iGEM 2022 — Team boku-vienna

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{% block title %}Notebook{% endblock %}
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## Notebook

You can find a short overview over our activities over this iGEM Season

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### March-May

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Brainstorming and Literature Research ( **[Everyone](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): The entire team took the time from the first meeting to the beginning of lab work to define our project, do literature research, discuss ideas and solidify roles and responsibilities. We consulted our advisors and PIs every step of the way.

Fundraising and Contacting of sponsors ( **[Sandra, Tatyana, Zahra, Jakob](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): As we settled on project idea, we immediately brainstormed possible sponsors and interested parties. Under the leadership of Sandra, we began contacting sponsors, went to project pitches, collected feedback, and integrated it into our project design.

Communication and Collaborations ( **[Sandra, Magdalena, Neha, Paula, Tatyana](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Parallel to all other activities, our team has began brainstorming a social media strategy, possible collaborations, and looked for iGEM teams to collaborate with on the ideas they propose.

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### June

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Lab Safety Instructions ( **[Sandra, Magdalena, Julia, Tatyana, Anna, Irfan, Paula](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): The team member that were set to work in the wet lab recieves their safety instructions so they could work at the lab responsibly.

Fundraising and Contacting of sponsors ( **[Sandra, Tatyana, Zahra, Jakob](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Our work on securing sponsorships continues.

Communication and Collaborations ( **[Sandra, Magdalena, Neha, Paula, Tatyana](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Our first posts on social media are being developed. Template for the newsletter is made. Team logo and project logo is designed. First collaboration meetings commense.

Plasmid Design (**[Julia, Tatyana](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): After having settled on potetnial coding sequences, we began the work of codon-optimization, ordering parts and conceptualizing protocols for our lab work in July.

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### July

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##### Lab Work Timeline

#### 04.07.-10.07

Media Preparation and Pouring of Plates (**[Paula, Magdalena & Irfan](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): In the beginning, it was important to stock up on all the important things like media and agar that we could immediately use once we had our necessary strains.

Inventory (**[Paula, Julia & Anna](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): We had to take inventory of everything we had available before beginning work

#### 11.07.-17.07.

Media Preparation and Pouring of Plates (**[Paula, Magdalena & Irfan](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): In the beginning, it was important to stock up on all the important things like media and agar that we could immediately use once we had our necessary strains.

Gel Pouring  (**[Paula, Julia, Magdalena &Irfan](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Every week, the wetlab team stocked up on clean-up and analytical gels.

Spider Silk: PCR Amplification (**[Paula, Julia](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Part T, S#1, S#2 & S#3

Gel Electrophoresis, Sequencing (**[Paula, Julia & Magdalena](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Analysis of results of PCRs

#### 18.07-24.07.

Spider Silk: PCR Amplification (**[Anna, Julia & Magdalena](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): MaSp\_A, MaSp\_B, MaSp\_C.

Media Preparation and Pouring of Plates (**[Anna, Magdalena & Irfan](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Throughout the project, it was important to keep up continuous inventory.

Gel Pouring  (**[Julia, Magdalena &Irfan](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Every week, the wetlab team stocked up on clean-up and analytical gels.

Gel Electrophoresis, Sequencing (**[Anna, Julia & Magdalena](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Analysis of results of PCRs

#### 25.07.-31.07

Spider Silk: PCR Amplification (**[Julia](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): MaSp\_A, MaSp\_B, MaSp\_C.

Media Preparation and Pouring of Plates (**[Anna, Magdalena & Irfan](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Throughout the project, it was important to keep up continuous inventory.

Gel Pouring  (**[Anna, Magdalena &Irfan](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Every week, the wetlab team stocked up on clean-up and analytical gels.

Gel Electrophoresis, Sequencing (**[Julia & Anna](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Analysis of results of PCRs

##### Other Activities in July

Collaborations: In Person Meet-up (**[Magdalena & Zahra](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): European iGEM meetup in Hamburg.

Partnerships (**[Tatyana, Magdalena and Sandra](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): First meetings of our various fruitful partnerships.

Communication (**[Neha & Magdalena](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Continuous work on our Social Media.

### August

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#### 01.08.-07.08

Amplifying Gelatin parts via PCR (**[Magdalena](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**).

