iGEM 2021 — Team DTU-Denmark

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# Team:DTU-Denmark/Notebook

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# Notebook

February
March
April
May
June
July
August
September
October

## February

### February 15th 2021: 1st General meeting

On this day we had our fisrt online team meeting! We met our supervisors and introduced ourselves to eachother.
Our supervisors introduced us to the world of iGEM and we split into 3 teams to do our homework for next time: brainstorm about 2 project ideas per group.

### February 22nd 2021: 2nd General meeting

On this day we formed our subgroups and aligned expectations within the groups.
Following this we pitched our developed project ideas to eachother. Our homework after this meeting was to further discuss and develop our ideas. Team building also started to plan activities for us to get to know eachother better.

## March

### March 1st 2021: 3rd General meeting

On this day we presented our further developed project ideas to eachother and brainstormed together, how they could be even better. We established expectations from drylab and wetlab teams. Human practices and PR subgroups gave an overview of interviews and stakeholders contacted.

### March 8th 2021: 4th General meeting

On this day, we worked on further developing our projects ideas and voted on the different ones, to decide which one we will work with. We started creating a budget for our project with the help of the economy subgroup. The PR/SoME subgroup got access to all social media platforms and started working on potential collaboration ideas.

### March 15th 2021: 5th General meeting

Based on the project assessment template the scoring from each group was validated. Working on methane assimilation got the most votes! We discussed the different time slots to participate in the Ambassador call organized by iGEM.

### March 22nd 2021: 6th General meeting

We met in person for the first time to identify potential areas of interest in the project. Decided to assimilate methane in 2 organisms via co-culturing with pMMO subunit being able to metabolize methane. Admin updates to create iGEM account and get the team code. Decided to use Benchling as a platform to document details of our project.
We joined the European Ambassador meeting with other iGEM teams from Tubingen, Boku Vienna, Moscow to discuss status of project and covid crisis affecting project workflow.

Souvenir from the Ambassador meeting:

![](https://static.igem.org/mediawiki/2021/1/18/T--DTU-Denmark--ambassador-meeting.jpg)

## April

### April 8th 2021: 7th General meeting

On this day a new supervisor joined our team: Benjamin Schwetz. We discussed the compartmentalisation of our project into two parts: i) methane to methanol and ii) methanol utilization. Furthermore we made a brief project description. Our economy team applied for our first of many grants! This was the day when we have found out that we can start working in the lab on June 1st, which we were really excited about and started planning right away! We also discussed the on-going hearings in the Danish parliament about gene editing and agreed that we should be ready for upcoming political discussions.

### April 12th 2021: 8th General meeting

On this day we had a discussion about the possibilities of co-culturing two microorganisms - one converting methane to methanol (mutant methanotrophic bacteria), another to uptake methanol and produce protein of interest (mutant yeast). The idea of joining a political festival in Bornholm was brought up for the first time. We said good bye to Annette, who decided to leave our team.

### April 26th 2021: 9th General meeting

On the 9th meeting we decided to split into 3 groups to design the required parts simultaneously. Each group has established separate meetings on top of the general meeting. We decided to use Golden Gate Assembly as one of our cloning methods and Emil gave a plasmid crash course for the whole team to create the best possible designes. We also discussed digital content for Folkemødet.

This month we could also meet in person for the first time!

![](https://static.igem.org/mediawiki/2021/1/13/T--DTU-Denmark--first-meeting.jpg)

## May

### May 1st 2021: Saturday meeting

We decided to meet up on the weekend to work on our constructs: Looked into plasmid based USER cloning and discussed the strategies for using available strains and plasmids codon optimised for *K. phaffii* , humans, *S. cerevisiae* .

### May 5th 2021: 11th General meeting

On this day, wetlab and drylab made the last preparations to enter the lab: we ordered NEB kits, did the codon optimisation of our constructs, brainstormed more technical questions and had a meeting with Tomas Strucko. The human practices subgroup was preparing for Folkemødet and our economy group sent more grant applications. We made a logo competition within the team, this is how we decided on our final logo. The PR subgroup had a meeting about editing our website and reached out to Mikkel Schwarz for the team photoshoot.

![](https://static.igem.org/mediawiki/2021/d/dc/T--DTU-Denmark--logo.png)

### May 10th 2021: 12th General meeting

On our 12th general meeting we discussed strategies on education and reaching out to elementary and high schools. Wetlab and drylab were looking for methanotroph promoters and did some research on glucose repression. We reached out to Unibio and scheduled a meeting with Solange. In the beginning of the project we picked Mambalgin-1 as our protein of interest. The human practices subgroup further planned the GMO discussions at Folkemødet and started planning discussions with high school students.

### May 17th 2021: 13th General meeting

This week wetlab and drylab went through the USER protocol and started determining integration sites in  *K. phaffii*  genome. Before going to the lab we had to fill out a lab safety form and we all also signed a team contract stating the expectations in the duration of the project. Karen and Karl were working hard on preparing for Folkemødet, furthermore we also hired Mikkel Schwarz for our photoshoot and promotion video.

### May 24th 2021: 14th General meeting

August joined our team as a supervisor and after discussions with him, we decided to change the protein of interest to hemoglobin, we had discussion on using soy or human hemoglobin, as human hemoglobin could also possibly be used therapeutically, but ethically it is not fit for food production, thus we picked soy leghemoglobin as final protein for the proof-of-concept. When it comes to our human practices subgroup, Karl and Karen were working on the video advertising for Folkemødet and we also had a meeting with Kenneth from Novozymes.

