work with image data from papers for now until we can get images of our own fungi.

**Wetlab:**

* ***Morphology*:** We designed insertion primers and ordered them. received training for the microscope.
* ***Protein secretion*:** Our experiments were delayed by water damage. So we had to restart USER cloning, and make new PCR fragments. The sequencing data showed problems with *glaA* insert. We did Colony PCR troubleshooting by restriction enzyme digest but it didn’t work out.

## July 20, 2020 - July 26, 2020

**General:**
We are working on the storyboard for the promotion video.

**Outreach:**
We had a meeting with FCB-UANL. Some of the team went to a Danish iGEM meetup at SDU.

**Drylab:**
We continued to test out different image analysis tools on paper data sets and sent out requests for more data sets from other papers.

**Wetlab:**

* ***Morphology*:** We did spore dissection of *A. niger* colonies with successful knockouts. We extracted gDNA for amplifying gene sequences. We did mini-prep for template plasmid for insertion experiments. We fixated *A. niger* with TM agar and observed its growth to give data to the Drylab team.
* ***Protein secretion*:** We had positive *A. niger* transformants through Tissue PCR, these were reinoculated on new plates. We struggled growing the *E. coli* transformed with the SigP switching plasmids. We did monosporic purification to form homokaryons of the *ΔglaA A. niger* strain.

![](https://static.igem.org/mediawiki/2020/e/ef/T--DTU-Denmark--Danish_igem_meetup.png)

### All the participants at the danish iGEM meetup. Thanks to SDU for hosting!

## July 27, 2020 - August 2, 2020

**Outreach:**
We had some follow-up meetings on collaborations.

**Drylab:**
Claus is be back next week, so we can get some microcopy instruction and start taking pictures. Therefore, we started planning our microscopy. We also talked to people from SDU regarding 3D printing of microscope chambers.

**Wetlab:**

* ***Morphology*:** We did lots of PCRs for insertion of genes, and the majority of these didn’t work. We were deciding to drop weak and possible medium promoters due to lack of time. We had a very productive meeting together with the whole wetlab team on rough planning on project collaboration between both subgroups.
* ***Protein secretion*:** We did a re-inoculation of spores from *ΔglaA* as they were overgrown. We also did Tissue PCR with lots of positives, seven of these were re-inoculated on plates. The PhusionU PCR didn't work again, We will be changing our strategy for this PCR and ordered new primers. We also did Spore dissection and inoculation of isolated spores on 5-FOA to get the plasmid out. With our troubleshooting PCR’s starting mid week

**Admin:**
We selected our iGEM track: Manufacturing.

![](https://static.igem.org/mediawiki/2019/d/d3/HQ18_TrackIcon_Manufacturing.svg)

### We chose to compete in the manufacturing track.

# August notebook

## August 3, 2020 - August 9, 2020

**Outreach:**
We met with Aakii from PF, the DTU students’ association, regarding our participation in the Masters’ introduction week.

**Drylab:**
We selected and ordered stain (calcufluor-white) and perfusion chambers for microscopy. We scheduled time with Claus for microscope instructions. We also started working on our own image analysis tool as the majority of image analysis tools were not flexible enough to tune to our needs.

**Wetlab:**

* ***Morphology*:** We re-designed primers for non-inducible promoter systems. We plated KO strains on 5-FOA plates and made a stock of our KO strains. We started protoplastation of KO strains to prepare for double KOs.
* ***Protein secretion*:** We did Spore dissection but the needle broke so we had a short delay. We also did USER cloning, transformation in *E. coli*, Colony PCR and sent SignalPs for sequencing. We will do Spore dissection and inoculation on 5-FOA to get the plasmid out.

## August 10, 2020 - August 16, 2020

**Drylab:**
We attempted our first microscopic slides with help from the wetlab team. We held a Wiki meeting for assigning tasks to all team members and the wetlab team came up with the idea of computing wide-functioning synthetic signal peptides.

**Wetlab:**

* ***Morphology*:** We have run the Biolector for three of the knockout strains, We prepared to make stock and protoplasts of all the knockouts. We did amplification of parts for re-insertion vectors.
* ***Protein secretion*:** We mastered spore dissection and inoculated isolated spores on 5-FOA with 10/10 *ΔglaA A. niger* strains. We did the Starch-iodine assay and saw minor differences between K/O and WT, We had 7/10 positive sequences for SigP switching. However, there is a point mutation in the glucoamylase gene, but it is silent. We also did PCRs for linearizing SigP fragments, Protoplasting the *ΔglaA A. niger* strain and transformation with the linearized SigP fragments.

![](https://static.igem.org/mediawiki/2020/2/26/T--DTU-Denmark--hyphae_stains.png)

### Digitized image of *A. niger* hyphae.

## August 17, 2020 - August 23, 2020

**General:**
We filmed our promotion video. We also talked about options for meetups for the Giant Jamboree.

**Outreach:**
We will participate in Naturvidenskabsfestivalen, where several pairs of team members will present workshops on filamentous fungi at primary schools in the greater Copenhagen area.

**Drylab:**
We continued working on achieving better microscopic images - thanks to the wetlab team for preparing the slides! We met with Henrik Nielsen to learn more about his program SignalP and how we could apply it for predicting synthetic signal peptides.

**Wetlab:**

* ***Morphology*:** We made protoplasts of knockouts (4 strains), we started double knockouts and tried to assemble the promoter-terminator-backbone plasmid, We’re not sure if it is correct - it might be the primers that bind to several places. All genes are ready for insertion,we checked the double knockouts and created promoter-gene-term plasmid for insertion. plasmid.
* ***Protein secretion*:** We did protoplastation of *ΔglaA A. niger* strain, we made digestion of pGIA2p1 to linearize instead of PCR linearization and transformed protoplast with CRISPR knockout vectors (from morph) and 1 pGIA2p1 (to check if restriction digestion worked) for total of 33 transformations (biological triplicate and controls included). We did further protoplasting of the *ΔglaA A. niger* strain to start integrating the remaining signal peptides and formation of homokaryons on all double K/Os.

**Human Practice:**
We contacted people from DSM and Chr. Hansen.

**Admin:**
We started planning a trip to a summer house in our autumn break to write the bulk of our wiki.

## August 24, 2020- August 30, 2020

**Outreach:**
We presented a workshop for DTU Masters' introduction week.

**Drylab:**
We improved at growing our fungi for microscopic images, but we had some issues with contamination, so we started thinking about how to solve this. We started working on SignalPrepper, following ideas from Henrik Nielsen.

**Wetlab:**

* ***Morphology*:** We made a construct of promoter-term-backbone. (strong and weak) and sent it for sequencing, we also transformed A. niger for double knockout. We got positive colonies for double knockouts isolated (Tissue PCR and re-inoculation) and we did further transformation of the knockouts that did not work. We picked the double knockouts that were positive.
* ***Protein secretion*:** We reinoculated positive 6 morph K/O + *ΔglaA* strains. We got the last sequencing data. We had some weird contamination, we investigated the contamination and reinoculated our strains on 4 types of media and asked Jakob to help investigate. We did further protoplasting the *ΔglaA* strain to start integrating the remaining signal peptides.

![](https://static.igem.org/mediawiki/2020/f/f8/T--DTU-Denmark--masterdays_presentation.png)

### Master days presentation about our project, iGEM and synthetic biology for new master students on DTU.

# September notebook

## August 31, 2020 - September 6, 2020

**General:**
The promo video is on it’s way!

**Drylab:**
We worked on improving our microscopy, and we continued working on the image analysis tool. Furthermore, we finished SignalPrepper and gave the resulting best scoring synthetic signal peptides to the Wetlab team.

**Wetlab:**

* ***Morphology*:** We did USER cloning of plasmid with genes, we isolated two doubles knockout strains and held a measurement meeting where we planned measurement steps with signalP-team. We ran biolector with double and single knockouts and did Glucoamylase and Bradford assay.
* ***Protein secretion*:** For the SigP switching inserts, we observed fungal growth and we suspect some positive colonies (Tissue PCR). We did monosporic purification of double knockouts (*ΔglaA* + 5x morph vectors) and reinoculated on 5-FOA. We made more protoplasts of *ΔglaA*. We did PCRs to have synthetic signal peptides (predicted by SignalPrepper) insert into the pGIA2p1 glaA plasmid.

**Human Practice:**
We met with Kenneth from Novozymes.

**Team Building:**
The team had a great evening trying to escape the clutches of a criminal mastermind in an Escape room. We, of course, prevailed!

![](https://static.igem.org/mediawiki/2020/c/c0/T--DTU-Denmark--fungus_unstained.png)

### Unstained picture of *A. niger*

## September 14, 2020 - September 20, 2020

**General:**
We participated in the Nordic iGEM conference and presented our preliminary work. The final cut of our promo video was ready for upload!

**Drylab:**
We took many pictures this week including the *ΔchsC\_ΔspaA* double knockout. Work on the image analysis model progressed, particularly we created target segmentation, cut the images into tiles, made a ‘data loader’, and started setting up the model itself. Work on the simulation model also progressed as we decided to implement substrate diffusion, lateral branching and chemotropism.

**Wetlab:** We started the first round of bioreactor runs! We also did Glucoamylase and BCA assays of morphology strains.

* ***Morphology*:** We found out that the Biolector runs from last week were contaminated. We re-did Glucoamylase assay.
* ***Protein secretion*:** We did USER cloning of five synthetic signal peptides for A. niger insertion. We made protoplasts of four of double knockouts , consisting of *RacA*, *Gul1*, *SpaA* or *chsC* and *ΔglaA*. We transformed the five synthetic signal peptides and the last native ones (*pmeA* and *faeA*).

## September 21, 2020 - September 27, 2020

**General:**
We worked on the concepts for the presentation video.

**Outreach:**
This week was Naturvidenskabsfestivalen and though we could not visit schools we gave several presentations virtually.

**Drylab:**
We continued our work on getting good microscopic images of all mutant strains, but some mutants were difficult to grow in such a way that we could get good pictures. We also continued working on the image analysis and the simulation models.

**Wetlab:**
We ran Bioreactor experiments with the reference strain, *ΔchsC* and *ΔspaA*. We performed glucoamylase assay and BCA performed the samples form these runs.

* ***Morphology*:** We did the Germination assay with help from the Drylab team. We repeated the Biolector experiment.
* ***Protein secretion*:** We sequenced five synthetic sigP constructs, 3 of which were positive and were linearized for transformation. The three positive synthetic signal peptides were transformed into the *ΔglaA* protoplasts.

