iGEM 2021 — Team RUBochum

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# Team:RUBochum/Notebook

* ![](https://static.igem.org/mediawiki/2021/b/b4/T--RUBochum--LogoEndversion.jpg)
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* Project

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  + [Results](https://2021.igem.org/Team%3ARUBochum/Results)
  + [Implementation](https://2021.igem.org/Team%3ARUBochum/Implementation)
  + [Contribution](https://2021.igem.org/Team%3ARUBochum/Contribution)
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* Laboratory

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* Human Practices

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* Team

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  + [Sponsors](https://2021.igem.org/Team%3ARUBochum/Sponsors)
* Excellence

  + [Featured](https://2021.igem.org/Team%3ARUBochum/Featured)
  + [Entrepreneurship](https://2021.igem.org/Team%3ARUBochum/Entrepreneurship)
* [Judging Form](https://igem.org/2021_Judging_Form?id=3860)

![RUBochumCoverPictureNotebook](https://static.igem.org/mediawiki/2021/2/22/T--RUBochum--Headpicture18.jpeg)

# Notebook

* 24.02.2021: First team meeting: Organisation and brainstorming for project ideas

* 14.03.2021: 2nd meeting: Exchange about project and researching ideas. Topics: Different synthetic foods (milk and egg production) or fungus sensors

* 19.03.2021: 3rd meeting: Agreement on a project - production of platypus yogurt, creation of subteams

* 23.03.2021: 4th meeting: Researching on milk ingredients and their production, finding a PI and financial resources (sponsors)

* 02.04.2021: 5th meeting: Further planning. Interim status exchange in all subgroups

* 11.04.2021: 6th meeting: Information exchange

* 18.04.2021: 7th meeting: Information exchange

* 25.04.2021: 8th meeting: Information exchange and writing of articles, creating a  [web page](https://www.mbt.ruhr-uni-bochum.de/mbt/lehre/igem.html.en)

* 30.04.2021: 9th meeting: Information exchange

* 05.05.2021: First mentor meeting

* 07.05.2021: 10th meeting: Information exchange. From now on 3 regular meetings per week.

* 14.05.2021: German meet-up, 11th meeting

* 17.05.2021: First meeting with our PI, Prof. Dr. Dirk Tischler. Laboratory work is guaranteed, starting mid July

* 19.05.2021: 2nd mentor meeting

* 21.05.2021: 12th meeting: Information exchange

* 25.05.2021: First meeting with team Darmstadt: Information exchange and help

* 28.05.2021: 13th meeting: Information exchange

* 29.05.2021: RUBochum Game night

* 02.06.2021: 3rd mentor meeting

* 04.06.2021: 14th meeting: Information exchange and introduction into methods of bioinformatics

* 05.07.2021: Start of laboratory work; continues every day until 07.10.2021

* 07.06.2021: Development meeting for primers and bioinformatic analysis

* 11.06.2021: 15th meeting: Information exchange

* 18.06.2021: team Darmstadt Pubquiz, 16th meeting: Information exchange, 4th mentor meeting

* 19.06.2021: team Bielefeld HTML meeting

* 25.06.2021: 3rd Meeting with team Darmstadt: Information exchange and help, 17th meeting: Information exchange

* 28.06.2021: Safety instruction for the laboratory and meeting with Dr. Julia Götsch from Promega

* 02.07.2021: 18th meeting: Information exchange

* 03.07.2021: Russian meet-up

* 07.07.2021: 5th mentor meeting

* 09.07.2021: 19th meeting: Information exchange

* 16.07.2021: 20th meeting: Information exchange

* 22.07.2021: Meeting with Dr. Thomas Gassler to get more insight into Pichia pastoris expression systems

* 23.07.2021: 21st meeting: Information exchange

* 28.07.2021: 6th mentor meeting

* 06.08.2021: 22nd meeting: Information exchange

* 09.08.2021: Meeting with Geoff Williams for the Australian Platypus Conservancy (APC) talking about dairy industry and its impact on platypuses

* 11.08.2021: 7th mentor meeting

* 13.08.2021: 4th meeting with team Darmstadt: Information exchange and help, 23rd meeting: Information exchange

* 18.08.2021: Meeting with Dr. Knut Madden from Biogrammatics talking about food industry and the optimization of Pichia protocols
  Meeting with Prof. Dr. Christopher Grefen about education and genetic engineering

* 20.08.2021: 24th meeting: Information exchange

* 25.08.2021: Meeting with Prof. Dr. Markus Piotrowski about education and genetic engineering in plants, 8th mentor meeting

* 26.08.2021: Meeting with Dr. Michael Krohn from the BrainAG to talk about Brazzein, the flavour and synthetic food industry

* 27.08.2021: 25th Meeting: Information exchange

* 31.08.2021: Meeting with Prof. Dr. Julia Bandow to talk about MLP and different screening methods of antimicrobial molecules

* 01-03.09.2021: Participation at the German Conference for Synthetic Biology

* 03.09.2021: 26th Meeting: Information exchange

* 07.09.2021: Meeting with Dr. Breanna Duffy from New Harvest about cellular agriculture, synthetic foods and entrepreneurship.