## June

### June 2nd 2021: 15th General meeting

During the first week of June we all finished the lab safety test and training, so we were ready to go to the lab. We received assistance from Niels, from the 2020 DTU iGEM team for our drylab modeling work. The human practices team had 2 great interview with Kenneth and with Irini, and we officially got accepted to Folkemødet. On Friday morning we had our first common breakfast, which became a habit on Fridays during the summer months.

### June 9th 2021: 16th General meeting

This week we had a meeting with Ampliqon to finalize the details of our sponsorship and talked about a possible facility visit. We ordered the gRNAs for our 4 identified integration sites and made cryostocks for  *K. phaffi* GS115 Δku70 strain. Our drylab team had a meeting with Nikolaus Sonnenschein from DTU Bioengineering for Genomic Scale Metabolic modeling to analyze potential things to do with the GS115 model. Possible risks associated with using pMMO were validated and it was agreed to look for yeast which have native methane activity or to keep sMMO as a backup.

### June 16th 2021: 17th General meeting

Wetlab started working on pDIV151 purification and PCR amplification for all of our fragments, the general workflow of all the experiments were made and documentation on Benchling started. Drylab discussed the aspects of the GSM model to further optimize the existing model. This week we had our portraits taken by Mikkel, furthermore Karen and Karl gave an amazing presentation at Folkemødet.

### June 23rd 2021: 18th General meeting

This week the wetlab group realized that our gRNA primers did not have the right sequence, thus we ordered new primers, which finally resulted in successful experiments. Drylab was working on the GSM model based on Niko's advices. Our economy team booked the summer house for our wiki freeze week and the PR team was working on writing the children's book.

### June 29th 2021: 19th General meeting

Wetlab had great news this week, the gRNA primers finally worked, and started doing the USER cloning experiments, while drylab worked on further perfecting the GSM model. We filmed our presentation video this week, which required a lot of preparation during the days leading up to the filming. Our PR team made a nice post card for a collaboration with iGEM Düsseldorf and started planning the Ampliqon facility visit with iGEM SDU.

## July

### July 6th 2021: 20th General meeting

Our wetlab team had to troubleshoot this week for the failed USER transformations with the help of August and Louise, and started plannig the methanol kill curve experiment. Drylab started working on the gene knock-out using cobrapy and trying to optimize protein production and they ran the first attempts using GSM model which show that including sorbitol and/or glycerol in the medium gives a better protein production. Furthermore our human practices team applied to participate in Naturvidenskabelig festival.

### July 13th 2021: 21st General meeting

This week, we have found the cause of USER cloning failing - wrong combination of RE & nicking enzymes and with this information in mind we corrected our protocols and continued with our experiments. Drylab this week was working on starting training on Kivy for app development and on growth curves with R script. We successfully submitted the promotion video and signed up for the iGEM Nordic conference.

### July 20th 2021: 22nd General meeting

Our wetlab team successfully carried out USER cloning for IS1C1 and IS2C1. One out of four available crRNA candidates per IS were tested. We got great colony PCR results for crRNA candidates targeting IS4 and IS5, also confirmed by sequencing. Drylab continued working on documenting everything for the GSM model. Human practices had a busy week, with interviews with Unibio and Novozymes. This week we posted our last team member introduction on all our social media platforms and translated our promotion video into all of our teammember's native and spoken languages!

### July 27th 2021: 23rd General meeting

This week we designed primers and homology arms and ordered them for further experiments, we also started planning yeast transformation for the following week. Our team started preparing for the Nordic meetup. Drylab and wetlab started collaborating more closely, in order to improve our models. Human practices was working on future meetings and signed up for the iGEM CRISPR conference.

## August

### August 3rd 2021: 24th General meeting

This week during the general meeting we decided to split up the wetlab team into 3 subgroups: pMMO, Hemoglobin and AOX knockout groups to be more efficient. Drylab had a final discussion about the GSM model with Niko and Benjamin, finalized the kinetic model and prepared it for presentation. Human practices hosted a GMO discussion with other iGEM teams and started discussing collaboration possibilities with the Gröningen iGEM team. Our initial thought was to collaborate by model verification and/or app development. We started planning the high school visit as part of our education outreach. Unfortunately we had to say goodbye to Cristina, because she decided to go on exchange in the US.

### August 10th 2021: 25th General meeting

The pMMO subgorup started working on gRNA vector construction, two candidates for each chromosome based on advice from our supervisors. The hemoglobin subgroup created an overview of required inserts, figuring out measurement methods for leghemoglobin content. Discussed different promoters to determine expression levels, while the AOX groups started working on methanol shake flask experiments. Drylab was working hard on the Kivy app development and finished the GSM model! Our team finally registered for the Giant Jamboree and had a meeting with Jens Preben Morth about protein expression and pMMO integration.

### August 17th 2021: 26th General meeting

This week the hemoglobin subgroup performed their first Golden Gate Assembly with the new kits, the pMMO subgroup was working on PCR amplification of new primers for homology arms and plasmid extraction, while the AOX group, after many failed USER clonings, retried with new generic PCR fragments and newly linearized plasmids. They also started experiments to investigate if K. phaffii can be frozen down immediately before electroporation and used at a later time. Drylab was working on uploading parts to the registry and on the app development. Our team also started working on ideas for the presentation video.

### August 24th 2021: 27th General meeting

This week the pMMO subgroup had some troubles with the Golden Gate Assembly and started trouble shooting with the help of our supervisors. AOX subgroup started working on yeast colony PCRs and they confirmed their first successful knockout:  *aox2.*  The hemoglobin subgroup was working hard on Golden Gate Assembly and they sent their first constructs for sequencing. This time around drylab started working on the structure of the wiki and the struggles with the navbar began. Later this week we had an amazing facility visit to Ampliqon in Odense with the SDU iGEM team.