**Human Practice:**
We set up a presentation with Novozymes for a week before the wiki freeze.

## September 28, 2020 - October 4, 2020

**Outreach:**
We had a meeting with FCB-UANL.

**Drylab:**
We took a lot of microscope images, worked on the simulation, and discussed benchmarking with Dennis from Novozymes. For the simulation we worked with the code to model a substrate gradient and to allow for directional changes in hyphal growth.

**Wetlab:**
We ran Bioreactors with reference strain, *ΔracA* and *ΔchsC\_Δgul* and performed Glucoamylase and BCA assays on them. The biolectors reactors runs will be repeated during the next weeks, as pH was not controlled during fermentation. We ran the Biolector with the morphology strains in YPD + MM with maltose instead of glucose.

* ***Morphology*:** We did the Germination assay with help from the Drylab team. We repeated the Biolector experiment.
* ***Protein secretion*:** We did monosporic purification of the 3 positive synthetic signal peptides.

![](https://static.igem.org/mediawiki/2020/e/ef/T--DTU-Denmark--bioreactor_setup.jpg)

### Bioreactors for lab-scale testing of our K/O strains.

# October notebook

## October 5, 2020 - October 11, 2020

**Drylab:**
We finished most of our image analysis work, now only missing a few mutants. The simulation model is almost ready, just need to get the results looking their best and then run all the simulations for the different mutants.

**Wetlab:**
We ran Bioreactors with reference strain, *ΔracA* and *ΔchsC\_Δgul* and performed Glucoamylase and BCA assays on them. The biolectors reactors runs will be repeated during the next weeks, as pH was not controlled during fermentation. We ran the Biolector with the morphology strains in YPD + MM with maltose instead of glucose.

* ***Morphology*:** We ran Bioreactors with *ΔchsC\_ΔspaA* and *ΔspaA\_Δgul-1*, *gul-1*, ATCC 1015, *racA*, and *ΔchsC\_Δgul-1*. We also ran germination assays and when, separation columns arrived, determined total protein secretion from bioreactors.
* ***Protein secretion*:** We performed flower plate experiments, where we sadly saw no expression in SigP strains.

## October 12, 2020 - October 18, 2020

**General:**
The team spent the week together in a summer house working on data analysis, the wiki, and presentations.

## October 19, 2020 - October 25, 2020

**General:**
The team spent the week working on the wiki. We also presented our project for DTU Bioengineering - the section we worked at during our project.

**Drylab:**
We got a final round of microscope images for image analysis and did some bug fixing in the simulation model.

**Human Practice:**
We presented our project to Novozymes for a final round of feedback from the industry stakeholders.

## October 26, 2020 - November 1, 2020

**General:**
The team spent the first part of this week finishing the wiki, then finishing up the presentation video and the judging form. We also presented our project at the DTU blue dot ceremony.

![](https://static.igem.org/mediawiki/2020/0/05/T--DTU-Denmark--summer_house.jpg)

### Summerhouse meeting regarding the work we had done throughout the day.

![](https://static.igem.org/mediawiki/2020/4/4c/T--DTU-Denmark--presentation_movie_filming.jpg)

### Filming day for our presentation video. Thanks for watching!

[![](https://static.igem.org/mediawiki/2020/a/ac/T--DTU-Denmark--Nextpage.png)](https://2020.igem.org/Team%3ADTU-Denmark/Experiments)

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# Team:DTU-Denmark/Engineering

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[Contribution](https://2020.igem.org/Team%3ADTU-Denmark/Contribution)
[Engineering](https://2020.igem.org/Team%3ADTU-Denmark/Engineering)

---

[Implementation](https://2020.igem.org/Team%3ADTU-Denmark/Implementation)
[Proof of concept](https://2020.igem.org/Team%3ADTU-Denmark/Proof_Of_Concept)

Labwork

[Design](https://2020.igem.org/Team%3ADTU-Denmark/Design)
[Experiments](https://2020.igem.org/Team%3ADTU-Denmark/Experiments)
[Measurement](https://2020.igem.org/Team%3ADTU-Denmark/Measurement)
[Safety](https://2020.igem.org/Team%3ADTU-Denmark/Safety)

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[Model](https://2020.igem.org/Team%3ADTU-Denmark/Model)
[Software](https://2020.igem.org/Team%3ADTU-Denmark/Software)

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[Human Practices](https://2020.igem.org/Team%3ADTU-Denmark/Human_Practices)

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[Parts](https://2020.igem.org/Team%3ADTU-Denmark/Parts)
[Part Collection](https://2020.igem.org/Team%3ADTU-Denmark/Part_Collection)

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[Team](https://2020.igem.org/Team%3ADTU-Denmark/Team)
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[Notebook](https://2020.igem.org/Team%3ADTU-Denmark/Notebook)
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[Inclusion](https://2020.igem.org/Team%3ADTU-Denmark/Inclusion)

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[Sponsorship](https://2020.igem.org/Team%3ADTU-Denmark/Sponsorship)

[Awards](https://2020.igem.org/Team%3ADTU-Denmark/Awards)
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* [Engineering morphology](#section2)* [Engineering protein secretion](#section3)* Back to top

## To RESHAPE *Aspergillus niger* we have used the tools of synthetic biology. We have designed, built and tested a total of eight parts for gene knockout in the *A. niger* genome. Additionally, we have designed and built a new parts collection to add novel signal peptides to our protein products, containing 15 parts. In total we added 76 parts to the iGEM registry benefitting further teams choosing to work with *A. niger*.

![](https://static.igem.org/mediawiki/2020/3/3e/T--DTU-Denmark--Engineering.png)

# Engineering morphology:

 ![](https://static.igem.org/mediawiki/2020/8/84/T--DTU-Denmark--research.png)
We wanted to ease the work with *Aspergillus niger* and thereby improve it as a cell factory. Quickly, we figured out that morphology has a huge impact on *A. niger's* performance as a cell factory. Its way of growing in filamentous structures can be difficult in large fermentation tanks as the filaments can tangle around stirrers and probes. Furthermore, morphology is closely linked to the production, and depending on the product of interest different types of morphology are preferred. We chose to focus on protein production, thereby changing the morphology to ease the work with *A. niger* without hurting its ability for protein production.

To do this, we went into the literature to find genes affecting morphology. We found seven genes that were proven to alter the morphology, either when down/up regulated or completely knocked-out. An overview of the genes is found in the [design page.](https://2020.igem.org/Team%3ADTU-Denmark/Design)We decided for the initial work to only do knockouts of the seven genes as this would also be the first step in making up/down regulated strains.

 ![](https://static.igem.org/mediawiki/2020/3/33/T--DTU-Denmark--design.png)
To make our new morphology strains we used CRISPR-Cas9 to meditate gene knockouts in the *A. niger* genome. The CRISPR-Cas9 system we used was developed and optimized for *Aspergilli* by Uffe Mortensens lab (Nødvig et. al., 2015; Nødvig et al., 2018). The system is described in detail in our [design page.](https://2020.igem.org/Team%3ADTU-Denmark/Design)

To use the system a SingleGuideRNA (sgRNA) with the sequence matching the gene to be knocked-out needs to be designed. sgRNA consists of gRNA, that attaches to Cas9, and crRNA, that is the sequence matching the cutting site of the gene of interest. By identifying two crRNA regions for each of the genes, we managed to design seven CRISPR-Cas9 plasmids.

The design of the seven CRISPR-Cas9 plasmids for knockout of morphology genes are found in the iGEM registry with the following part numbers:

* CRISPR\_chsC\_KO [BBa\_K3385014](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3385014)
* CRISPR\_spaA\_KO [BBa\_K3385018](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3385018)
* CRISPR\_gul-1\_KO [BBa\_K3385022](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3385022)
* CRISPR\_arfA\_KO [BBa\_K3385026](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3385026)

* CRISPR\_racA\_KO [BBa\_K3385030](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3385030)
* CRISPR\_aplD\_KO [BBa\_K3385034](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3385034)
* CRISPR\_pkaR\_KO [BBa\_K3385038](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3385038)

 ![](https://static.igem.org/mediawiki/2020/a/a0/T--DTU-Denmark--build.png)
From our design we constructed the seven CRISPR-Cas9 plasmids. The sgRNAs are made using primer extending PCR amplification with primers containing the crRNA sequence. The plasmids were assembled using USER cloning. For a complete overview of the build process visit our [experiments page.](https://2020.igem.org/Team%3ADTU-Denmark/Experiments)

 ![](https://static.igem.org/mediawiki/2020/7/7c/T--DTU-Denmark--new_updated_colors.svg)

The plasmids were transformed into *A. niger*, meditating knockouts of the genes. Six of the seven gene knockouts resulted in viable strains. From these initial six strains we again used the CRISPR-Cas9 plasmids resulting in three double knockout strains. In total we managed to make nine new morphology strains.

 ![](https://static.igem.org/mediawiki/2020/8/8b/T--DTU-Denmark--test_learn.png)
To characterize the new strains, we observed their macroscopic morphology on different solid medias as well as through the microscope. We measured their growth in a BioLector and 1L bioreactors. The strains protein production was also assessed from bioreactor samples. Read the details of all the measurements in the [measurement page.](https://2020.igem.org/Team%3ADTU-Denmark/Measurement)

We learned how the gene knockouts affected the strains behavior. You will find all the data we obtained for the new morphology strains and our conclusions in our [results page.](https://2020.igem.org/Team%3ADTU-Denmark/Results)

 ![](https://static.igem.org/mediawiki/2020/3/37/T--DTU-Denmark--improve.png)
While some of the strains showed increased protein production and good growth data, other gene knockouts did not benefit the strains on these parameters. This was expected from our initial research. The original design for up and down regulating the genes was also made but never built. You can read about this design on our [design page.](https://2020.igem.org/Team%3ADTU-Denmark/Design)

# Engineering protein secretion:

 ![](https://static.igem.org/mediawiki/2020/8/84/T--DTU-Denmark--research.png)
We wanted to improve protein secretion in *A. niger*. To do this we wanted to research the rather abundant area of signal peptides. In literature we found the top secreted proteins in *A. niger* and identified their signal peptides. Furthermore, our drylab team developed a tool that predicts signal peptides, [SignalPrepper.](https://2020.igem.org/Team%3ADTU-Denmark/Software)From this prediction we got five synthetic signal peptides that we also wanted to test.