Golden Gate Cloning Backbone 1 (**[Julia](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): First Trials; MaSp insert + secretion factor

Transformation into *E.coli* (**[Julia](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): BB1 with MaSp insert

Media Preparation and Pouring of Plates (**[Tatyana, Magdalena & Irfan](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Throughout the project, it was important to keep up continuous inventory.

Gel Pouring  (**[Tatyana, Julia, Magdalena &Irfan](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Every week, the wetlab team stocked up on clean-up and analytical gels.

Mini-Prep, Restriction Digest & Sequencing (**[Julia](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): MaSp insert backbone 1

#### 08.08.-14.08

Amplifying Gelatin parts via PCR (**[Magdalena](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**).

Golden Gate Cloning Backbone 1 (**[Julia](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): MaSp insert + secretion factor

Transformation into *E.coli* (**[Julia](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): BB1 MaSp insert

Mini-Prep, Restriction Digest & Sequencing (**[Julia](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): MaSp insert backbone 1

**09.08:** Brick Experiments (**[Sandra](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): 31 bricks created according to  **[protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%7D%7D)**; size 1x1x1

#### 15.08.-21.08

Amplifying Gelatin parts via PCR (**[Magdalena](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**)

Golden Gate Cloning Backbone 1 (**[Julia](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): MaSp insert + secretion factor

Transformation into *E.coli* (**[Julia](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): BB1 MaSp insert

Mini-Prep, Restriction Digest & Sequencing (**[Julia](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): MaSp insert backbone 1

Media Preparation and Pouring of Plates (**[Magdalena & Irfan](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Throughout the project, it was important to keep up continuous inventory.

Gel Pouring  (**[Tatyana & Irfan](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Every week, the wetlab team stocked up on clean-up and analytical gels.

**17.08.:** Material Testing (**[Paula](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): According to**protocol**. Testing of bricks from 9.8. Results described **[here](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%7D%7D)**

Brick Experiments (**[Sandra & Paula](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**) 106 Bricks created **[according to protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%7D%7D)**; size 1x1x1 and 3.5x3.5x2.5.

#### 22.08.-28.08.

Amplifying Gelatin parts via PCR (**[Magdalena](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**).

Golden Gate Cloning: Backbone 2 and Backbone 3 (**[Julia](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): 4 variants of BB2 and 2 variants of BB3 for *P.pastoris*

Transformation into *E.coli* (**[Julia](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): BB2 & BB3 MaSp insert

Mini-Prep, Restriction Digest (**[Julia](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): MaSp insert BB2 & BB3

**23.08.:**Material Testing (**[Paula](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): According to**protocol**. Testing of bricks from 19.8. Results described **[here](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%7D%7D)**

Media Preparation and Pouring of Plates (**[Magdalena & Irfan](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Throughout the project, it was important to keep up continuous inventory.

Gel Pouring  (**[Tatyana & Irfan](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Every week, the wetlab team stocked up on clean-up and analytical gels.

#### 29.08.-31.08.

Trial: Fusing gelatin insert via PCR (**[Magdalena](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**).

Golden Gate Cloning: Backbone 2 and Backbone 3 (**[Julia](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): 4 variants of BB2 and 2 variants of BB3 for *P.pastoris*

Transformation into *E.coli* (**[Julia](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): BB2 & BB3 MaSp insert

Mini-Prep, Restriction Digest (**[Julia](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): MaSp insert BB2 & BB3

**30.08.:** Bio-Glue (**[Sandra](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Zoom conference at 14:00 with **[Prof. Gübitz](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27attributions%27%29%7D%7D)** for possible collaboration and advice on the use of lignin.

##### Other Activities in August

Fundraising (**[Sandra, Zahra & Tatyana](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Project pitches, conversations with potential sponsors. Arrangement of future material testing.

Collaborations: In Person Meet-up (**[Magdalena, Paula & Neha](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Junior Jam in Münster.

Collaborations: Podcast Appearance (**[Tatyana & Julia](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): iGEM Exeter "Test Tube" Podcast.

Partnerships (**[Tatyana, Magdalena and Sandra](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Progress on ongoing partnerships.

Communication (**[Neha & Magdalena](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Continuous work on our Social Media.

### September

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#### 01.09.-04.09.

Trial: Fusing gelatin insert via PCR (**[Magdalena](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**).