### August 31st 2021: 28th General meeting

This week the AOX group conducted their last knockout, while the hemoglobin group started experiencing some problems with the sequencing results. The pMMO group obtained colonies for construct 6 and designed new primers for better sequencing. Drylab was working in close collaboration with the Gröningen iGEM team and continued working on the wiki. Our team started having meetings about the presentation video with Mikkel.

## September

### September 7th 2021: 29th General meeting

Our wetlab subgroups were experiencing difficulties with colony PCR and sequencing this week and were working hard brainstorming and troubleshooting with the help of our supervisors. Drylab further developed the GSM model with the option to include different organisms, protein sequence of choice and different activators and repressors and started planning new methanol kill curve experiments. Our whole team was working hard on the manuscript for the presentation video and we decided on the final track of choice.

### September 14th 2021: 30th General meeting

Wetlab was working hard this week to produce nice results, but they experienced some further problems with  *K. phaffii*  transformation and false positives. Drylab continued working on the methanol kill curve experiment and started putting more time into the collaboration with iGEM Gröningen. Signe, Karen and Emil went to Roskilde to hold a workshop to high school students with great success! Furthermore our human practices team had a meeting with Alexandre Huchelmann, member of the European Commision, DG Health and Food Safety.

### September 21st 2021: 31st General meeting

The pMMO group was successful with assembling construct 3 and 6, which later got confirmed by sequencing as well, however they were unsuccessful with the transformation of  *K. phaffii* . Hemoglobin subgroup has success with construct 6, while the AOX group experienced problems with false positives. Human practices group finished working on the children's book this week and started looking into publishing it. Drylab was running further Biolector experiments to test different carbon sources for the GSM model.

### September 28th 2021: 32nd General meeting

AOX subgroup successfully created a double knockout, verified by sequencing and started setting up the experiments for the last knock out. The hemoglobin subgroup was testing many colonies hoping to find one with the right plasmid, while pMMO subgroup had to order new primers because of an incorrect restriction site in the gblock sequences. Drylab made the last modifications on the app and worked hard to prepare the wiki for the wiki freeze week.

## October

### October 5th 2021: 33rd General meeting

For the pMMO subgroup E. coli colony PCR of Pmo\_A and Pmo\_C resulted in no bands on the gel.
Venus was consistently missing from constructs and yeast colony PCR of construct 3 and 6 sequencing results did not confirm genomic integration. AOX group conducted their last transformation, but unfortunately the triple knockout was unsuccessful. For the hemoglobin subgroup results showed that the inserts were not present in the constructs sent for sequencing, additionally *K. phaffii* transformation with construct 6 was unsuccessful. This week during the weekens we filmed our presentation video, participated in an SBE event and started planning the wiki freeze week!

### October 12th 2021: 34th General meeting

This week we stopped all lab work and started focusing on collecting every information for the wiki. Started working on the design and animations and on Saturday we left for the wiki freeze week. In the last week before wiki freeze our team took a trip to a summer house to design and perfect our wiki page while enjoying each other’s company!

![](https://static.igem.org/mediawiki/2021/1/1d/T--DTU-Denmark--car-trip1.png)
![](https://static.igem.org/mediawiki/2021/2/23/T--DTU-Denmark--car-trip2.png)
![](https://static.igem.org/mediawiki/2021/f/f2/T--DTU-Denmark--car-trip3.png)
![](https://static.igem.org/mediawiki/2021/9/99/T--DTU-Denmark--cooking.png)
![](https://static.igem.org/mediawiki/2021/6/63/T--DTU-Denmark--nap.png)

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# Team:DTU-Denmark/Engineering

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## Engineering K. Phaffii for methane utilisation

Our project, Pheast, endeavored to design and test an engineered cell-factory to use methane as a feedstock, using the methylotrophic yeast, *Komagataella phaffii* GS115 (*K. phaffii*). Currently, there is a big incentive for developing efficient gene editing tools such as CRISPR/Cas9-based platforms for engineering *K. phaffii*), as this yeast is a popular production chassis for industrial enzymes and biotherapeutics, due to its recombinant protein production abilities and low secretion of endogenous proteins[1] [2] [3] [4]. This project consisted of several milestones, and iterations through the engineering cycle to reach our goal of developing an efficient toolbox for the genetic engineering of *K. phaffii*. For an efficient use of such a toolbox it is necessary to do both deletions and insertions of genes of interest. Our toolbox does both.

1 / 8

[![](https://static.igem.org/mediawiki/2021/3/35/T--DTU-Denmark--Engineering_Design_1.gif)](https://2021.igem.org/Team%3ADTU-Denmark/Design)

2 / 8

[![](https://static.igem.org/mediawiki/2021/a/a8/T--DTU-Denmark--Engineering_Build_1.gif)](https://2021.igem.org/Team%3ADTU-Denmark/Experiments)

3 / 8

[![](https://static.igem.org/mediawiki/2021/2/28/T--DTU-Denmark--Engineering_Test_1.gif)](https://2021.igem.org/Team%3ADTU-Denmark/Results)

4 / 8

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5 / 8

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6 / 8

[![](https://static.igem.org/mediawiki/2021/1/1a/T--DTU-Denmark--Engineering_Build_2.gif)](https://2021.igem.org/Team%3ADTU-Denmark/Experiments)

7 / 8

![](https://static.igem.org/mediawiki/2021/6/60/T--DTU-Denmark--Engineering_Test_2.gif)

8 / 8

![](https://static.igem.org/mediawiki/2021/4/43/T--DTU-Denmark--Engineering_Learn_2.gif)

❮
❯

### Summary

To validate the use of our CRISPR/Cas9 toolbox for genomic integration and gene deletion K. phaffii we went through two iterations of the engineering cycle. At the end of the second cycle, and after several rounds of troubleshooting, we experimentally validated our designed CRISPR based toolbox.