![](https://static.igem.org/mediawiki/2020/3/33/T--DTU-Denmark--design.png)
We were able to characterize our signal peptides by their ability to facilitate export of glucoamylase enzyme (encoded by *glaA*) as its activity can easily be measured by an assay. We designed an expression cassette for integration in *A. niger*. This cassette was based on an integration platform designed by the DTU-Denmark 2019 team. We also used one of their promoters. The initial construct is seen in the figure to the right. The signal peptide can easily be replaced with primer extending PCR amplification and USER cloning as illustrated in the figure below.

![](https://static.igem.org/mediawiki/2020/e/ef/T--DTU-Denmark--Engineering_SP_niko.svg)

 ![](https://static.igem.org/mediawiki/2020/9/98/T--DTU-Denmark--USER_cloning_NIKO.svg)

The design of the 15 expression constructs for testing the signal peptides are found in the iGEM registry with the following part numbers:

* T1G3R\_SglaA\_glaA [BBa\_K3385057](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3385057)
* T1G3R\_SfaeA\_glaA [BBa\_K3385058](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3385058)
* T1G3R\_SpmeA\_glaA [BBa\_K3385059](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3385059)
* T1G3R\_SpgaI\_glaA [BBa\_K3385060](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3385060)
* T1G3R\_SxlnD\_glaA [BBa\_K3385061](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3385061)
* T1G3R\_SlacA\_glaA [BBa\_K3385062](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3385062)
* T1G3R\_SeglA\_glaA [BBa\_K3385063](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3385063)
* T1G3R\_SeglB\_glaA [BBa\_K3385064](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3385064)

* T1G3R\_ScbhA\_glaA [BBa\_K3385065](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3385065)
* T1G3R\_ScbhB\_glaA [BBa\_K3385066](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3385066)
* T1G3R\_Spower\_glaA [BBa\_K3385067](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3385067)
* T1G3R\_Spilot\_glaA [BBa\_K3385068](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3385068)
* T1G3R\_Spineapple\_glaA [BBa\_K3385069](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3385069)
* T1G3R\_Speanut\_glaA [BBa\_K3385070](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3385070)
* T1G3R\_Spuppy\_glaA [BBa\_K3385071](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3385071)

Additionally, a CRISPR-Cas9 plasmid, as the ones designed for the morphology gene knockouts, was designed to knockout the gene *glaA*:

* CRISPR\_glaA\_KO [BBa\_K3385010](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3385010)

 ![](https://static.igem.org/mediawiki/2020/a/a0/T--DTU-Denmark--build.png)
From our design we built the 15 expression constructs and the CRISPR-Cas9 plasmid. We first knocked-out the *glaA* gene in *A. niger.* Thereafter, we successfully inserted 11 of the 15 expression constructs into the genome of *A. niger.*

 ![](https://static.igem.org/mediawiki/2020/1/10/T--DTU-Denmark--test.png)
Unfortunately, we did only reach the initial part of this step. Due to time constrictions we did not obtain conclusive data for the eleven constructed strains but we hope to be able to continue this work soon!

[![](https://static.igem.org/mediawiki/2020/a/ac/T--DTU-Denmark--Nextpage.png)](https://2020.igem.org/Team%3ADTU-Denmark/Design)

References

##### 1. Nødvig, C., Nielsen, J., Kogle, M., & Mortensen, U. (2015). A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi. *PLOS ONE*, 10(7). doi: 10.1371/journal. Pone.0133085 2. Nødvig, C., Hoof, J., Kogle, M., Jarczynska, Z., Lehmbeck, J., Klitgaard, D., & Mortensen, U. (2018). Efficient oligo nucleotide mediated CRISPR-Cas9 gene editing in Aspergilli. *Fungal Genetics And Biology*, 115, 78-79. doi: 10.1016/j.fgb.2018.01.004

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# Team:DTU-Denmark/Experiments

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[Engineering](https://2020.igem.org/Team%3ADTU-Denmark/Engineering)

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[Implementation](https://2020.igem.org/Team%3ADTU-Denmark/Implementation)
[Proof of concept](https://2020.igem.org/Team%3ADTU-Denmark/Proof_Of_Concept)

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[Design](https://2020.igem.org/Team%3ADTU-Denmark/Design)
[Experiments](https://2020.igem.org/Team%3ADTU-Denmark/Experiments)
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[Human Practices](https://2020.igem.org/Team%3ADTU-Denmark/Human_Practices)

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* Back to top

# Experiments

The different steps of genetic modification of *A. niger*  are given in the flowsheet below. The protocol for each step a can be downloaded below.

![](https://static.igem.org/mediawiki/2020/d/d8/T--DTU-Denmark--FLOWCHART_FINAL_FINAL_FINAL.png)

**Click on the protocols below to download them.**

[PCR amplification of Biobricks](https://static.igem.org/mediawiki/2020/0/06/T--DTU-Denmark--PCR_PROTOKOLs1.pdf)

 [Gel electrophoresis and Gel purification](https://static.igem.org/mediawiki/2020/9/9f/T--DTU-Denmark--Gel_Electrophoresis_Purification.pdf)

 [PacI & Nt. BbvCI digestion of backbone](https://static.igem.org/mediawiki/2020/a/a4/T--DTU-Denmark--PacI%2BNt.BbvCI_Digestion_USER.pdf)

 [USER cloning of Biobricks and plasmid](https://static.igem.org/mediawiki/2020/4/40/T--DTU-Denmark--USER_cloning.pdf)

 [Transform into E. coli](https://static.igem.org/mediawiki/2020/e/ec/T--DTU-Denmark--Transformation_of_Ecoli.pdf)

 [Make E. coli competent cells](https://static.igem.org/mediawiki/2020/3/3c/T--DTU-Denmark--Ecoli_Competent_Cells.pdf)

 [Screen transformants through Colony PCR](https://static.igem.org/mediawiki/2020/4/4e/T--DTU-Denmark--Screen_Colony_PCR.pdf)

[Purification of plasmids](https://static.igem.org/mediawiki/2020/f/fe/T--DTU-Denmark--Purification_of_plasmids.pdf)

 [Grow A. niger on plates](https://static.igem.org/mediawiki/2020/7/7a/T--DTU-Denmark--A.niger_growth.pdf)

 [Make protoplasts](https://static.igem.org/mediawiki/2020/b/b0/T--DTU-Denmark--Make_protoplasts.pdf)

 [Transform plasmid into A. niger](https://static.igem.org/mediawiki/2020/c/c0/T--DTU-Denmark--Transformation_of_Aniger.pdf)

 [Screen transformants through Tissue PCR](https://static.igem.org/mediawiki/2020/8/83/T--DTU-Denmark--Screen_Tissue_PCR.pdf)

 [Monosporic purification](https://static.igem.org/mediawiki/2020/d/de/T--DTU-Denmark--Monosporic_Purification.pdf)

 [Make a stock](https://static.igem.org/mediawiki/2020/f/f0/T--DTU-Denmark--Make_Stock.pdf)

# Protocols specific for our project

[Sample preparation for microscope slides](https://static.igem.org/mediawiki/2020/1/11/T--DTU-Denmark--Microscope_Slides.pdf)

 [Glucoamylase Assay](https://static.igem.org/mediawiki/2020/7/74/T--DTU-Denmark--Glucoamylase_Assay.pdf)

 [Bioreactor](https://static.igem.org/mediawiki/2020/9/93/T--DTU-Denmark--Bioreactor_Protocol.pdf)

[BCA Assay](https://static.igem.org/mediawiki/2020/d/d1/T--DTU-Denmark--BCAassay.pdf)

 [BioLector](https://static.igem.org/mediawiki/2020/b/b6/T--DTU-Denmark--BioLector_protocol.pdf)

 [DNA extraction](https://static.igem.org/mediawiki/2020/9/9c/T--DTU-Denmark--Genomic_Sequencing.pdf)

*Some of these protocols have been adapted from our lab at DTU bioengineering, and others we have developed ourselves through the course of this project.*

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# Team:DTU-Denmark/Results

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[Implementation](https://2020.igem.org/Team%3ADTU-Denmark/Implementation)
[Proof of concept](https://2020.igem.org/Team%3ADTU-Denmark/Proof_Of_Concept)

Labwork

[Design](https://2020.igem.org/Team%3ADTU-Denmark/Design)
[Experiments](https://2020.igem.org/Team%3ADTU-Denmark/Experiments)
[Measurement](https://2020.igem.org/Team%3ADTU-Denmark/Measurement)
[Safety](https://2020.igem.org/Team%3ADTU-Denmark/Safety)

Modelling

[Model](https://2020.igem.org/Team%3ADTU-Denmark/Model)
[Software](https://2020.igem.org/Team%3ADTU-Denmark/Software)

[Results](https://2020.igem.org/Team%3ADTU-Denmark/Results)
[Human Practices](https://2020.igem.org/Team%3ADTU-Denmark/Human_Practices)

Parts

[Parts](https://2020.igem.org/Team%3ADTU-Denmark/Parts)
[Part Collection](https://2020.igem.org/Team%3ADTU-Denmark/Part_Collection)

People

[Team](https://2020.igem.org/Team%3ADTU-Denmark/Team)
[Collaboration](https://2020.igem.org/Team%3ADTU-Denmark/Collaborations)
[Science communication](https://2020.igem.org/Team%3ADTU-Denmark/Education)
[Notebook](https://2020.igem.org/Team%3ADTU-Denmark/Notebook)
[Attributions](https://2020.igem.org/Team%3ADTU-Denmark/Attributions)
[Inclusion](https://2020.igem.org/Team%3ADTU-Denmark/Inclusion)

---

[Sponsorship](https://2020.igem.org/Team%3ADTU-Denmark/Sponsorship)

[Awards](https://2020.igem.org/Team%3ADTU-Denmark/Awards)
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* [The reference strain](#section2)
* [The new strains](#section3)
* [Comparison](#section13)
  + [Simulations](#section15)
  + [Growth](#section16)
  + [Protein secretion](#section17)
  + [Summary](#section18)
* [Signal Peptides](#section14)
* Back to top

## During the project the whole RESHAPE team has been working hard in the lab and behind the computers to RESHAPE *Aspergillus niger*. We managed to make nine new morphology strains, a library of 11 signal peptide mutant strains and nine mycelium growth simulations based on real life data. Here you will find all our results, characterizations of our new morphology strains and the Mycemulator simulations. To find out more about the specific experiments please visit our [*Measurement page.*](https://2020.igem.org/Team%3ADTU-Denmark/Measurement)

# The reference strain

The ATCC 1015 strain of *Aspergillus niger* was used as our reference strain. It has no known mutations from the wild type *A. niger*.