* 08.09.2021: 9th mentor meeting

* 10.09.2021: 5th meeting with team Darmstadt: Information exchange and help

* 12.09.2021: Travelling to the Lippe State Museum Detmold to inspect stuffed platypus

* 14.09.2021: Interview for the regional newspaper WAZ

* 17.09.2021: 27th meeting: Information exchange

* 22.09.2021: Teaching genetic engineering at school
  The WDR (West German broadcasting) is filming inside our laboratory
  * 10th mentor meeting

  * 24.09.2021: 28th meeting: Information exchange

  * 25.09.2021: Meeting iGEM Teams in Paris - iGEM Sorbonne and GO Paris-Saclay

  * 29.09.2021: Taking part in the German Association for Synthetic Biology's "Biohackers Watch Party" and discussion about entrepreneurship with Dr. Britta Winterberg from Formo, Dr. Max Mundt Startup Origin.bio and Dr. Reyk Horland, Co-founder of the TissUse GmbH

  * 30.09.2021: Live interview and TV feature at the WDR Lokalzeit Ruhr

  * 03.10.2021: 29th meeting: Information exchange

  * 06.10.2021: 11th mentor meeting

  * 08.10.2021: 30th meeting: Information exchange

  * 13.10.2021: 6th meeting with team Darmstadt: Information exchange and help
    Taking part in the German Association for Synthetic Biology's "Biohackers Watch Party" and discussion with Dr. Dorothea Hämmerer, the journalist Janina Otto and science YouTuber Doktor Whatson

  * 15.10.2021: 31st Meeting: Information exchange

  * 18.10.2021: Meeting with Dr. Britta Winterberg about Formo, synthetic milk and entrepreneurship
    12th mentor meeting

  * 22.10.2021: 32nd meeting: Information exchange

  * 27.10.2021: Meeting with Zoltan Toth-Czifra, founder of RealDealMilk

# Laboratory

* July to August:

+ Getting the lab space organised
+ Introduction to laboratory methods
+ Preparation of buffers and media
+ Preparation of competent cells E. coli DH5α
+ Preparation of genes and primers

* August to September:

+ PCR and cloning of all constructs
+ Transformations
+ Check-PCRs and sequencing
+ Preparation of P. pastoris competent cells X33

* September to October

+ Preparation of P. pastoris competent cells X33 repetition
+ Multiple transformations with the final construct into P. pastoris
+ Heterologous overexpression

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# Team:RUBochum/Engineering

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  + [Parts](https://2021.igem.org/Team%3ARUBochum/Parts)
  + [Proof of Concept](https://2021.igem.org/Team%3ARUBochum/Proof_Of_Concept)
  + [Results](https://2021.igem.org/Team%3ARUBochum/Results)
  + [Implementation](https://2021.igem.org/Team%3ARUBochum/Implementation)
  + [Contribution](https://2021.igem.org/Team%3ARUBochum/Contribution)
  + [Platypusfacts](https://2021.igem.org/Team%3ARUBochum/Platypusfacts)
* Laboratory

  + [Notebook](https://2021.igem.org/Team%3ARUBochum/Notebook)
  + [Safety](https://2021.igem.org/Team%3ARUBochum/Safety)
* Human Practices

  + [(Integrated) Human Practices](https://2021.igem.org/Team%3ARUBochum/Human_Practices)
  + [Public Engagement and Education Outreach](https://2021.igem.org/Team%3ARUBochum/Communication)
* Team

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  + [Attributions](https://2021.igem.org/Team%3ARUBochum/Attributions)
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  + [Sponsors](https://2021.igem.org/Team%3ARUBochum/Sponsors)
* Excellence

  + [Featured](https://2021.igem.org/Team%3ARUBochum/Featured)
  + [Entrepreneurship](https://2021.igem.org/Team%3ARUBochum/Entrepreneurship)
* [Judging Form](https://igem.org/2021_Judging_Form?id=3860)

![RUBochumCoverPictureEngineering](https://static.igem.org/mediawiki/2021/2/2e/T--RUBochum--Headpicture6.jpeg)

**Brazzein**
Brazzein is a protein which originates from the fruit of the climbing plant *pentadiplandra brazzeana*.
It’s 500 to 2000 times sweeter than a common sugar molecule.
As an ingredient of our yogurt it could be a tasty and healthy alternative to other sugar substitutes due to its different metabolic pathway and the high demand for amino acids in the human body.