---

## References

##### [1] Yang, Z., & Blenner, M. (2020). Genome editing systems across yeast species. Current Opinion in Biotechnology, 66, 255-266. [2] Need for Crispr-based tools in k phaffi 2021: Cai, P., Duan, X., Wu, X., Gao, L., Ye, M., & Zhou, Y. J. (2021). Recombination machinery engineering facilitates metabolic engineering of the industrial yeast Pichia pastoris. Nucleic Acids Research, 49(13), 7791–7805. [3] Gao, J., Jiang, L., & Lian, J. (2021). Development of synthetic biology tools to engineer Pichia pastoris as a chassis for the production of natural products. Synthetic and systems biotechnology, 6(2), 110-119. [4] Karbalaei, M., Rezaee, S. A., & Farsiani, H. (2020). Pichia pastoris: A highly successful expression system for optimal synthesis of heterologous proteins. Journal of Cellular Physiology, 235(9), 5867–5881. https://doi.org/10.1002/jcp.29583 [5] 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 [6] 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 [7] Garcia Vanegas, K., Lehka, B. J., & Mortensen, U. H. (2017). SWITCH: a dynamic CRISPR tool for genome engineering and metabolic pathway control for cell factory construction in Saccharomyces cerevisiae. Microbial Cell Factories, 16(25), 25.

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# Team:DTU-Denmark/Experiments

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  Of Concept](https://2021.igem.org/Team%3ADTU-Denmark/Proof_Of_Concept)
  [Implementation](https://2021.igem.org/Team%3ADTU-Denmark/Implementation)

  * Labwork

    [Design](https://2021.igem.org/Team%3ADTU-Denmark/Design)
    [Safety](https://2021.igem.org/Team%3ADTU-Denmark/Safety)
    [Experiments](https://2021.igem.org/Team%3ADTU-Denmark/Experiments)

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      [All Parts](https://2021.igem.org/Team%3ADTU-Denmark/Parts)
      [Part Collection](https://2021.igem.org/Team%3ADTU-Denmark/Part_Collection)
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      [Integrated HP](https://2021.igem.org/Team%3ADTU-Denmark/Human_Practices)
      [Sustainable](https://2021.igem.org/Team%3ADTU-Denmark/Sustainable)
    * Outreach

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# Our Experimental Workflow

For the experimental design, the following workflow was carried out to build the toolbox for the methanotrophic *K. phaffii* cell factory.

**Click on the elements in the workflow to download the protocols for the different experiments!**

![Workplace](https://static.igem.org/mediawiki/2021/3/33/T--DTU-Denmark--flow.png)

**Click the on the button below to read about our Media Protocols!**

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# Overview

The goal of this project was to develop a publicly available toolbox for genetic engineering of *K.phaffii* due to its potential as an industrial cell factory. For this, we divided the project into three parts as the major milestones. First, we wanted to design CRISPR Cas9 tools to engineer *K. phaffii*. Second was to use the designed CRISPR Cas9 tools for genomic deletion and finally in genomic deletion in *K. phaffii* GS115 [1].

CRISPR-Cas9 tools for K. phaffii
Genomic Deletion
 Genomic Integration

## CRISPR/Cas9 tools for *K. phaffii*

The CRISPR-Cas9 system offers a powerful tool for genetic engineering, as it enables marker-free, scar less genome editing. We designed 16 different gRNAs for 4 integration sites and carried out USER cloning. The cloned plasmids were transformed into *E. coli* and colony PCR was carried out to identify transformants harbouring the correctly assembled plasmids. Based on the results of colony PCR, plasmids were purified from the *E.coli* transformants and sent for commercial Sanger Sequencing.

To assemble the sgRNA plasmids, 2 fragments from the pDIV153 plasmid were PCR amplified and an analytical gel was run to check the correct band sizes of these fragments. Fragment 1 was expected to have a band size of ~700 bps and Fragment 2, which are our designed gRNAs, were expected to have a band size of ~500 bps. (Fig. 1 and 2)

![](https://static.igem.org/mediawiki/2021/0/04/T--DTU-Denmark--figure1_fragment1.png)![](https://static.igem.org/mediawiki/2021/c/ca/T--DTU-Denmark--figure2_fragment2.png)

The PCR reactions containing our desired fragments were Dpn1 digested to remove residual methylated template DNA. The pDIV151 backbone was extracted from *E. coli* colonies, provided by our supervisor Louise Kastberg, following extraction these plasmids were linearized using AsiSI and Nb.BsmI. (Fig. 3)

![](https://static.igem.org/mediawiki/2021/c/c7/T--DTU-Denmark--figure3_linear_backbone.png)

USER cloning was carried out to assemble the pDIV151 plasmids containing fragment 1 and fragment 2. Instead of running reactions with 4 candidate gRNAs per integration site, we were advised to select 2 candidates per integration site based on the distance for the target, therefore we selected gRNA\_Chr\_1\_Cand\_1, gRNA\_Chr\_1\_Cand\_4, gRNA\_Chr\_2\_Cand\_1, gRNA\_Chr\_2\_Cand\_2, gRNA\_Chr\_4\_Cand\_1, gRNA\_Chr\_4\_Cand\_3, gRNA\_Chr\_3\_Cand\_1 and gRNA\_Chr\_3\_Cand\_2.