ATCC 1015 and the nine *A. niger* mutant strains were inoculated and grown on four different growth media. The morphology of a fungus differs depending on the environmental conditions it is grown under, which is the reasoning behind choosing to grow the *A. niger* strains on four different media. We have described the four media types further in the [*Measurement page.*](https://2020.igem.org/Team%3ADTU-Denmark/Measurement#section1)

The strains were grown on Yeast Extract Peptone Dextrose (YPD), Transformation Media (TM), Creatine Sucrose Agar (CREA) and Czepek Yeast Extract Agar (CYA).
![](https://static.igem.org/mediawiki/2020/e/e4/T--DTU-Denmark--WT_allplates.png)

# Microscope pictures and Simulation model

![](https://static.igem.org/mediawiki/2020/8/88/T--DTU-Denmark--WT_simulation.png)

*Left: Confocal microscope picture of ATCC 1015 at 10X magnification. Right: Growth simulation for 12h performed using the Mycemulator.*

Microscopic images were analyzed by the image analysis tool extracting growth parameters which were then fed to the Mycemulator. A simulation of ATCC 1015 growing for 12 hours (using the experimental growth rate from our BioLector data) is seen above.

**Parameters specific for simulating ATCC 1015:**

* Branching frequency: 0.0169257
* Gamma distribution parameters used for curvature angles: (1.6114694, 2.0238222)
* Beta distribution parameters used for branching angles: (4.2886834, 0.9839464)
* Experimental growth rate: 0.2757333

# The new morphology strains

## We made nine new strains with the tools of synthetic biology. Here you will find the characterization of them.

## **Click on one to learn more!**

![](https://static.igem.org/mediawiki/2020/c/c2/T--DTU-Denmark--chsC_tryit.png)

*ΔchsC*

**Theoretical expectation:** *chsC* encodes a chitin synthase. It is involved in hyphal wall integrity and in conidiophore development. Deletion of the gene was expected to give more dense pelleted growth and an increased citric acid production.

**Summary:** The strain presents a more hyperbranched morphology, but protein secretion is slightly decreased, and the growth rate is lower than the reference strain. By itself it’s not the best candidate, however, it has potential when combined with other genes.

![](https://static.igem.org/mediawiki/2020/d/d3/T--DTU-Denmark--spyder_chsC.png)

*Radar chart showing 6 different parameters of ΔchsC normalized to the reference values from ATCC 1015 (shown in yellow). Read about the axis in the [Summary](#section18) section.*

# Plates

The strain was grown on Yeast Extract Peptone Dextrose (YPD), Transformation Media (TM), Creatine Sucrose Agar (CREA) and Czepek Yeast Extract Agar (CYA).
![](https://static.igem.org/mediawiki/2020/8/8b/T--DTU-Denmark--chsC_allplates.png)

# Microscope pictures and Simulation model

![](https://static.igem.org/mediawiki/2020/a/aa/T--DTU-Denmark--chsC_simulation.png)

*Left: Confocal microscope picture of ΔchsC at 10X magnification after app. 24h growth. Right: Growth simulation for 12h performed using the Mycemulator.*

Microscopic images were analyzed by the image analysis tool extracting growth parameters which were then fed to the Mycemulator. A simulation of *ΔchsC* growing for 12 hours (using experimental growth rate from our BioLector data) is seen above.

**Parameters specific for simulating *ΔchsC*:**

* Branching frequency: 0.0251938
* Gamma distribution parameters used for curvature angles: (1.5962901, 4.3710747)
* Beta distribution parameters used for branching angles: (2.294856826, 0.9438161)
* Experimental growth rate: 0.21145

# BioLector

Comparing the growth kinetics of the *ΔchsC* mutant with the reference strain ATCC 1015 in the BioLector, the mutant exhibits a longer lag phase followed by an equivalently long exponential growth phase. The growth rate for the mutant is slightly lower than for the reference strain, at μMax0.21h-1 versus μMax0.28h-1.

![](https://static.igem.org/mediawiki/2020/c/c2/T--DTU-Denmark--chsC_growth_lector.png)

*Growth profile of ΔchsC over 72 hours, measuring absorbance at 620 nm. Plotted against the reference strain (ATCC 1015) shown in yellow.*

# Bioreactor

Looking at the growth of the *ΔchsC* mutant in the bioreactors, it showed a shorter lag phase followed by a longer exponential growth phase when comparing it with the reference strain ATCC 1015. The growth rate is lower than the reference strain, at μMax0.23h-1 and μMax0.22h-1 versus μMax0.42h-1 for both reference duplicates. The fermentation was accidentally run with overpressure and is therefore only directly comparable with the *ΔspaA* mutant strain and with one run of the ATCC 1015 fermentations, due to these bioreactors also being run with overpressure. Glucose consumption starts slowly but increases quickly after the mutant stops growing.

![](https://static.igem.org/mediawiki/2020/0/0a/T--DTU-Denmark--chsC_growth_reactor.png)

*Growth curve obtained from the off-gas analysis (CO2-44 (%)) of ΔchsC plotted against the reference strain (ATCC 1015) in a logarithmic scale. Right axis: Glucose consumption by ΔchsC during the fermentation in g/L (dark green).*

![](https://static.igem.org/mediawiki/2020/a/af/T--DTU-Denmark--chsC_micro_reactor.png)

*Light microscope picture of ΔchsC after 22 hours of fermentation with a 20X magnification.*

Microscope pictures of the bioreactor sample for *ΔchsC* had a very different morphology from the brightfield microscopy pictures seen above. It showed an increase in agglomeration along with a more hyperbranched phenotype.

# Protein secretion

As seen in the figure below glucoamylase activity increases over time during the fermentation. The mutant exhibits lower glucoamylase activity compared to the reference strain.

 ![](https://static.igem.org/mediawiki/2020/1/16/T--DTU-Denmark--gluco_chsC.png)

*Glucoamylase activity in UA/mL of ΔchsC from eight samples taken during the fermentation. Plotted against the reference strain (ATCC 1015) shown in yellow.*

The final protein concentration is also significantly lower for the mutant strain than for reference strain, which is consistent with the reduced glucoamylase activity level. This is shown in the bar chart below.

 ![](https://static.igem.org/mediawiki/2020/a/af/T--DTU-Denmark--chsC_gluco.png)

*ΔchsC bioreactor duplicates compared to reference strain (ATCC 1015) of the last time-point samples from the fermentations. Green: Glucoamylase activity in UA/mL. Blue: Specific activity in UA/mg calculated from the activity and the protein concentration. Purple: Protein concentration in mg/mL.*

![](https://static.igem.org/mediawiki/2020/a/a9/T--DTU-Denmark--racA_tryit.png)

*ΔracA*

**Theoretical expectation:** *racA* encodes a Rho GTPase which controls actin dynamics and it is thereby also involved in polarity regulation. The knockout mutant was expected to have a hyperbranched morphology.

**Summary:** The strain showed a slight increase in protein secretion and a hyperbranching pelleted morphology when run in the bioreactor. While it closely follow the behavior of the reference strain, its morphology on solid media was very different. As it had a decreased sporulation it made the strain hard to work with.

![](https://static.igem.org/mediawiki/2020/c/c8/T--DTU-Denmark--spyder_racA.png)

*Radar chart showing 6 different parameters of ΔracA normalized to the reference values from ATCC 1015 (shown in yellow). Read about the axis in the [Summary](#section18) section.*

# Plates

The strain was grown on Yeast Extract Peptone Dextrose (YPD), Transformation Media (TM), Creatine Sucrose Agar (CREA) and Czepek Yeast Extract Agar (CYA).
![](https://static.igem.org/mediawiki/2020/3/38/T--DTU-Denmark--racA_allplates.png)

# Microscope pictures and Simulation model

![](https://static.igem.org/mediawiki/2020/0/09/T--DTU-Denmark--racA_simulation.png)

*Left: Confocal microscope picture of ΔracA at 10X magnification after app. 24h growth. Right: Growth simulation for 12h performed using the Mycemulator.*

Microscopic images were analyzed by the image analysis tool extracting growth parameters which were then fed to the Mycemulator. A simulation of *ΔracA* growing for 12 hours (using experimental growth rate from our BioLector data) is seen above.

**Parameters specific for simulating *ΔracA*:**

* Branching frequency: 0.0426064
* Gamma distribution parameters used for curvature angles: (0.8423059, 16.2365375)
* Beta distribution parameters used for branching angles: (1599535.5508791, 2.3452173)
* Experimental growth rate: 0.15865

# BioLector

Comparing the growth kinetics of the *ΔracA* mutant with the reference strain ATCC 1015 in the BioLector, the mutant exhibits a longer lag phase and an almost double as long exponential growth phase. The growth rate for the mutant is lower than for the reference strain, at μMax0.16h-1 versus μMax0.28h-1.

![](https://static.igem.org/mediawiki/2020/a/ac/T--DTU-Denmark--racA_growth_lector.png)

*Growth profile of ΔracA over 72 hours, measuring absorbance at 620 nm. Plotted against the reference strain (ATCC 1015) shown in yellow.*

# Bioreactor

Looking at the growth of the *ΔracA* mutant in bioreactors, it showed an equivalently long lag phase as the reference strain ATCC 1015 followed by a slightly shorter exponential growth phase. *ΔracA* also has a growth rate almost equivalent to the growth rate of the reference strain, at μMax0.36h-1 and μMax0.32h-1 versus μMax0.37h-1 and μMax0.33h-1. Glucose levels start decreasing two thirds of the way through growth but are stil not completely depleted at the time of the last sample.

![](https://static.igem.org/mediawiki/2020/8/8c/T--DTU-Denmark--racA_growth_reactor.png)

*Left axis: Growth curve obtained from the off-gas analysis (CO2-44 (%)) of ΔracA plotted against the reference strain (ATCC 1015) in a logarithmic scale. Right axis: Glucose consumption by ΔracA during the fermentation in g/L (dark green).*

![](https://static.igem.org/mediawiki/2020/3/36/T--DTU-Denmark--bioreactor_micro_racA.png)

*Light microscope picture of ΔracA after 23 hours of fermentation with a 20X magnification.*

The microscope picture from the bioreactor sample for *ΔracA* showed a pelleted morphology making it hard to compare with the brightfield microscopy pictures seen above. It showed a very hyperbranched pelleted morphology. When run in the bioreactor, the pelleted morphology was observable on a macroscopic level.