Golden Gate Cloning: Backbone 2 and Backbone 3 (**[Julia](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): 4 variants of BB2 and 2 variants of BB3 for *P.pastoris*

Transformation into *E.coli* (**[Julia](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): BB2 & BB3 MaSp insert

Mini-Prep, Restriction Digest (**[Julia](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): MaSp insert BB2 & BB3

Media Preparation and Pouring of Plates (**[Tatyana, Magdalena & Irfan](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Throughout the project, it was important to keep up continuous inventory.

Gel Pouring  (**[Tatyana & Irfan](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Every week, the wetlab team stocked up on clean-up and analytical gels.

#### 05.09.-11.09.

Trial: Fusing gelatin insert via PCR (**[Magdalena](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**).

Golden Gate Backbone 3 (**[Julia](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): 4 expression casettes for MaSp *P.pastoris*

Mini-Prep, Restriction Digest (**[Julia](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): 4x MaSp BB3

Transformation into *E.coli* (**[Julia](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): 4x MaSp BB3

**08.09.:** Bio-Glue (**[Paula](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Zoom conference at 14:00 with **[Ing. Sandra Bischof](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27attributions%27%29%7D%7D)** for possible collaboration and advice on the use of lignin.

Media Preparation and Pouring of Plates (**[Tatyana, Magdalena & Irfan](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Throughout the project, it was important to keep up continuous inventory.

Gel Pouring  (**[Tatyana & Irfan](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Every week, the wetlab team stocked up on clean-up and analytical gels.

#### 12.09.-18.09.

Trial: Fusing gelatin insert via Gibson Assembly (**[Magdalena](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**).

Golden Gate Backbone 3 (**[Julia](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): 4 expression casettes for MaSp *P.pastoris*

Mini-Prep, Restriction Digest (**[Julia](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): 4x MaSp BB3

Golden Gate Backbone 1 (**[Julia](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): for PHB *P. pastoris*

Transformation into *E.coli* (**[Julia](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): PHB BB1

Mini-Prep, Restriction Digest, Sequencing (**[Julia](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): PHB insert BB1

Media Preparation and Pouring of Plates (**[Tatyana & Magdalena](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Throughout the project, it was important to keep up continuous inventory.

Gel Pouring  (**[Tatyana & Magdalena](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Every week, the wetlab team stocked up on clean-up and analytical gels.

#### 19.09.-25.09.

Trial: Fusing gelatin insert via NEBs Gibson Assembly (**[Magdalena](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**).

Golden Gate Backbone 2 (**[Julia](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): for PHB in *P. pastoris*

Transformation of *P. pastoris* (**[Julia](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): 1xMaSp BB3, 2 variants

Transformation into *E.coli* (**[Julia](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): PHB in BB2

Mini-Prep, Restriction Digest (**[Julia](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): PHB insert BB2

**25.09.:** Brick Experiments (**[Sandra & Paula](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): 12 Bricks created **[according to protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%7D%7D)**; size 3.5x3.5x2.5.

Media Preparation and Pouring of Plates (**[Tatyana, Zahra & Magdalena](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Throughout the project, it was important to keep up continuous inventory.

Gel Pouring  (**[Tatyana & Zahra](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Every week, the wetlab team stocked up on clean-up and analytical gels.

#### 26.09.-30.09.

Golden Gate Backbone 1 (**[Magdalena & Zahra](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): for gelatin in BB1.

Golden Gate Backbone 3 (**[Julia](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): for PHB in *P. pastoris*

Transformation into *E.coli* (**[Julia](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): PHB in BB3

Mini-Prep, Restriction Digest (**[Magdalena, Julia & Zahra](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): PHB insert BB3; Gelatin in BB1

Transformation of *P. pastoris* (**[Julia](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): 1xMaSp BB3, 2 variants; 4x MaSp; PHB

**28.09.:** Bio-Glue (**[Paula](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Visit in the laboratory in BOKU Tulln; 6 bricks created with added BioGlue; 100ml each.

Media Preparation and Pouring of Plates (**[Tatyana, Magdalena & Irfan](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Throughout the project, it was important to keep up continuous inventory.

Gel Pouring  (**[Tatyana & Irfan](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Every week, the wetlab team stocked up on clean-up and analytical gels.

##### Other Activities in September

Project Modeling (**[Neha & Jakob](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Brainstorming, structuring, and making of our models.

Fundraising (**[Sandra, Zahra & Tatyana](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Project pitches, conversations with potential sponsors. Arrangement of future material testing.

Collaborations: In Person Meet-up (**[Magdalena](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Conference in Düsseldorf.