### *E. coli* transformation

Transformed *E. coli* DHα were plated on LB-Amp plates and incubated at 37 °C overnight.

![](https://static.igem.org/mediawiki/2021/d/d1/T--DTU-Denmark--figure4_grna1_5.png)

We did an initial transformation of our constructs containing gRNA\_Chr\_1\_Cand\_1 and gRNA\_Chr\_2\_Cand\_1 which resulted in 1-3 colonies. (Fig. 4) Furthermore a single colony grew on the negative control. We tested all 4 colonies using colony PCR to see if they were carrying the inserts of fragment 1 and 2.

![](https://static.igem.org/mediawiki/2021/2/24/T--DTU-Denmark--figure5_grnaX6.png)

A subsequent transformation was carried out with the remaining gRNA candidates denoted gRNA\_Chr\_1\_Cand\_4, gRNA\_Chr\_2\_Cand\_2, gRNA\_Chr\_4\_Cand\_1, gRNA\_Chr\_4\_Cand\_3, gRNA\_Chr\_3\_Cand\_1 and gRNA\_Chr\_3\_Cand\_2 on the picture above. As Fig. 5 shows, all of our constructs resulted in more than 30 colonies on each plate. There was no colony on the negative control and more than 300 colonies on the positive control. Following this, colony PCR was conducted to check if the inserts contain the correct fragments.

### Colony PCR

Colony PCR was used as a screening method to find successfully assembled plasmids from the transformants prior to sequencing.

3 colonies from each plate were picked for colony PCR. However, since gRNA\_Chr\_1\_Cand\_1 had only one colony, it was subjected to colony PCR. Following this, the reaction mixtures were run on analytical gel (see Fig. 6 below). The expected band size of the correctly assembled plasmids is about 1300 bps.

![](https://static.igem.org/mediawiki/2021/0/09/T--DTU-Denmark--figure6_colPCR_1_5.png)

Regarding gRNA\_Chr\_1\_Cand\_1 and gRNA\_Chr\_2\_Cand\_1, out of the 4 tested transformant colonies, gel check of constructs indicated that gRNA\_Chr\_1\_Cand\_1 and gRNA\_Chr\_2\_Cand\_1.2 were the successfully assembled constructs.

![](https://static.igem.org/mediawiki/2021/7/78/T--DTU-Denmark--figure7_colPCR.png)

As it can be seen on Fig. 7 above, the following colonies also carried the correctly assembled plasmids: gRNA\_Chr\_2\_Cand\_2.2, gRNA\_Chr\_2\_Cand\_2.3, gRNA\_Chr\_4\_Cand\_3.1, gRNA\_Chr\_4\_Cand\_3.2, gRNA\_Chr\_4\_Cand\_3.3, gRNA\_Chr\_3\_Cand\_1.1, gRNA\_Chr\_3\_Cand\_1.3, gRNA\_Chr\_3\_Cand\_2.1, gRNA\_Chr\_3\_Cand\_2.2, gRNA\_Chr\_3\_Cand\_2.3.

![](https://static.igem.org/mediawiki/2021/9/90/T--DTU-Denmark--figure8_colPCR.png)

We decided to repeat colony PCR for gRNA candidates gRNA\_Chr\_1\_Cand\_2, gRNA\_Chr\_1\_Cand\_3, gRNA\_Chr\_1\_Cand\_4 and gRNA\_Chr\_4\_Cand\_1.
gRNA\_Chr\_1\_Cand\_3.2, gRNA\_Chr\_1\_Cand\_4.1, gRNA\_Chr\_1\_Cand\_4.2, gRNA\_Chr\_1\_Cand\_4.3, gRNA\_Chr\_4\_Cand\_1.1 turned out to be colonies harboring the desired constructs. (Fig. 8)

### Sequencing

*E. coli* transformants harbouring correctly constructed plasmids based on colony PCR results were subjected to Sanger Sequencing.
After successful sequencing results (see Fig. 9) cryostocks were made for all versions of the pDIV151 plasmids with gRNAs for further experimentation.

![](https://static.igem.org/mediawiki/2021/3/39/T--DTU-Denmark--figure9_grnaseq.png)

The template in the alignment above is the in silico plasmids containing the designed crRNA targeting the respective protospacer in the *K. phaffii* genome aligned with the sequencing result of the gRNA plasmid([visit our part collection](https://2021.igem.org/Team%3ADTU-Denmark/Part_Collection)).

In our attempt to design the toolbox for the methanotrophic *K. phaffii* cell factory, we were successful in identifying neutral integration sites and gRNAs which target the intergenic regions of the four chromosomes that can be utilized in the future when engineering *K. phaffii*.

## Genomic Integration

In this step, we aimed to assemble the different repair templates, to insert into the genome of *K. phaffii* GS115 Δku70 strain, using Golden Gate assembly. Before we went ahead with the assembly of inserts, we checked the gRNA efficiency without providing the repair templates to the *K. phaffii* GS115 Δku70 strain. Once this was validated, the subgroups tried to assemble the inserts.