# Protein secretion

The glucoamylase activity increases over time during the fermentation as seen in the figure below. The two duplicates showed a large difference in glucoamylase activity, one being equal to the reference strain whereas the other had an increased activity.

![](https://static.igem.org/mediawiki/2020/e/ea/T--DTU-Denmark--gluco_racA.png)

**Glucoamylase activity in UA/mL of ΔracA from 8 samples taken during the fermentation. Plotted against the reference strain (ATCC 1015) shown in yellow.**

A difference were also seen between the duplicates for the total protein secretion. Therefore, when calculating the specific activity both showed similar result with a value slightly higher than the reference strain. The difference the duplicates might be explained by a deviation in setting up the technical duplicates.

 ![](https://static.igem.org/mediawiki/2020/6/6e/T--DTU-Denmark--racA_gluco.png)

*ΔracA bioreactor duplicates compared to reference strain (ATCC 1015) of the last time-point samples from the fermentations. Green: Glucoamylase activity in UA/mL. Blue: Specific activity in UA/mg calculated from the activity and the protein concentration. Purple: Protein concentration in mg/mL.*

![](https://static.igem.org/mediawiki/2020/6/61/T--DTU-Denmark--chsC_gul-1_tryit.png)

![](https://static.igem.org/mediawiki/2020/2/20/T--DTU-Denmark--novel_work.svg)

*ΔchsC\_Δgul-1*

**Theoretical expectation:** This is a double knock-out mutant, where both the genes *chsC*, which encodes a chitin synthase, and *gul-1*, which encodes an mRNA binding protein involved in cell wall remodeling, are knocked out. There does not exist any prior knowledge in the literature about knocking both of these genes simultaneously out in *A. niger*.

**Summary:** The strain presents a very hyperbranched morphology, caused by an additive effect of both genes. Protein secretion is higher than in the reference strain. It showed a macroscopic pelleted morphology when run in the bioreactor.

![](https://static.igem.org/mediawiki/2020/4/40/T--DTU-Denmark--spyder_chsC_gul-1.png)

*Radar chart showing 6 different parameters of ΔchsC\_Δgul-1 normalized to the reference values from ATCC 1015 (shown in yellow). Read about the axis in the [Summary](#section18) section.*

# Plates

The strain was grown on Yeast Extract Peptone Dextrose (YPD), Transformation Media (TM), Creatine Sucrose Agar (CREA) and Czepek Yeast Extract Agar (CYA).
![](https://static.igem.org/mediawiki/2020/6/62/T--DTU-Denmark--chsC_gul1_allplates.png)

# Microscope pictures and Simulation model

![](https://static.igem.org/mediawiki/2020/7/76/T--DTU-Denmark--chsC_gul1_simulation.png)

*Left: Confocal microscope picture of ΔchsC\_Δgul-1 at 10X magnification after app. 24h growth. Right: Growth simulation for 12h performed using the Mycemulator.*

Microscopic images were analyzed by the image analysis tool extracting growth parameters which were then fed to the Mycemulator. A simulation of *ΔchsC\_Δgul-1* growing for 12 hours (using experimental growth rate from our BioLector data) is seen above.

**Parameters specific for simulating *ΔchsC\_Δgul-1*:**

* Branching frequency: 0.0280344
* Gamma distribution parameters used for curvature angles: (1.272886, 6.5509939)
* Beta distribution parameters used for branching angles: (2.9856469, 0.9208045)
* Experimental growth rate: 0.1381667

# BioLector

Comparing the growth kinetics of the *ΔchsC\_Δgul-1* mutant with the reference strain ATCC 1015 in the BioLector, the mutant exhibits an equivalently long lag phase followed by a twice as long exponential growth phase. The growth rate for the mutant is lower than for the reference strain, at μMax0.14h-1 versus μMax0.28h-1.

![](https://static.igem.org/mediawiki/2020/5/56/T--DTU-Denmark--chsC_gul1_growth_lector.png)

*Growth profile of ΔchsC\_Δgul-1 over 72 hours, measuring absorbance at 620 nm. Plotted against the reference strain (ATCC 1015) shown in yellow.*

# Bioreactor

Looking at the growth of the *ΔchsC\_Δgul-1* mutant in bioreactors, it showed a slightly shorter lag phase and a shorter exponential growth phase compared to the reference strain ATCC 1015. *ΔchsC\_Δgul-1* also has a higher growth rate than the reference strain, at μMax0.42h-1 for both duplicates versus μMax0.37h-1 and μMax0.33h-1. Glucose levels start decreasing during growth but are first depleted over 12 hours after the strain enters the stationary phase.

![](https://static.igem.org/mediawiki/2020/7/71/T--DTU-Denmark--chsC_gul1_growth_reactor.png)

*Left axis: Growth curve obtained from the off-gas analysis (CO2-44 (%)) of ΔchsC\_Δgul-1 plotted against the reference strain (ATCC 1015) in a logarithmic scale. Right axis: Glucose consumption by ΔchsC\_Δgul-1 during the fermentation in g/L (dark green).*

![](https://static.igem.org/mediawiki/2020/e/e1/T--DTU-Denmark--bioreactor_micro_chsC_gul-1.png)

*Light microscope picture of ΔchsC\_Δgul-1 after 23 hours of fermentation with a 20X magnification.*

Microscope picture from the bioreactor sample for *ΔchsC\_Δgul-1* has a similar morphology to that of the brightfield microscopy pictures seen above. It showed a hyperbranched pelleted morphology.
When run in the bioreactor, the pelleted morphology was observable on a macroscopic level.

# Protein secretion

As seen in the figure below, glucoamylase activity increases during the fermentation and reaches a higher level than the reference strain.

![](https://static.igem.org/mediawiki/2020/2/21/T--DTU-Denmark--gluco_chsC_gul1.png)

*Glucoamylase activity in UA/mL of ΔchsC\_Δgul-1 from 8 samples taken during the fermentation. Plotted against the reference strain (ATCC 1015) shown in yellow.*

This mutant presents similar tendency in the results as the single knock-out strain *Δgul-1*, with similar number in total protein secretion but increased activity per total protein when compared to the reference strain. As commented in *Δgul-1* results, this might be due to a different protein secretion profile that has a similar amount of protein being secreted but where the family of glucoamylases are preferentially produced. However, these results are lower when compared directly to *Δgul-1*, but significantly higher than when compared to *ΔchsC*, showing more properties of the *Δgul-1 strain*.

 ![](https://static.igem.org/mediawiki/2020/a/a1/T--DTU-Denmark--chsC_gul1_gluco.png)

*ΔchsC\_Δgul-1 bioreactor duplicates compared to reference strain (ATCC 1015) of the last time-point samples from the fermentations. Green: Glucoamylase activity in UA/mL. Blue: Specific activity in UA/mg calculated from the activity and the protein concentration. Purple: Protein concentration in mg/mL.*

![](https://static.igem.org/mediawiki/2020/1/1d/T--DTU-Denmark--spaA_tryit.png)

*ΔspaA*

**Theoretical expectation:** It has been suggested that *spaA* encodes a molecule which is involved in polarity maintenance and thereby polar growth. The mutant was expected to have a dense hyperbranched morphology.

**Summary:** The strain presents a shorter lag phase than the reference strain and the highest growth rate in the BioLector, although this is not seen in the bioreactor. It does not have a great increase in hyperbranching and protein secretion is very similar to the reference strain.

![](https://static.igem.org/mediawiki/2020/6/6e/T--DTU-Denmark--spyder_spaA.png)

*Radar chart showing 6 different parameters of *ΔspaA* normalized to the reference values from ATCC 1015 (shown in yellow). Read about the axis in the [Summary](#section18) section.*

# Plates

The strain was grown on Yeast Extract Peptone Dextrose (YPD), Transformation Media (TM), Creatine Sucrose Agar (CREA) and Czepek Yeast Extract Agar (CYA).
![](https://static.igem.org/mediawiki/2020/4/44/T--DTU-Denmark--spaA_allplates.png)

# Microscope pictures and Simulation model

![](https://static.igem.org/mediawiki/2020/5/50/T--DTU-Denmark--spaA_simulation.png)

*Left: Confocal microscope picture of ΔspaA at 10X magnification af app. 24h growth. Right: Growth simulation for 12h performed using the Mycemulator.*

Microscopic images were analyzed by the image analysis tool extracting growth parameters which were then fed to the Mycemulator. A simulation of *ΔspaA* growing for 12 hours (using experimental growth rate from ou BioLector data) is seen above.

**Parameters specific for simulating *ΔspaA*:**

* Branching frequency: 0.0182251
* Gamma distribution parameters used for curvature angles: (1.6948611, 2.8805343)
* Beta distribution parameters used for branching angles: (5.1349854, 1.2365410)
* Experimental growth rate: 0.345225

# BioLector

Comparing the growth kinetics of the *ΔspaA* mutant with the reference strain ATCC 1015 in the BioLector, the mutant exhibits a shorter lag phase and a shorter exponential growth phase. The growth rate for the mutant is higher than for the reference strain, at μMax0.35h-1 versus μMax0.28h-1.

![](https://static.igem.org/mediawiki/2020/e/e3/T--DTU-Denmark--spaA_growth_lector.png)

*Growth profile of ΔspaA over 72 hours, measuring absorbance at 620 nm. Plotted against the reference strain (ATCC 1015) shown in yellow.*

# Bioreactor

Looking at the growth of the *ΔspaA* mutant in bioreactors, it showed a short lag phase followed by a long exponential growth phase compared with the reference strain ATCC 1015. The *ΔspaA* mutant has a lower growth rate than the reference, at μMax0.19h-1 and μMax0.20h-1 versus μMax0.42h-1 for both duplicates. The fermentation was accidentally run with overpressure and is therefore only directly comparable with the *ΔchsC* mutant strain and with one run of the ATCC 1015 fermentations, due to these bioreactors also being run with overpressure. Glucose levels decrease slowly in the beginning and then decrease fast after the mutant enters stationary phase.