Collaborations: Podcast Appearance (**[Tatyana & Julia](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): iGEM Exeter "Test Tube" Podcast.

Partnerships (**[Tatyana & Magdalena](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Progress on ongoing partnerships.

Communication (**[Neha, Tatyana & Magdalena](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Continuous work on our Social Media.

Wiki Programming (**[Tatyana](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Programming the layout of the wiki, writing Wiki Texts

Wiki Writing (**[Everyone](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): writing wiki Texts

### October

---

#### 01.10-02.10.

Golden Gate Backbone 1 (**[Magdalena](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): for gelatin in BB1.

Golden Gate Backbone 3 (**[Julia](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): for PHB in *P. pastoris*

Transformation into *E.coli* (**[Julia](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): PHB

Mini-Prep, Restriction Digest (**[Magdalena, Julia & Zahra](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): PHB insert BB3; gelatin in BB1

Transformation of *P. pastoris* (**[Julia](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): 1xMaSp BB3, 2 variants; 4x MaSp; PHB

Engineered *P.pastoris* cultivation (**[Julia](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): 1x MaSp insert, 2 variants

Media Preparation and Pouring of Plates (**[Tatyana, Zahra & Irfan](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Throughout the project, it was important to keep up continuous inventory.

Gel Pouring  (**[Tatyana & Zahra](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Every week, the wetlab team stocked up on clean-up and analytical gels.

#### 03.10.-09.10.

Golden Gate Backbone 3 (**[Magdalena & Zahra](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): for gelatin in BB3.

Transformation into *E.coli* (**[Magdalena](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): gelatin in BB3

Mini-Prep, Restriction Digest (**[Magdalena & Zahra](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): gelatin in BB3

Engineered *P.pastoris* cultivation (**[Julia](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): 1x MaSp insert, 2 variants; 4x MaSp insert; PHB

SDS-PAGE (**[Julia](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): supernatant of *P.pastoris* + 1x MaSp variant inserts culture.

**10.10.**Brick Creation and Material Testing (**[Paula](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Mixture 4 brick creation; Brick testing.

Media Preparation and Pouring of Plates (**[Tatyana, Zahra & Irfan](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Throughout the project, it was important to keep up continuous inventory.

Gel Pouring  (**[Tatyana & Zahra](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Every week, the wetlab team stocked up on clean-up and analytical gels.

##### Other Activities in September

Project Modeling (**[Neha & Jakob](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Finishing the models, describing the results.

Partnerships (**[Tatyana & Magdalena](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Last meetings of ongoing partnerships.

Communication (**[Neha, Tatyana & Magdalena](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Continuous work on our Social Media.

Wiki Programming (**[Tatyana](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): Programming the layout of the wiki, writing wiki texts

Wiki Writing (**[Everyone](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%7D%7D)**): writing wiki texts

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/4423/wiki/images-for-pages/eingineering.png)

Designed in **Freepik**

## Engineering Success

---

Our project had many challenging aspects, and many of these required their own engineering cycles. However, our work on the major strain spider silk is a great representative of our work with a common challenge in our project: difficult inserts.

## Major Strain Spider Silk: From Insert to Plasmid

---

#### Background

Spider Silk is one of the three biopolymers used as components of our bricks. Spiders produce seven different versions of Spider Silk in their nets. For our biopolymer we chose the “major strain spider silk (MaSp)” (Fig.1). This version of spider silk is very robust and therefore used to enhance the stability of our bricks. During this project, three different plasmids containing the major spider silk gene were developed to receive information as to which type of plasmid ensures the highest expression rates.

![](https://static.igem.wiki/teams/4423/wiki/model/spider-silk-overview.png)

Fig. 1: Different types of Spider Silk (Source: Eisoldt et al., 2011)

---

#### The Insert

##### Design

Major strain spider silk consists of four major parts, the first T-sequence, two S-sequences and another T-sequence at the end (Fig. 2). Due to a high number of repeats in the sequence, synthesising the whole sequence is not possible in one try. The engineering design was therefore split into two steps, allowing us to order the insert in smaller parts, which needed to be combined to gain the whole sequence for the major strain spider silk.