### *K. phaffii* transformation without repair templates

*K. phaffii* transformation was an essential step for us to validate the efficiency of gRNAs since the design of the homology arms will be based on the transformation results. Since the Δku70 strain is non-homologous end-joining deficient, the transformation plates are suspected to result in none to few transformant colonies (significantly fewer colonies compared to the wild type). This was successfully validated in 6 designed gRNAs: gRNA\_Chr\_1\_Cand\_1, gRNA\_Chr\_2\_Cand\_1, gRNA\_Chr\_2\_Cand\_2, gRNA\_Chr\_4\_Cand\_3, gRNA\_Chr\_3\_Cand\_1, gRNA\_Chr\_3\_Cand\_2, thus enabling us to go ahead and design homology arms necessary to assemble the different inserts. As a positive control, wild type strain was subjected to transformation to observe the presence of colonies and the negative control was only water. (Fig. 1)

![](https://static.igem.org/mediawiki/2021/8/86/T--DTU-Denmark--fig1eff.png)

Once the gRNA efficiency was validated, the different inserts were PCR amplified before performing golden gate assembly (GGA). These included homology arms for the respective gRNAs, promoters, coding sequence and terminator.

**Click on the tabs pMMO and Hemoglobin below to get more information on these two specific!**

pMMO
Hemoglobin

# pMMO integration

For pMMO integration, homology arms, GAP1 promoter, TEF promoter, TEF terminator, PmoA, PmoB, PmoB fused to Venus and PmoC with both signal peptide and without signal peptide were amplified, as can be seen in Fig. 2,3,4,5 and 6. The PCR amplified products were purified and are ready for Golden Gate Assembly followed by *E.coli* transformation.

![](https://static.igem.org/mediawiki/2021/f/fb/T--DTU-Denmark--fig2HAnew.png)![](https://static.igem.org/mediawiki/2021/a/a5/T--DTU-Denmark--fig3gap1.png)![](https://static.igem.org/mediawiki/2021/0/04/T--DTU-Denmark--fig4pmobnew.png)![](https://static.igem.org/mediawiki/2021/9/96/T--DTU-Denmark--fig5pmos.png)![](https://static.igem.org/mediawiki/2021/3/39/T--DTU-Denmark--fig6pmos.png)

PCR amplification of homology arms for gRNA\_Chr\_2\_Cand\_1 and gRNA\_Chr\_2\_Cand\_2 was repeated to introduce BsmBI compatibility instead of BsaI, as an internal BsaI was overlooked in the ordered gBlock of all PmoB versions, which was not the same sequence as we worked with in Benchling.

### Golden Gate Assembly

The aim of our Golden Gate Assembly experiments was to create the repair templates used in co-transformation of *K. phaffii* GS115 Δku70 with our previously constructed pDIV151\_gRNA plasmids.

8 different repair templates were designed:

* Construct 1 containing PmoB\_Venus
* Construct 2 containing PmoB\_SP with Venus
* Construct 3 containing PmoA\_SP
* Construct 4 containing PmoA
* Construct 5 containing PmoC
* Construct 6 containing PmoC\_SP
* Construct 7 containing PmoB
* Construct 8 containing PmoB\_SP

We attempted to integrate all the constructs with the repair templates. However, the constructs with PmoB versions failed due to unexpected BsaBI recognition site within the ordered gBlock. Results from other constructs are presented here.

### *E. coli* transformation

DH5α competent *E. coli* cells were transformed and plated on YPD-NTC plates and incubated at 37°C overnight.

![](https://static.igem.org/mediawiki/2021/3/39/T--DTU-Denmark--fig7GGA236.png)

Looking at the construct plates (Fig. 7), we observed 7 small colonies on construct 2, 2 big colonies on construct 3 and 1 big colony on construct 6 plates, while negative control plates had no colonies and the positive control plates had many small colonies.

Based on these observations, we decided to pick the following colonies for colony PCR: 2 , 3.1, 3.2 and 6.

### *E. coli* colony PCR

To screen for correctly assembled pGGAselect\_construct 2/3/6 plasmids prior to plasmid extraction from main cultures, the following colonies were picked for colony PCR:

* pGGAselect\_Construct 2
* pGGAselect\_Construct 3.1
* pGGAselect\_Construct 3.3
* pGGAselect\_Construct 6

The expected band sizes of the constructs is the following:

| Construct | Expected band size |
| --- | --- |
| pGGAselect\_Construct 2 | 3014 bp |
| pGGAselect\_Construct 3 | 1793 bp |
| pGGAselect\_Construct 6 | 1832 bp |

Fig. 8 shows the results of the colony PCR. When it comes to construct 3.2 and 6 the brightest bands can be observed around 1800 bps, which is promising, therefore we decided to send these for sequencing in the next step.

![](https://static.igem.org/mediawiki/2021/9/9d/T--DTU-Denmark--fig8colpcr236.png)

### *E. coli* sequencing

![](https://static.igem.org/mediawiki/2021/5/52/T--DTU-Denmark--fig9seqa.png)![](https://static.igem.org/mediawiki/2021/c/c5/T--DTU-Denmark--fig10seqc.png)

The red lines in the sequencing results represent a couple of mismatches. This could have been due to the PCR amplification carried out prior to sequencing. It is also to be noted that, these identified mismatches have not been documented to have any effect on the expression of the gene of interest encoded to the repair template. Hence based on the sequencing results (Fig. 9 and 10) we further proceeded with amplification of repair templates before starting *K. phaffii*  transformation.

### *K. phaffii* transformation

Once the repair templates had been assembled in pGGAselect and success confirmed by sequencing, the repair templates were PCR amplified using primers that bind to HAU (FW) and HAD (RV)(Fig. 11), then column purified to be ready for co-transformation with a constructed site-specific Cas9-gRNA plasmid.