![](https://static.igem.org/mediawiki/2020/4/48/T--DTU-Denmark--spaA_growth_reactor.png)

*Left axis: Growth curve obtained from the off gas analysis (CO2-44 (%)) of ΔspaA plotted against the reference strain (ATCC 1015) in a logarithmic scale. Right axis: Glucose consumption by ΔspaA during the fermentation in g/L (dark green).*

![](https://static.igem.org/mediawiki/2020/2/2a/T--DTU-Denmark--bioreactor_micro_spaA.png)

*Light microscope picture of ΔspaA after 22 hours of fermentation with a 20X magnification.*

The microscope picture from the bioreactor sample for *ΔspaA* has a similar morphology to that of the brightfield microscopy pictures seen above.

# Protein secretion

Glucoamylase activity for the mutant strain shows a similar tendency to the reference strain, with one of the duplicates having lower final value.

![](https://static.igem.org/mediawiki/2020/2/22/T--DTU-Denmark--gluco_spaA.png)

*Glucoamylase activity in UA/mL of ΔspaA from 8 samples taken during the fermentation. Plotted against the reference strain (ATCC 1015) shown in yellow.*

Protein secretion is not consistent but when computing the specific activity per mg of protein, it can be seen that the mutant again presents similar values to the reference strain.

 ![](https://static.igem.org/mediawiki/2020/0/0e/T--DTU-Denmark--spaA_gluco.png)

*ΔspaA bioreactor duplicates compared to reference strain (ATCC 1015) of the last time-point samples from the fermentations. Green: Glucoamylase activity in UA/mL. Blue: Specific activity in UA/mg calculated from the activity and the protein concentration. Purple: Protein concentration in mg/mL.*

![](https://static.igem.org/mediawiki/2020/f/f6/T--DTU-Denmark--pkaR_tryit.png)

![](https://static.igem.org/mediawiki/2020/2/20/T--DTU-Denmark--novel_work.svg)

*ΔpkaR*

**Theoretical expectation:** *pkaR* encodes a regulatory subunit in Protein Kinase A (PKA) which is involved in stress response and regulation of morphology and growth. The gene has been characterized in *Neurospora crassa* and a homolog was found in *A. niger*.

**Summary:** The strain shoed lower growth rate in the BioLector along with an increase in branching frequency. Protein secretion could not be studied as it was not run in the bioreactor due to time limitations.

![](https://static.igem.org/mediawiki/2020/0/0a/T--DTU-Denmark--spyder_pkaR.png)

*Radar chart showing 6 different parameters of ΔpkaR normalized to the reference values from ATCC 1015 (shown in yellow). Read about the axis in the [Summary](#section18) section. \*Indicates that not all data was obtained to fill out the chart.*

# Plates

The strain was grown on Yeast Extract Peptone Dextrose (YPD), Transformation Media (TM), Creatine Sucrose Agar (CREA) and Czepek Yeast Extract Agar (CYA).
![](https://static.igem.org/mediawiki/2020/9/99/T--DTU-Denmark--pkaR_allplates.png)

# Microscope pictures and Simulation model

![](https://static.igem.org/mediawiki/2020/5/5c/T--DTU-Denmark--pkaR_simulation.png)

*Left: Confocal microscope picture of ΔpkaR at 10X magnification after app. 24h growth. Right: Growth simulation for 12h performed using the Mycemulator.*

Microscopic images were analyzed by the image analysis tool extracting growth parameters which were then fed to the Mycemulator. A simulation of *ΔpkaR* growing for 12 hours (using the experimental growth rate from our BioLector data) is seen above.

**Parameters specific for simulating *ΔpkaR*:**

* Branching frequency: 0.0312627
* Gamma distribution parameters used for curvature angles: (1.6546373, 8.6411386)
* Beta distribution parameters used for branching angles: (1.4343473, 0.5666034)
* Experimental growth rate: 0.2090333

# BioLector

Comparing the growth kinetics of the *ΔpkaR* mutant with the reference strain ATCC 1015 in the BioLector, the mutant exhibits a similar lag phase and a longer exponential growth phase. The growth rate for the mutant is lower than for the reference strain, at μMax0.21h-1 versus &muMax0.28h-1.

![](https://static.igem.org/mediawiki/2020/5/50/T--DTU-Denmark--pkaR_growth_lector.png)

*Growth profile of ΔpkaR over 72 hours, measuring absorbance at 620 nm. Plotted against the reference strain (ATCC 1015) shown in yellow.*

# Bioreactor and Protein secretion

*ΔpkaR* was not run in the bioreactor due to time limitations. Data for growth in bioreactor and protein secretion for this strain were therefore not obtained.

![](https://static.igem.org/mediawiki/2020/8/8d/T--DTU-Denmark--chsC_spaA_tryit.png)

![](https://static.igem.org/mediawiki/2020/2/20/T--DTU-Denmark--novel_work.svg)

*ΔchsC\_ΔspaA*

**Theoretical expectation:** This is a double knock-out mutant, where both *chsC*, which encodes a chitin synthase, and *spaA*, which might be involved in polarity maintenance and polar growth, are knocked out. There does not exist any prior knowledge in the literature about knocking both of these genes out simultaneously in *A. niger*.

**Summary:** The strain presents the highest branching frequency when compared to the rest of the mutants, even higher than what would be expected by an additive effect of both genes. However, growth rate and protein secretion are slightly lower than the reference strain.

![](https://static.igem.org/mediawiki/2020/6/6d/T--DTU-Denmark--spyder_chsC_spaA.png)

*Radar chart showing 6 different parameters of ΔchsC\_ΔspaA normalized to the reference values from ATCC 1015 (shown in yellow). Read about the axis in the [Summary](#section18) section.*

# Plates

The strain was grown on Yeast Extract Peptone Dextrose (YPD), Transformation Media (TM), Creatine Sucrose Agar (CREA) and Czepek Yeast Extract Agar (CYA).
![](https://static.igem.org/mediawiki/2020/b/b1/T--DTU-Denmark--chsC_spaA_allplates.png)

# Microscope pictures and Simulation model

![](https://static.igem.org/mediawiki/2020/f/f8/T--DTU-Denmark--chsC_spaA_simulation.png)

*Left: Confocal microscope picture of ΔchsC\_ΔspaA at 10X magnification after app. 24h growth. Right: Growth simulation for 12h performed using the Mycemulator.*

Microscopic images were analyzed by the image analysis tool extracting growth parameters which were then fed to the Mycemulator. A simulation of *ΔchsC\_ΔspaA* growing for 12 hours (using the experimental growth rate from our BioLector data) is seen above.

**Parameters specific for simulating *ΔchsC\_ΔspaA*:**

* Branching frequency: 0.0476127
* Gamma distribution parameters used for curvature angles: (1.5398610, 5.8823202)
* Beta distribution parameters used for branching angles: (5.6843956, 1.2075437)
* Experimental growth rate: 0.264

# BioLector

Comparing the growth kinetics of the *ΔchsC\_ΔspaA* mutant with the reference strain ATCC 1015 in the BioLector, the mutant exhibits a slightly shorter lag phase followed by a slightly longer exponential growth phase. The growth rate for the mutant is almost equivalent to the growth rate for the reference strain, at μMax0.26h-1 versus μMax0.28h-1.

![](https://static.igem.org/mediawiki/2020/f/f4/T--DTU-Denmark--chsC_spaA_growth_lector.png)

*Growth profile of ΔchsC\_ΔspaA over 72 hours, measuring absorbance at 620 nm. Plotted against the reference strain (ATCC 1015) shown in yellow.*

# Bioreactor

Looking at the growth of the *ΔchsC\_ΔspaA* mutant in bioreactors, it showed a eqvuivalently long lag phase as the reference strain ATCC 1015, followed by a longer exponential growth phase. *ΔchsC\_ΔspaA* has a lower growth rate than the reference strain, at μMax0.29h-1 and μMax0.27h-1 versus μMax0.37h-1 and μMax0.33h-1. Glucose levels start decreasing half way through growth and are depleted shortly after the strain enters the stationary phase.

![](https://static.igem.org/mediawiki/2020/2/24/T--DTU-Denmark--chsC_spaA_growth_reactor.png)

**Left axis: Growth curve obtained from the off-gas analysis (CO2-44 (%)) of ΔchsC\_ΔspaA plotted against the reference strain (ATCC 1015) in a logarithmic scale. Right axis: Glucose consumption by ΔchsC\_ΔspaA during the fermentation in g/L (dark green).**

![](https://static.igem.org/mediawiki/2020/f/f5/T--DTU-Denmark--bioreactor_micro_chsC_spaA.png)

*Light microscope picture of ΔchsC\_ΔspaA after 24 hours of fermentation with a 20X magnification.*

The microscope picture from the bioreactor sample for *ΔchsC\_ΔspaA* has a very similar morphology to that of the brightfield microscopy pictures seen above. It showed a hyperbranched morphology with a slight increase in agglomeration.

# Protein secretion

Glucoamylase activity follows that reference strain with one duplicates higher level of activity.

![](https://static.igem.org/mediawiki/2020/f/f4/T--DTU-Denmark--gluco_chsC_spaA.png)

*Glucoamylase activity in UA/mL of ΔchsC\_ΔspaA from eight samples taken during the fermentation. Plotted against the reference strain (ATCC 1015) shown in yellow.*

Total protein secretion presents a 2-fold increase compared to the reference strain. However, specific activity per total protein is higher in the reference strain, suggesting that protein secretion profile has changed with a decrease in the glucoamylase family.
 ![](https://static.igem.org/mediawiki/2020/6/6f/T--DTU-Denmark--chsC_spaA_gluco.png)

*ΔchsC\_ΔspaA bioreactor duplicates compared to reference strain (ATCC 1015) of the last time-point samples from the fermentations. Green: Glucoamylase activity in UA/mL. Blue: Specific activity in UA/mg calculated from the activity and the protein concentration. Purple: Protein concentration in mg/mL.*

![](https://static.igem.org/mediawiki/2020/5/50/T--DTU-Denmark--gul-1_tryit.png)

![](https://static.igem.org/mediawiki/2020/2/20/T--DTU-Denmark--novel_work.svg)

*Δgul-1*

**Theoretical expectation:** *gul-1* was found in literature about the fungi *Neurospora crassa* where it encodes an mRNA binding protein involved in remodeling of the cell wall. A homolog was found in *A. niger*. The expected morphology of the *Δgul-1* mutant was hyperbranched pellets.