![](https://static.igem.wiki/teams/4423/wiki/engineering/fig2.png)

Fig.2: MaSp coding sequence

**Step 1:** In a first step, only the **T-sequence** in the beginning and the first of the two S-sequences were created using polymerase chain reaction (PCR)(Fig. 3). The S-sequence could be ordered at the company IDT, when split into three different parts (S#1, S#2 and S#3). The T-sequence on the other hand could not be ordered at the company IDT. Thus, various primers, resulting in the T-sequence when combined, were ordered at the company **IDT**.

![](https://static.igem.wiki/teams/4423/wiki/engineering/fig2.png)

Fig.3: T-sequence in MaSp

In a first trial run a PCR was done containing all the components for part 1 of the engineering design. However, no band in the expected size could be seen on the electrophoresis performed after the PCR.

After discussing these results and brainstorming solutions, sequence-T, Part S#1, Part S#2 and Part S#3 were amplified by PCR separately. The reaction resulting in the T-sequence consisted of the **primers** “FW1 A1T”, “BW1 A1T”, “FW2 A1T” and “BW2 A1T”. For the parts S#1, S#2 and S#3 the template sequences were mixed with the according primers resulting in the three following reactions. Template “A1S#1” and primers “FW2 A1T” and “A1Spart1and2BW” for the part S#1, template “A1S#2” and primers “A1Spart1and2FW” and “A1Spart2and3BW” for the part S#2 as well as template “A1S#3” and primers “A1Spart2and3FW” and “A1Spart3BW”. To combine these products in a second reaction, the primers “FW1 A1T”, “BW2 A1T”, “A1Spart1and2FW”, “A1Spart1and2BW”, “A1Spart2and3FW”, “A1Spart2and3BW”, and “A1Spart3BW” were used. The success of this experiment was proven by sequencing the cleaned PCR product at the company “Microsynth”.

**Step 2:** The PCR product of step one was now amplified with three different sets of primers to gain the parts A, B and C together creating the insert used in the following Golden Gate cloning to create the designed plasmids. These primers also contained recognition sites for the restriction enzyme BsaI, resulting in sticky ends with the ability to combine the PCR products to the correct sequence, as well as the possibility to add the secretion factor and clone the whole insert into a backbone 1 with the fusion sites 2 and 3.

Part A was constructed with the use of the primers “A1T FW1” on the on hand and “PCR-2-BW” on the other hand (Fig. 4), resulting in a N-terminal sticky end with the overhang sequence “GAGA”, fitting the C-terminal sticky end of the secretion factor for *Pichia pastoris*, and a C-terminal sticky end with the overhang sequence “CGCT”, fitting the N-terminal sticky end of part B. This part consists of the N-terminal T-sequence as well as the first of the two S-sequences.

![](https://static.igem.wiki/teams/4423/wiki/engineering/fig4.png)

Fig.4: MaSp A

For part B the primers “PCR-3-FW” and “PCR-3-BW” were used for the creation of a PCR-product (Fig.5) containing only one S-sequence including the N-terminal sticky end with the overhang sequence “CGCT”, fitting the C-terminal sticky end of part A and the C-terminal sticky end with the overhang sequence “GGCG”, fitting the N-terminal sticky end of part C. Part B is used as the second of the two S-sequences in the complete insert.

![](https://static.igem.wiki/teams/4423/wiki/engineering/fig5.png)

Fig.5: MaSp B

The primers “PCR-4-FW” and “PCR-4-BW” were necessary to produce the small PCR product C, only including the C-terminal T-sequence (Fig. 6). This PCR product contains the C-terminal fusion site 3, needed for the cloning in backbone 1, as well as the N-terminal sticky end with the overhang sequence “GGCG”, fitting the C-terminal sticky end of part B.

The resulting parts A, B and C were determined to contain the expected sequences by sending them to the company “Mikrosynth” for sequencing, after cleaning up the PCR-products.

![](https://static.igem.wiki/teams/4423/wiki/engineering/fig6.png)

Fig.6: MaSp C

---

#### Golden Gate Cloning

##### Backbone 1

The cloning in backbone 1 (Fig. 7)combines the three parts of the main strain spider silk sequence with the secretion signal for *Pichia pastoris*. This secretion signal contains the N-terminal fusion site 2, used for cloning into backbone 1 and the C-terminal sticky end with the overhang sequence “GAGA”, fitting the N-terminal sticky end of part A, when cut with the restriction enzyme BsaI. To later gain inside on the success of the Golden Gate reaction, the backbone 1 contains a kanamycine resistance gene.