![](https://static.igem.org/mediawiki/2021/7/71/T--DTU-Denmark--fig11RT236.png)

Previously prepared electrocompetent *K. phaffii* GS115 Δku70 1M sorbitol preserves with a determined competence of 6.5x106 CFU/mL after electroporation were used for transformation
[(yeast competent cells protocol).](https://static.igem.org/mediawiki/2021/e/e1/T--DTU-Denmark--Y_competent.pdf)
After 3 days of incubation at 30°C colonies were obtained on plates for both constructs 3.2 and 6, respectively (Fig. 12). This result is important since there has not been a known documentation to the best of our knowledge for yeast electrocompetent cells transformation for *K. phaffii* making it novel. We were inspired by the *S. cerevisiae* electrocompetent cell transformation and successfully obtained visible results showing [yeast competent cells protocol](https://static.igem.org/mediawiki/2021/e/e1/T--DTU-Denmark--Y_competent.pdf) works effciently.

![](https://static.igem.org/mediawiki/2021/9/9c/T--DTU-Denmark--fig12kpt36.png)

### *K. phaffii* colony PCR

Following the transformation of *K. phaffii*, yeast colony PCR was carried out to confirm if PmoA\_SP and PmoC\_SP had been successfully integrated in the genome.

![](https://static.igem.org/mediawiki/2021/c/c7/T--DTU-Denmark--fig13kpt36.png)

Based on Fig. 13, we sent PCR amplified DNA fragments from the respective integration sites in transformant colonies for sequencing, however they all turned out to be false positives.

Although we were not successful in integrating the repair templates in the K. phaffii genome, the fact that we obtained some colonies, when providing a repair template, indicates that integration is possible. Through multiple iterations it is certainly possible to obtain such an engineered strain and this will provide new opportunities in synthetic biology using K. phaffii to produce different products of interest.

# Hemoglobin

For Leghemoglobin integration, homology arms for IS1C1, AOX1 promoter, AOX1 promoter 99% similarity, AOX1 promoter 89% similarity, FLD1 promoter, Leghemoglobin, Venus and TEF terminator were amplified, as seen on Fig. 14. The PCR amplified products were purified and are ready for GGA followed by *E.coli* transformation.

![](https://static.igem.org/mediawiki/2021/1/12/T--DTU-Denmark--fig14hemamp.png)

### Golden Gate Assembly

The aim of our Golden Gate Assembly experiments was to create the repair templates used in co-transformation of *K. phaffii* GS115 Δku70 with our previously constructed pDIV151\_gRNA plasmids.

4 different constructs with repair templates for Leghemoglobin were designed:

* Construct 1 containing Leghemoglobin with AOX1
* Construct 2 containing Leghemoglobin with AOX1\_99
* Construct 3 containing Leghemoglobin with AOX1\_89
* Construct 4 containing Leghemoglobin with FLD1

Furthermore, a fluorescent protein collection was also designed:

* Construct 5 containing codon optimized Venus with AOX1
* Construct 6 containing codon optimized Venus with AOX\_99
* Construct 7 containing codon optimized Venus with AOX\_89
* Construct 8 containing codon optimized Venus with FLD1
* Construct 9 containing GFP with AOX1
* Construct 10 containing mOrange with AOX1
* Construct 11 containing VENUS from kit with AOX1

### *E. coli* transformation

DH5α competent *E. coli* cells were transformed and plated on LB-CAM plates and incubated at 37°C overnight.

![](https://static.igem.org/mediawiki/2021/e/e4/T--DTU-Denmark--fig15hemgga.png)![](https://static.igem.org/mediawiki/2021/e/e0/T--DTU-Denmark--fig16hemgga.png)

As it can be seen on Fig. 15 and 16, we managed to obtain colonies on the following construct plates: 1,2,3,6,7,8. Therefore we proceeded to further investigate these colonies with colony PCR.

### *E. coli* colony PCR

Colony PCR has been carried out using Q5 high-fidelity DNA polymerase. Afterwards, a gel check was performed 100V, 20 minutes on 1% agarose.

![](https://static.igem.org/mediawiki/2021/f/f4/T--DTU-Denmark--fig17cpcrgga.png)![](https://static.igem.org/mediawiki/2021/f/f8/T--DTU-Denmark--fig18cpcrgga.png)

As Fig. 17 shows, we had 3 visible bands, which corresponds to construct 3, 7 and 8. The protocol was run on purified plasmids with construct 6 from 3 different colonies. From the above analysis it is clear that all three colonies possessed the desired plasmids with the inserts (Fig. 18), therefore we sent these for sequencing.

### Sequencing

Sequencing results of construct 3,7 and 8 were false positives and show that the sequenced plasmids did not contain the GGA constructs.
However, sequencing results from construct 6 was correctly assembled as seen in Fig. 19 and we used only this one construct for electroporation of *K. phaffii*.

![](https://static.igem.org/mediawiki/2021/5/5a/T--DTU-Denmark--fig19hemseq.png)

### *K. phaffii* transformation

With the confirmed sequencing result from construct 6, *K. phaffii* transformation was attempted.

![](https://static.igem.org/mediawiki/2021/3/37/T--DTU-Denmark--fig20kftnew.png)

The outcome of the controls were unexpected, as the positive control was not supposed to have any colonies, we suspect that we exchanged these by accident.
After 3 days of incubation, we had 3 colonies on our construct 6 plates (Fig. 20), these were named 6A, 6B and 6C and were used in the next step: yeast colony PCR.