**"Fact: Brazzein can be up to thousand times sweeter, is extremely stable, is suitable as a balanced food."**
Dr. Michael Krohn, executive Vice President as well as head of Research & Development at BrainAG - translated from German

**Caseine**
Milk contains different proteins like caseins and whey proteins.
We are producing the caseins of the milk of the platypus and bovine.
Besides having the ability to carry calcium and phosphate, they are crucial for the production of cheese and yoghurt.
The casein proteins can be divided into four different subgroups (α-s1-, α-s2-, β and κ-casein) and occur in all mammals.

|  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Proteins || [*αs1 casein Bos taurus*](http://parts.igem.org/Part%3ABBa_K3860000) || [αs2 casein *Bos taurus*](http://parts.igem.org/Part%3ABBa_K3860001) || [β casein *Bos taurus*](http://parts.igem.org/Part%3ABBa_K3860002) || [κ casein *Bos taurus*](http://parts.igem.org/Part%3ABBa_K3860003) || [αcasein *Ornithorhynchus anatinus*](http://parts.igem.org/Part%3ABBa_K3860004) || [β casein *Ornithorhynchus anatinus*](http://parts.igem.org/Part%3ABBa_K3860005) || [κ casein Ornithorhynchus anatinus](http://parts.igem.org/Part%3ABBa_K3860006) |

**Design**

All proteins were cloned with the alpha secretion signal behind the promoter region. The secretion should allow an easy purification.
Due to reports secretion should be a problem with caseins. An alternative approach would be the coupling of the different caseins to hinder the folding of disturbing structures.

* **References**
  [**Overview**](https://www.sciencedirect.com/book/9781845697587/handbook-of-food-proteins)
  [**Elucidation**](https://pubmed.ncbi.nlm.nih.gov/1688683/)

**Development of an optimized industrial process**
**Backbone**
The variety of proteins were produced in the pPICZαA vector which has the AOX1 promotor and is thus methanol dependent.
Three different types of primers were created to get multiple combinations of integrations into the multiple cloning site. Genes can be inserted with the alpha secretion signal or a polyhistidine-tag through a gibson assembly.
Following to the creation of the plasmids additionally chromosomal integrations were planned.
Therefore, GoldenPiCS system was used with the four integration loci RGI2, ENO1, NTS and AOX1tt.

![Fermentation strategy](https://static.igem.org/mediawiki/2021/0/0f/T--RUBochum--Strategy.png)
Figure 1: Principle of the heterologous fermentation process. Starting from multiple genotypes producing the whole product, the amount of genotypes is reduced inside the fermentation due to genetic optimization.

The goal was to create a homolog fermentation process with multiple genotypes.
In further work the amount of genotypes should be reduced to one. Plasmids and integrated genes get combined during this process.

**Purification of food proteins**
Cells were centrifuged and the supernatant was filtered size dependent. The copper chelating Phleomycin D1 can be denatured due to pH lowering during yoghurt production.
Due to chromosomal integration a selection marker and thus antibiotic purification will be redundant.

**Fatty acid syntheses**
**Fatty acids define the taste and consistency of milk**.
The lengths of the triacylglycerols vary between 4 to 24 C-atoms.
For the overproduction of fatty acids, the enzyme of the rate-determining step needs to be overproduced. In our case, we used the Acetyl-carboxylases complex from *E. coli* K12 which consists of 4 subunits.
Thioesterases terminate the elongation of fatty acids and thus determine their length.
To get the broad range of fatty acids, the Thioesterase II of *E. coli* was cloned into our back bone.
Due to the broad range of action of Thioesterase II, it’s also possible to get mostly fatty acids with a length of 6 to 18 C-atoms.

**Design**

The four subunits were connected to each other and fused with a glycine-rich linker.

Therefore the Gibson assembly was used.
A strain including the gene for the thioesterases was created.

* **References**
  [**Milk fatty acids**](https://lipidworld.biomedcentral.com/articles/10.1186/1476-511X-6-25#Sec3)
  [**Fatty acid syntheses**](https://www.jbc.org/article/S0021-9258%2820%2943396-4/fulltext)
  [**TesB**](https://www.ncbi.nlm.nih.gov/gene/945074)

**Monotreme Lactation Protein**
The Monotreme Lactation Protein (MLP) is a protein that is only found in the milk of platypus and other monotremes. MLP is highly expressed in platypus milk with antimicrobial properties.
The function of MLP is hypothesized to be the protection of the young from pathogenic infections, because monotremes don't possess teats.
 After hatching from their eggs, the young drink the milk directly from a mammary pad on the abdomen of the female platypus.
 So there is more chance of contamination with pathogens compared to other nursing strategies e.g. drinking the milk directly from teats.
 Studies showed that MLP demonstrated antimicrobial activities against *Staphylococcus aureus* and *Enterococcus faecalis*, which are classified as pathogens.
It didn't demonstrate any effect on *Escherichia coli*, *Salmonella enterica*, *Staphylococcus epidermidis*, and *Pseudomonas aeruginosa*.
MLP is also studied as a new candidate for antibiotics, because of this antimicrobial properties.

