Team:DTU-Denmark/Integrated Human Practices

As science affects the world, the world affects science. This page describes what this project is going to bring to the world, and what we have learned and used from others

Integrated Human Practices

From the very beginning of our project, our team shared a common goal – we wanted to create something meaningful. To this end, we have been in contact with important stakeholders and their advice has shaped our approach, and the project's direction.

Despite their relative anonymity, filamentous fungi are responsible for most of the industrially produced enzymes and are therefore exceptionally important to a lot of people’s everyday lives.
Project LEAP was founded in response to the acute lack of publicly available resources for synthetic biology work within filamentous fungi, and therefore aimed to expand the synthetic biology toolbox for these organisms.
Originally, the project aspired to create synthetic promoter libraries for filamentous fungi, yeast, and moss – but following valuable discussions with several companies and scientists, the team decided to develop a software that would enable the creation of promoters for any organism, and attempted to test the function of said software in Aspergilli.

Our stakeholder analysis led us to talk to 3 different companies: Zymergen, Novozymes, and Bolt Threads. We also talked to 3 fungal experts: Peter Richard (VTT, Finland), Jens Christian Frisvad (DTU, Denmark) and Jakob Blæsbjerg (DTU, Denmark). Additionally, we addressed the public by attending Science EXPO in Copenhagen, two biotechnology camps for high school students, and teaching synthetic biology in two high schools.
Fig. 1: Overview of the process for integrated human practices throughout the project.
The stakeholder analysis shows a square with four sections, which divides stakeholder’s into four categories: high interest and high power, high interest and low power, low interest, and high power, and low interest and low power.
Fig. 2: Stakeholder analysis

Stakeholder Analysis

In March, the team looked into the impact the project could have on different stakeholders, and therefore made a stakeholder analysis, as shown in figure 2. This stakeholder analysis reveals that companies such as Novozymes, Zymergen, and Bolt Threads are among the most important to our project, both in interest and power. This means that their opinions should be managed closely. Additionally, researchers such as Jens Frisvad (DTU, Denmark), Jakob Blæsbjerg (DTU, Denmark), and Peter Richard (VTT, Finland) could benefit from our project, making them important stakeholders. Although other iGEM teams do not have a lot of power, their interest could nevertheless be great and they should, therefore, be well informed. The public and the DTU BlueDot program will most likely not take much interest in the project as a promoter library can be a very technical concept and not immediately usable by non-specialists. However, as DTU BlueDot is a big sponsor of the team, they are important to keep satisfied.

Based on the stakeholder analysis, we decided to contact three different biotech companies; Novozymes, Zymergen, and Bolt Threads as they all work with genetically modified filamentous fungi. We asked them how our project could influence their work, and for suggestions regarding the experiments.

We also reached out to several scientists, including Jakob Blæsbjerg from DTU and Peter Richard from VTT (Technical Research Centre) in Finland. They provided us with protocols and advice on how to grow the fungi, and how to ensure we acquired reproducible and comparable results.

Even though the public is neither very powerful nor particularly interested in the project, at least according to our stakeholder analysis, we nevertheless decided to contact high schools in order to talk to young people about synthetic biology, our project and what good we believe it can do in the world. We also partook in several events: The annual UNF (Ungdommens Naturvidenskabelige Forening) Biotech Camp; Science Expo, a large science fair in Copenhagen; and the annual Biotech Academy Camp in order to increase their knowledge and interest in a topic like synthetic biology. This is described further on Education and Engagement.

The stakeholder analysis shows a square with four sections, which divides stakeholder’s into four categories: high interest and high power, high interest and low power, low interest, and high power, and low interest and low power.
Fig. 2: Stakeholder analysis

Click on the different bubbles to read more about what we learned from each stakeholder.

Interactive figure of what we learned from our stakeholders: Bolt Threads, Jens Frisvad, Jakob Blæsbjerg, Zymergen, Peter Richards, and Novozymes Zymergen Zymergen Peter Richards Peter Richards Novozymes Novozymes Bolt Threads Bolt Threads Jens Frisvad Jens Frisvad Jakob Blæsbjerg Jakob Blæsbjerg

Zymergen

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Kenneth Bruno and Grayson Wawrzyn from Zymergen visited DTU in May 2019. On this occasion, we met with them and discussed our project. As Zymergen works with i.a. filamentous fungi and know what other biotechnology companies look for in promoters, they were able to provide us with a lot of valuable advice on how to approach our project. Among other things, they advised us to use the Aspergillus niger strain ATCC 1015 since this strain is commonly used in industry and contains an auxotrophic selection marker.
Zymergen specialises in genetically engineering a broad range of non-standard hosts, and this directed us to open the software to be able to parse any given organism genome. Additionally, Zymergen is in the business of gene expression regulation/control, and a stable promoter ladder is therefore vital to their setup. Accordingly, we directed our experimental setup to also test the scalability of our synthetic promoters. Additionally, we learned that the industry is interested in scarless assemblies between the promoter region and the start codon, as well as promoters that are active in the stationary phase. We took all of this into consideration when designing the software to create the promoters and when assembling the plasmids.