### *K. phaffii* colony PCR

The primers used for the colony PCR target chromosome 1 and produce PCR products with the length of 719 bps, which corresponds to the band lengths for all 3 colonies, seen on the gel picture below. This means that the insertion was not successful, as that would have resulted in a band of ~3000 bps. (Fig. 21)

![](https://static.igem.org/mediawiki/2021/0/04/T--DTU-Denmark--fig21venus.png)

We aimed to integrate leghemoglobin as a proof of concept to show that the engineered *K. phaffii* can produce leghemoglobin which is the main flavoring and colouring ingredient in meat industry. A successful integration would be able to change the dynamics of meat industry and move the society towards circular economy. However, our attempt to do this was not fully accomplished due to the time constraints and road bumps associated with the Golden Gate Assembly of the repair template. We do believe that this approach of ours was critically evaluated at every point and with time, it is indeed possible to implement this system.

Throughout these experiments, the results we obtained have further strengthened the main goals of our project PHEAST. The design and strategy which we have currently developed can be used to dwell deeper into obtaining the methane consuming engineered *K. phaffii* cell-factory and bring our idea into reality.

## Genomic Deletion

In this step, we aimed to understand the effect on *K. phaffii* upon deletion of non vital genes involved in the metabolism of methanol to formaldehyde. This experiment was designed as a proof to understand the genes which were deleted does not completely seize the growth of the cells but reduces the growth rate.

2 gRNAs each targeting the *aox1*, *aox2*, and *adh2* genes were amplified and purified. (Fig. 1) Repair templates were obtained by annealing the single stranded in silico designed homology arms that target the 45 base pairs upstream and downstream of the targeted genes. Further, *K. phaffii* transformation was carried out and the knockouts were confirmed by colony PCR and sequencing.

### PCR amplification of gRNAs for *aox1*, *aox2*, and *adh2*

![](https://static.igem.org/mediawiki/2021/3/3f/T--DTU-Denmark--fig1frag2.png)

### *E. coli* transformation

The gRNAs were transformed into DH5α *E. coli* along with pDIV151 backbone using USER cloning. Colony PCR and sequencing results showed successful integration of gRNAs in the pDIV151 backbone. (Fig. 2 and 3)

![](https://static.igem.org/mediawiki/2021/5/52/T--DTU-Denmark--fig2cpcr.png)![](https://static.igem.org/mediawiki/2021/f/fb/T--DTU-Denmark--fig3seqgrna.png)

Repair templates were annealed and the efficiency of gRNAs were tested. As expected, the strain did not survive without a repair template. Since repair templates were provided to the *k. phaffii* strain, colonies were observed on all plates. (Fig. 4)

![](https://static.igem.org/mediawiki/2021/d/df/T--DTU-Denmark--fig4grnaeff.png)

### *K. phaffii* transformation

*K. phaffii* transformation was carried out to check for knockouts of *aox1*, *aox2*, and *adh2* simultaneously. Colony PCR indicated that we were successful in carrying out a single knockout in each of the genes and a double knockout of *aox1* and *adh2*. (Fig. 5)

![](https://static.igem.org/mediawiki/2021/4/42/T--DTU-Denmark--fig5ko.png)

Although promising results from colony PCR were obtained in the form of bands expected for *aox1*, *aox2*, and *adh2*, sequencing results were inconclusive for *aox1* and *aox2*. However, *adh2* gene deletion was confirmed via sequencing as seen below. (Fig. 6)

![](https://static.igem.org/mediawiki/2021/e/e0/T--DTU-Denmark--fig6adh2.png)

Due to time constraints we did not conduct any protein assays to investigate if the *K. phaffii* *adh2* mutant strain is incapable of producing the ADH2 protein, as expected.

Next step was to carry out the double knockout from the *adh2* mutant strain and we were successful in this since *aox1* gRNA candidates 1 and 2 showed successful colony PCR results. We expected to see a band at 500bps as seen in Fig. 7.

![](https://static.igem.org/mediawiki/2021/f/f7/T--DTU-Denmark--fig7aoxadh.png)

To validate the scar less deletion of *aox1* the colony PCR samples were sent for Sanger Sequencing. The results we received were inconclusive, and we did not have a chance to repeat the sequencing.
However, we wanted to test if the knockouts did indeed impair the growth of the *K. phaffii*  GS115 Δku70+Δ*adh2*+*aox1* mutant strain when grown on methanol as sole carbon source.

A final Biolector experiment elucidated that the confirmed knockout of *adh2* and the putative knockout of *aox1* does indeed impair the growth of the *K. phaffii* mutant in presence of increasing levels of methanol compared to the *K. phaffii*  GS115 Δku70 strain.

This verifies that our CRISPR-based toolbox for genomic integration/deletion performs well. Interestingly, as we can see in Fig. 8, the mutant strain shows slightly lower growth efficiency even without addition of methanol to the YP medium (look at the “MeOH: 0” graph), meaning that lack of ADH2 and possibly AOX1 expression somewhat impairs the overall performance of the strain. We came to this conclusion on the basis of the graphs which show that the growth rate for both strains strats to decline clearly at about 8% but it is very clear that the growth rate is much lower compared to the Δku70 strain.

![](https://static.igem.org/mediawiki/2021/b/b4/T--DTU-Denmark--fig8biolector.png)

Although we did not get to the point of understanding the difference in growth rate with 3 knockouts, we were successful in extracting and analyzing the growth rate patterns and the suspected difference as expected. This thus served as our first proof of concept that although the non vital genes were deleted, the growth rate is decline and not fully hampered and that the strain is functional.

---

## References

##### [1]De Schutter, K., Lin, Y. C., Tiels, P., Van Hecke, A., Glinka, S., Weber-Lehmann, J., ... & Callewaert, N. (2009). Genome sequence of the recombinant protein production host Pichia pastoris. Nature biotechnology, 27(6), 561-566.

![](https://static.igem.org/mediawiki/2021/0/05/T--DTU-Denmark--presult.png)