![MLP molecule](https://static.igem.org/mediawiki/2021/7/73/T--RUBochum--MLP1.png)
Figure 2: Molecular structure of MLP

We hypothesized that MLP is an alternative for milk pasteurization. Different tests, with more bacteria types and the most common from human gut flora, should give a clearer picture if MLP could be safe for human consumption.
If so, we could use MLP to skip the milk pasteurization step, or add it to common dairy products to expand their shelf life.
We also wanted to raise awareness for platypus and monotreme animals in general, because these ancient species are threatened by human pollution and climate change. Especially the platypus population in Tasmania is under pressure by the dairy industry.

**Design**
The sequence of the MLP was applied into the MCS with the no secretion signal and Hexahistidine-Tag for easy purification.

* **References**
  <https://academic.oup.com/gbe/article/6/10/2754/610436>
  [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4224336/](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4224336/%20)
  [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5947691/](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5947691/%20)
  [https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0053686](https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0053686 )

**Vanillin**
For the production of vanilla yoghurt, the vanillin synthesis needed to be integrated into the fungus.
In our case we started with the last step of the vanilly synthesis: the conversion of vanillyl alcohol to vanillin via the aryl-alcohol oxidase.
We chose the enzyme from Penicillium simplicissimum. The enzyme was optimized for P. pastoris and coupled to a 6X His-tag.
![Vanillin molecule](https://static.igem.org/mediawiki/2021/4/49/T--RUBochum--Vanilllin1.png)
Figure 3: Drawing of a vanillin molecule

* **References**
  [Aryl-alcohol oxidase](https://www.ncbi.nlm.nih.gov/protein/CAA75722.1/)

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# Team:RUBochum/Experiments

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:RUBochum/Results

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* [Home](https://2021.igem.org/Team%3ARUBochum)
* Project

  + [Description](https://2021.igem.org/Team%3ARUBochum/Description)
  + [Design/Engineering](https://2021.igem.org/Team%3ARUBochum/Engineering)
  + [Parts](https://2021.igem.org/Team%3ARUBochum/Parts)
  + [Proof of Concept](https://2021.igem.org/Team%3ARUBochum/Proof_Of_Concept)
  + [Results](https://2021.igem.org/Team%3ARUBochum/Results)
  + [Implementation](https://2021.igem.org/Team%3ARUBochum/Implementation)
  + [Contribution](https://2021.igem.org/Team%3ARUBochum/Contribution)
  + [Platypusfacts](https://2021.igem.org/Team%3ARUBochum/Platypusfacts)
* Laboratory

  + [Notebook](https://2021.igem.org/Team%3ARUBochum/Notebook)
  + [Safety](https://2021.igem.org/Team%3ARUBochum/Safety)
* Human Practices

  + [(Integrated) Human Practices](https://2021.igem.org/Team%3ARUBochum/Human_Practices)
  + [Public Engagement and Education Outreach](https://2021.igem.org/Team%3ARUBochum/Communication)
* Team

  + [Team](https://2021.igem.org/Team%3ARUBochum/Team)
  + [Attributions](https://2021.igem.org/Team%3ARUBochum/Attributions)
  + [Collaborations and Meetings](https://2021.igem.org/Team%3ARUBochum/Collaborations)
  + [Sponsors](https://2021.igem.org/Team%3ARUBochum/Sponsors)
* Excellence

  + [Featured](https://2021.igem.org/Team%3ARUBochum/Featured)
  + [Entrepreneurship](https://2021.igem.org/Team%3ARUBochum/Entrepreneurship)
* [Judging Form](https://igem.org/2021_Judging_Form?id=3860)

![RUBochumCoverPictureResults](https://static.igem.org/mediawiki/2021/1/17/T--RUBochum--Headpicture3.jpeg)

---

# Developing a cloning strategy for an industrial optimized process.Bioinformatic planning Protein Screening For the first production of the variety of proteins in *P. pastoris*, we decided to use plasmids as extrachromosomal bodies. Our cloning strategy was based on the pPICZα A, which has the α secretion signal in front of the MCS and the BleoR gene which encodes for phleomycin D1. The methanol-induced AOX1 promoter was used for the production of our proteins. By creating two different pairs of primers for the Gibson assembly, we performed diverse insertions into the MCS. ![Cloning Strategy](https://static.igem.org/mediawiki/2021/2/26/T--RUBochum--CloningStrategy.png)