Peter Richard

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We talked to Peter Richard, a principal scientist at VTT in Finland. He is an expert in using synthetic biology with regards to Aspergillus spp. and provided feedback on our approach, in addition to advice on making our results as applicable as possible. He advised us to grow the fungi under comparable conditions, i.e. that we should keep things such as growth phase, temperature, media, and reporter gene consistent and in mind when testing the promoters. We implemented this throughout the project, most notably in the characterization phase where a standardized media was used. RFP was used as a reporter and the growth was measured over time, enabling evaluation of the promoters during both the lag phase, exponential phase, stationary phase, and eventually the death phase. Furthermore, he recommended keeping the plasmid copy number in mind while running the experiments as this would enable us to correlate gene expression to each promoter. We, therefore, decided to integrate the constructs in the Aspergillus genome, but unfortunately, we did not have time to test our promoters further in this construct, as described in the results.

Novozymes

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In June, the team visited the Novozymes facilities in Lyngby to learn about their approach to working with filamentious fungi in the industry. Apart from a guided tour around the facilities, the team also had an inspiring talk with Mikako Sasa, Science Manager and fungi specialist, where we discussed different fungal species and their potential applications.

From this initial meeting, we learned that promoters are valuable assets for a company such as Novozymes, and that our promoters would be even more valuable if they were consistent across different scales and conditions. This allows the user to worry less about the implementation of the promoters and enables them to focus on optimizing pathways and fermentation conditions.

In August, we came back and talked with Mary Ann Stringer and Kirk Matthew Schnorr, both scientists at Novozymes. Here, we learned more about what Novozymes looks for in a desirable promoter. As they are in the business of producing enzymes, they are usually looking for strong promoters, however, ideally they would want a promoter that was inactive in the exponential phase and highly active in the stationary phase. This would limit the stress on the organism while growing and building biomass, and maintain the high production rates when the amount of biomass is sufficiently large. This is consistent with what we had previously heard from Zymergen, and we decided to prioritize promoters that fulfilled these criteria.

At this meeting, we also learned about their concept of “Tank Years”. This refers to the number of times a tank can be run in a year and takes into consideration cleaning, sterilization, inoculating, running, and extraction of the product. Novozymes is interested in producing as much as possible, as quickly as possible, and this means that the production time in the tanks should be minimal, the cleaning easy, and the enzyme titers maximized.
We have taken this into account when designing our bioreactor experiments, where a conscious effort was taken to minimize downtime between runs.

Bolt Threads

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We have been in contact with the bio-materials company Bolt Threads which among other things work with filamentous fungi. We were in contact with Edyta Szewczyk, senior scientist at Bolt Threads, who gave us a lot of feedback and ideas. Especially three key points were emphasized: Filamentous fungi takes a long time to grow, which means we need to plan ahead; good reporter systems are essential for detecting transformants and expression, and; targeted genomic integration is a good solution to control for copy-number and genomic context. We have tackled these point by, 1) transforming our fungi as soon as possible, 2) using fluorescence (both GFP and RFP) to measure the expression, and 3) testing genomic integration methods which can be seen on Design page.

Jens Frisvad

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We reached out to one of the leading research scientists in filamentous fungi, Jens Christian Frisvad. Among other things, he told us to look at a several different fungal species, and not only the standard ones, as these might be able to expand the range of products that are possible to produce industrially. By basing our software on genomes from many different Aspergillus spp. our promoters are ideally applicable in several different species. Additionally, our software allows for the application of any list of genomes and the creation of promoters for any given species, enabling the creation of promoter libraries for non-standard organisms.

Jakob Blæsbjerg

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Following the discussion with Zymergen, we decided to contact Jakob Blæsbjerg Hoof, associate professor at the Department of Biotechnology and Biomedicine. He told us that using a fluorescence marker was the easiest approach to select for transformants, and that we should add a secretion tag to the fluorescent protein in order to prevent accumulation inside the cell. This was later backed up by Bolt Threads. Additionally, Blæsbjerg provided us with the ATCC 1015 Aspergillus niger strain, in addition to a protease-deficient variation (prtT KO), which were used in the experiment as well as protocols for protoplasting and transformation.



The logos of our three biggest supporters, DTU Blue Dot, Novo Nordisk fonden and Otto Mønsted fonden The logos of all of our sponsors, DTU, BioNordica, Eurofins Genomics, Qiagen, NEB New England biolabs, IDT Integrated DNA technologies and Twist bioscience