**Summary:** Protein secretion in the strain was highly increased and had a slightly lower growth rate in the bioreactor when compared to the reference. It presents an almost 2-fold increase in branching frequency, making this mutant a very good candidate for an improved cell factory for protein production.

![](https://static.igem.org/mediawiki/2020/d/de/T--DTU-Denmark--spyder_gul-1.png)

*Radar chart showing 6 different parameters of Δgul-1 normalized to the reference values from ATCC 1015 (shown in yellow). Read about the axis in the [Summary](#section18) section.*

# Plates

The strain was grown on Yeast Extract Peptone Dextrose (YPD), Transformation Media (TM), Creatine Sucrose Agar (CREA) and Czepek Yeast Extract Agar (CYA).
![](https://static.igem.org/mediawiki/2020/c/ca/T--DTU-Denmark--gul-1_allplates.png)

# Microscope pictures and Simulation model

![](https://static.igem.org/mediawiki/2020/e/e8/T--DTU-Denmark--gul1_simulation.png)

*Left: Confocal microscope picture of Δgul-1 at 10X magnification after app. 24h growth. Right: Growth simulation for 12h performed using the Mycemulator.*

Microscopic images were analyzed by the image analysis tool extracting growth parameters which were then fed to the Mycemulator. A simulation of *Δgul-1* growing for 12 hours (using experimental growth rate from our BioLector data) is seen above.

**Parameters specific for simulating *Δgul-1*:**

* Branching frequency: 0.0300301
* Gamma distribution parameters used for curvature angles: (0.6220764, 8.0102895)
* Beta distribution parameters used for branching angles: (3.2414641, 0.8869618)
* Experimental growth rate: 0,314625

# BioLector

Comparing the growth kinetics of the *Δgul-1* mutant with the reference strain ATCC 1015 in the BioLector, the mutant exhibits a shorter lag phase followed by a slightly shorter exponential growth phase. The growth rate for the mutant is higher than for the reference strain, at μMax0.32h-1 versus μMax0.28h-1.

![](https://static.igem.org/mediawiki/2020/3/35/T--DTU-Denmark--gul-1_growth_lector.png)

*Growth profile of Δgul-1 over 72 hours, measuring absorbance at 620 nm. Plotted against the reference strain (ATCC 1015) shown in yellow.*

# Bioreactor

Looking at the growth of the *Δgul-1* mutant in bioreactors, it showed a lag phase equivalent to that of the reference strain ATCC 1015 followed by a longer exponential growth phase. *Δgul-1* has a slightly lower growth rate than the reference strain, at μMax0.31h-1 and μMax0.32h-1 versus μMax0.37h-1 and μMax0.33h-1. Glucose levels start decreasing two thirds of the way through growth and keep decreasing for over 10 hours after the strain enters the stationary phase.

![](https://static.igem.org/mediawiki/2020/c/c2/T--DTU-Denmark--gul-1_growth_reactor.png)

*Left axis: Growth curve obtained from the off-gas analysis (CO2-44 (%)) of Δgul-1 plotted against the reference strain (ATCC 1015) in a logarithmic scale. Right axis: Glucose consumption by Δgul-1 during the fermentation in g/L (dark green).*

![](https://static.igem.org/mediawiki/2020/e/e7/T--DTU-Denmark--bioreactor_micro_gul-1.png)

*Light microscope picture of Δgul-1 after 23 hours of fermentation with a 20X magnification.*

The microscope picture from the bioreactor sample for *Δgul-1* has a similar morphology to that of the brightfield microscopy pictures seen above. It showed a hyperbranched phenotype in a more dispersed network.

# Protein secretion

Glucoamylase activity has a significant 2-fold increase in this mutant compared with the reference strain. Such high glucoamylase activities were reach that the total activity was not measured. From the flat curve it could be expected that the glucoamylase activity would have reached even higher levels have it been measured.

![](https://static.igem.org/mediawiki/2020/9/97/T--DTU-Denmark--gluco_gul1.png)

*Glucoamylase activity in UA/mL of Δgul-1 from 8 samples taken during the fermentation. Plotted against the reference strain (ATCC 1015) shown in yellow.*

The total protein secretion was to the reference strain and thereby the specific activity was substantially higher. This might be due to a different protein secretion profile that has a similar amount of protein being secreted but where the family of glucoamylases are preferentially produced.

 ![](https://static.igem.org/mediawiki/2020/9/9e/T--DTU-Denmark--gul-1_gluco.png)

*Δgul-1 bioreactor duplicates compared to reference strain (ATCC 1015) of the last time-point samples from the fermentations. Green: Glucoamylase activity in UA/mL. Blue: Specific activity in UA/mg calculated from the activity and the protein concentration. Purple: Protein concentration in mg/mL.*

![](https://static.igem.org/mediawiki/2020/b/be/T--DTU-Denmark--aplD_tryit.png)

*ΔaplD*

**Theoretical expectation:** *aplD* encodes a gamma-adaptin involved in endosomal cargo transport and affects filamentous growth and pellet formation. Deletion of the gene was expected to result in a hyperbranched morphology.

**Summary:** From the different experiments it can be assumed that *ΔaplD* plays an important role in proper growth. Therefore, the *ΔaplD* mutant is not viable.

![](https://static.igem.org/mediawiki/2020/4/44/T--DTU-Denmark--spyder_aplD.png)

*Radar chart showing 6 different parameters of ΔaplD normalized to the reference values from ATCC 1015 (shown in yellow). Read about the axis in the [Summary](#section18) section. \*Indicates that not all data was obtained to fill out the chart.*

# Plates

The strain was grown on Yeast Extract Peptone Dextrose (YPD), Transformation Media (TM), Creatine Sucrose Agar (CREA) and Czepek Yeast Extract Agar (CYA).
![](https://static.igem.org/mediawiki/2020/c/c7/T--DTU-Denmark--aplD_allplates.png)

# Microscope pictures and Simulation model

Unfortunately, we did not get a microscopy picture of *ΔaplD* where it was possible to see branching angles, curvature of mycelia, etc. Thus we could not analyze the images properly and simulate the growth.

# BioLector

Comparing the growth kinetics of the *ΔaplD* mutant with the reference strain ATCC 1015 in the BioLector, the mutant exhibits a much longer lag phase followed by a three times longer exponential growth phase. The growth rate for the mutant is also much lower than for the reference strain, at μMax0.10h-1 versus μMax0.28h-1. Additionally, only one out of the three replicates showed any growth at all.

![](https://static.igem.org/mediawiki/2020/6/66/T--DTU-Denmark--aplD_growth_lector.png)

*Growth profile of ΔaplD over 72 hours, measuring absorbance at 620 nm. Plotted against the reference strain (ATCC 1015) shown in yellow.*

# Bioreactor and Protein secretion

*ΔaplD* was not run in the bioreactor due to time limitations. Data for growth in bioreactor and protein secretion for this strain were therefore not obtained.

![](https://static.igem.org/mediawiki/2020/1/1b/T--DTU-Denmark--spaA_gul-1_tryit.png)

![](https://static.igem.org/mediawiki/2020/2/20/T--DTU-Denmark--novel_work.svg)

*ΔspaA\_Δgul-1*

**Theoretical expectation:** This is a double knock-out mutant, where both *spaA*, which might be involved in polarity maintenance and polar growth, and *gul-1*, which encodes an mRNA binding protein involved in cell wall remodeling, are knocked out. There does not exist any prior knowledge in the literature about simultaneously knocking out both of these genes in *A. niger*.

**Summary:** The strain presents higher branching frequency and protein secretion than the reference strain. Growth rate and lag phase are very similar to the reference. On solid media it presented a very slow growth making it harder to work with.

![](https://static.igem.org/mediawiki/2020/8/8c/T--DTU-Denmark--spyder_spaA_gul-1.png)

*Radar chart showing 6 different parameters of ΔspaA\_Δgul-1 normalized to the reference values from ATCC 1015 (shown in yellow). Read about the axis in the [Summary](#section18) section.*

# Plates

The strain was grown on Yeast Extract Peptone Dextrose (YPD), Transformation Media (TM), Creatine Sucrose Agar (CREA) and Czepek Yeast Extract Agar (CYA).
![](https://static.igem.org/mediawiki/2020/7/78/T--DTU-Denmark--spaA_gul1_allplates.png)

# Microscope pictures and Simulation model

![](https://static.igem.org/mediawiki/2020/1/16/T--DTU-Denmark--spaA_gul1_simulation.png)

*Left: Confocal microscope picture of ΔspaA\_Δgul-1 at 10X magnification after app. 24h growth. Right: Growth simulation for 12h performed using the Mycemulator.*

Microscopic images were analyzed by the image analysis tool extracting growth parameters which were then fed to the Mycemulator. A simulation of *ΔspaA\_Δgul-1* growing for 12 hours (using the experimental growth rate from our BioLector data) is seen above.

**Parameters specific for simulating *ΔspaA\_Δgul-1*:**

* Branching frequency: 0.0298207
* Gamma distribution parameters used for curvature angles: (1.6563537, 6.1423278)
* Beta distribution parameters used for branching angles: (3.0350945, 0.9824538)
* Experimental growth rate: 0.3264333

# BioLector

Comparing the growth kinetics of the *ΔspaA\_Δgul-1* mutant with the reference strain ATCC 1015 in the BioLector, the mutant exhibits a shorter lag phase followed by an equivalently long exponential growth phase. The growth rate for the mutant is higher than for the reference strain, at μMax0.33h-1 versus μMax0.28h-1.

![](https://static.igem.org/mediawiki/2020/d/de/T--DTU-Denmark--spaA_gul1_growth_lector.png)

*Growth profile of ΔspaA\_Δgul-1 over 72 hours, measuring absorbance at 620 nm. Plotted against the reference strain (ATCC 1015) shown in yellow.*

# Bioreactor

Looking at the growth of the *ΔspaA\_Δgul-1* mutant in bioreactors, it showed an equivalently long lag phase as the reference strain ATCC 1015 followed by a longer exponential growth phase. *ΔspaA\_Δgul-1* has a growth rate very close to that of the reference strain, at μMax0.32h-1 and μMax0.33h-1 versus μMax0.37h-1 and μMax0.33h-1. Glucose levels start decreasing half way through growth and are depleted simultaneously with the strain entering the stationary phase.

![](https://static.igem.org/mediawiki/2020/9/9c/T--DTU-Denmark--spaA_gul1_growth_reactor.png)

*Left axis: Growth curve obtained from the off-gas analysis (CO2-44 (%)) of ΔspaA\_Δgul-1 plotted against the reference strain (ATCC 1015) in a logarithmic scale. Right axis: Glucose consumption by ΔspaA\_Δgul-1 during the fermentation in g/L (dark green).*

![](https://static.igem.org/mediawiki/2020/1/14/T--DTU-Denmark--bioreactor_micro_spaA_gul-1.pngo_chsC_gul-1.png)

*Light microscope picture of ΔspaA\_Δgul-1 after 26 hours of fermentation with a 20X magnification.*

The microscope picture from the bioreactor sample for *ΔspaA\_Δgul-1* has a very similar morphology to that of the brightfield microscopy pictures seen above. It showed a hyperbranched morphology along with a more pelleted network.

# Protein secretion

Both duplicates display the same profile, with a rapid increase in glucoamylase activity during the last 10 hours of the fermentation.

![](https://static.igem.org/mediawiki/2020/b/b9/T--DTU-Denmark--gluco_spaA_gul1.png)

*Glucoamylase activity in UA/mL of ΔspaA\_Δgul-1 from 8 samples taken during the fermentation. Plotted against the reference strain (ATCC 1015) shown in yellow.*

The final protein secretion varies considerably, with one of them being very low, resulting in the specific activity being very high. Considering this is probably caused by some type of experimental error when measuring the protein concentration, we can assume that the specific activity is overestimated. However it is clear that the strain produces more glucoamylase compared with the reference strain.
 ![](https://static.igem.org/mediawiki/2020/f/fb/T--DTU-Denmark--spaA_gul-1_gluco.png)

*ΔspaA\_Δgul-1 bioreactor duplicates compared to reference strain (ATCC 1015) of the last time-point samples from the fermentations. Green: Glucoamylase activity in UA/mL. Blue: Specific activity in UA/mg calculated from the activity and the protein concentration. Purple: Protein concentration in mg/mL.*

# Comparison

# Simulations

As we see in the microscope the growth pattern of our strains are very different. This is also clear to see in our growth simulations from the Mycemultor. Take your time to enjoy these amazing simulations.

## ATCC 1015

![](https://static.igem.org/mediawiki/2020/b/b6/T--DTU-Denmark--sim_WT.gif)

## *Δgul-1*

![](https://static.igem.org/mediawiki/2020/2/21/T--DTU-Denmark--sim_gul1.gif)

## *ΔchsC\_Δgul-1*

![](https://static.igem.org/mediawiki/2020/3/31/T--DTU-Denmark--sim_chsC_gul1.gif)

## *ΔchsC*

![](https://static.igem.org/mediawiki/2020/c/cd/T--DTU-Denmark--sim_chsC.gif)

## *ΔracA*

![](https://static.igem.org/mediawiki/2020/8/8a/T--DTU-Denmark--sim_raca.gif)

## *ΔchsC\_ΔspaA*

![](https://static.igem.org/mediawiki/2020/3/3a/T--DTU-Denmark--sim_chsC_spaA.gif)

## *ΔspaA*

![](https://static.igem.org/mediawiki/2020/f/f7/T--DTU-Denmark--sim_spaA.gif)

## *ΔpkaR*

![](https://static.igem.org/mediawiki/2020/0/09/T--DTU-Denmark--sim_pkaR.gif)

## *ΔspaA\_Δgul-1*

![](https://static.igem.org/mediawiki/2020/b/b7/T--DTU-Denmark--sim_spaA_gul1.gif)

# Growth

The growth rates of all the mutants were determined from BioLector growth data. Seven of mutants were also run in 1L bioreactors and growth rates were determined from these as well. Unfortunately, we were not able to run the last two, *ΔaplD* and *ΔpkaR*, in the bioreactor due to time limitations.

Overall the growth rates obtained from the bioreactor runs were higher than the ones from the BioLector, with two exception *ΔchsC* and *ΔspaA*. The growth rates determined from the BioLector are for some of the mutants not comparable to the growth rates obtained from bioreactor runs. An example of this is *ΔspaA* which shows the highest value in the BioLector whereas its growth rate in the bioreactor is half of the one for the reference strain. These differences might be due to differences in fermentation conditions in the two setups. The bioreactor is in general better as it allows you to control more parameters including pH, agitation and oxygen level.

For growth rates obtained from the bioreactor, the duplicates show consistent results. All the mutants had similar growth rates to the reference strain with the exception of *ΔchsC* and *ΔspaA*.

![](https://static.igem.org/mediawiki/2020/5/52/T--DTU-Denmark--all_growth_lector.png)

*Bar chart of the mean growth rate with their corresponding standard deviation for all the mutant strains compared to the reference strain (ATCC 1015) obtained from the BioLector.*

![](https://static.igem.org/mediawiki/2020/d/d3/T--DTU-Denmark--growth_all_bioreactor.png)

*Bar chart of the growth rate for both duplicates for seven of the mutant strains compared to the reference strain (ATCC 1015) obtained from the bioreactor. The black line represents the conditions for the first three strains separated from the others due to them being with overpressure. The strains are therefore only comparable within the first three and within the last six strains.*

From the BioLector and bioreactor growth data, the length of the lag phases and exponential phases were determined. From the plot it can be observed that the replicates follow each other very closely. For all mutants it is seen that the lag phase is longer in the BioLector than in the bioreactors. This could be due to bioreactors having more optimal growth conditions compared with the BioLector, thereby resulting in a shorter adaptation phase.

From the bioreactor most strains show a similar lag phase to that of the reference strain. In industry a short lag phase would be favorable as it shorten the fermentation time without affecting the time of efficient production (in the exponential and stationary phases). In this sense, *ΔchsC* and *ΔspaA* show great promise as they have a significant shorter lag phases than the reference strain. So although they have a smaller growth rate, their total fermentation time is shorter than that of the reference strain (before entering stationary phase).

*ΔchsC\_Δgul-1* presents a slightly shorter lag phase and significantly shorter fermentation time (before entering stationary phase) due to its higer growth rate. This makes it a favorable strain.

![](https://static.igem.org/mediawiki/2020/6/60/T--DTU-Denmark--Lector_phases.png)

*Stacked bar chart of the duration in hours of lag and exponential phases with their corresponding standard deviation from all the mutant strains compared to the reference strain (ATCC 1015) obtained from the BioLector.*

![](https://static.igem.org/mediawiki/2020/7/73/T--DTU-Denmark--reactor_phases.png)

*Stacked bar chart of the duration in hours of lag and exponential phases from all the mutant strains that were run in the bioreactor compared to the reference strain (ATCC 1015). The black line represents the conditions for the first three strains separated from the others due to them being run with overpressure. The strains are therefore only comparable within the first three and within the last six strains.*

# Protein secretion

From the figure below it is seen that protein secretion and glucoamylase activity varies significantly between the strains. The strains with a knock out in the *gul-1* gene show significantly higher specific activity than the reference strain. *Δgul-1* showed a 3-fold increase where *ΔchsC\_Δgul-1* and *ΔspaA\_Δgul-1* showed an approximately 2-fold increase indicating that they are significantly better protein producers.

In this project we have chosen to assess protein production as an indicator of succesful engineering. However in industry it depends on the product of choice. A high protein producing strain might not be the best for production of secondary metabolites.

![](https://static.igem.org/mediawiki/2020/e/e7/T--DTU-Denmark--all_gluco.png)

*Protein secretion and glucoamylase activity for all strains run in the bioreactor for the last time-point samples. Green: Glucoamylase activity in UA/mL. Blue: Specific activity in UA/mg calculated from the activity and the protein concentration. Purple: Protein concentration in mg/mL. The black line represents the conditions for the first three strains separated from the others due to them being with overpressure. The strains are therefore only comparable within the first three and within the last six strains.*

# Summary

All our new strains are summarized in the radar charts seen below. The strains are evaluated on six criteria, all normalized to the reference strain:

* **RESHAPE score:** This is a subjective score given by the wetlab team. It is based on how the strain was to work with compared to the reference strain. Things taken into consideration were:

+ Sporulation, was it hard to make spore stocks?
+ How long did it take for it to grow on solid media?
+ How promising did we find it as a good cell factory for protein production?

* **Branching frequency:** Value analyzed by the Morphologizer from the microscopic pictures. High branching frequency is linked to hyperbranching.
* **Space fillingness:**
Value analyzed by the Morphologizer from the microscopic pictures. This value is an indicator of agglomeration in mycelium.* **Growth rate (BioLector):** Growth rates obtain from the BioLector.
* **Growth rate (Bioreactor):** Growth rates obtain from the bioreactor.
* **Protein secretion (specific activity):** Glucoamylase activity per mg secreted protein.

As seen from the radar charts all our strains behave differently due to their gene knock out. In this project we have seen the selected genes effect on morphology and how they affect *A. niger* performance as a cell factory. We have proven that the tools of synthetic biology can be a key player in controlling and improving the cell factories of the future.

![](https://static.igem.org/mediawiki/2020/d/dd/T--DTU-Denmark--spyder_all.png)

*Comparison of the nine radar charts from all the mutant strains normalized to the reference values from ATCC 1015 (shown in yellow). \*Indicates that not all data was obtained to fill out the chart.*

# Signal Peptides

![](https://static.igem.org/mediawiki/2020/8/86/T--DTU-Denmark--sigP_starchplates_tall.svg)

Eleven mutants with signal peptide glucoamylase insertions were constructed. Eight with natural signal peptides found in literature and three with synthetic signal peptides predicted by the SignalPrepper tool. To learn more about the way these strains were designed please see the [*Design page.*](https://2020.igem.org/Team%3ADTU-Denmark/Design)We wanted to test the secretion levels for the different signal peptide strains through the glucoamylase assay. Unfortunately we did not reach this point due to time limitations. Here you see the eight of the native signal peptide strains plated on minimal media starch plates. Iodine was added to the plates to show were the starch was consumed. From these pictures it can not be concluded how well the different strains secret glucoamylase.

![](https://static.igem.org/mediawiki/2020/2/26/T--DTU-Denmark--sigP_starchplates_big.svg)

[![](https://static.igem.org/mediawiki/2020/a/ac/T--DTU-Denmark--Nextpage.png)](https://2020.igem.org/Team%3ADTU-Denmark/Proof_Of_Concept)

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