![](https://static.igem.wiki/teams/4423/wiki/engineering/fig7.png)

Fig.7: BB1-MaSp

After the Golden Gate reaction, the resulting plasmid was transformed into chemically competent *E.coli* of the strain *DH10b* by heatshock treatment. For the determination of a successful reaction a negative control was added to the Golden Gate reaction. After plating the transformed *E.coli* on LB-media plates containing the antibiotic kanamycine and incubating over night at 37 degrees Celsius, cell growth could only be witnessed on the plates containing the *E.coli* with the correct plasmid. However, after extracting the plasmid, using the “MiniPrep” method, choosing the samples to send for sequencing by doing a restriction digest to get slight knowledge about the composition of the plasmid and sending the chosen samples for sequencing, slight mutations in the coding sequence could be noticed in all the samples.

Since only a few mutations occurred and the sequencing results of the PCR products clearly showed the expected sequences, the decision to just repeat the Golden Gate experiment was made. The second run proved to be very successful as every sample sent to sequencing matched the expected sequence.

---

##### Backbone 2

Backbone 2 was only needed for the plasmid containing the **four coding sequences**, since it can otherwise be skipped by using by using an empty backbone 3 with the fusion sites 1 and 4, created by digest with BbsI.

For the plasmid version including the coding sequence for main strain spider silk four times, four different backbone 2 plasmids were made. To avoid problems caused by inadequate binding of DNA in repeating regions, two promoters and three terminators were used in the four backbone 2 plasmids.

Every backbone 2, used for the main strain spider silk contains the fusion sites 1 and 4, created by digest with BbsI, as well as an ampicillin resistance used for the determination of a successful cloning. The used promoters contain the N-terminal fusion site 1 and the C-terminal fusion site 2, fitting the N-terminal fusion site 2 of the secretion factor, whereas the used terminators contain the N-terminal fusion site 3, fitting the C-terminal fusion site 3 of the main strain spider silk and the C-terminal fusion site 4, used for cloning in backbone 2, when digested with the restriction enzyme BbsI.

The difference between the used backbones lies in the fusion sites, created by a digest with BsaI. This digest is used for the cloning in backbone 3.

To assemble the backbone 2-versions, a Golden Gate reaction with the restriction enzyme BbsI, was performed. The reactions entailed the different promoters and terminators as well as the previously created backbone 1, containing the major strain spider silk insert including the secretion signal for *Pichia pastoris*.

The **first created backbone 2** consists of the promoter “pGAP”, the terminator “RPL2Att”, the MaSp insert including the secretion signal and a backbone 2 version building the fusion sites A and B after a digest with the restriction enzyme BsaI. The N-terminal fusion site A is used for the cloning in backbone 3 while the C-terminal fusion site B fits the N-terminal fusion site B of the second created backbone 2.

For the **second backbone 2-version** the promoter “pTEF”, the terminator “RPP1Btt” and the MaSp insert including the secretion signal were cloned in an empty backbone 2, building the N-terminal fusion site B, fitting the C-terminal fusion site B of the first backbone 2 and the C-terminal fusion site C, fitting the N-terminal fusion site C of the third backbone 2 version, when digested with the restriction enzyme BsaI.

The **third created backbone 2** consists of the promoter “pGAP”, the terminator “RPS25Att”, the MaSp insert including the secretion signal and a backbone 2 version building the fusion sites C and E after a digest with the restriction enzyme BsaI. The N-terminal fusion site C fits the C-terminal fusion site C of the second backbone 2 while the C-terminal fusion site E fits the N-terminal fusion site B of the fourth created backbone 2.

For the **fourth backbone 2-version** the promoter “pTEF”, the terminator “RPP1Btt” and the MaSp insert including the secretion signal were cloned in an empty backbone 2, building the N-terminal fusion site C, fitting the C-terminal fusion site C of the third backbone 2 and the C-terminal fusion site E, used for cloning in a backbone 3 with the fusion sites A and E, when digested with the restriction enzyme BsaI.

The Golden Gate products of the four backbone 2 versions were transformed into chemically competent *E.coli DH10b* and plated on LB-Ampicillin Agar plates. After an incubation period of 18 hours at 37°C, colony growth could only be witnessed on the plates containing the complete backbone 2 versions, but not on the control plates only containing the empty and, after the Golden Gate, linearized backbone 2 versions. As further proof for the successful cloning a control digest was performed using the restriction enzyme BsaI. After loading the digest on a 1% agarose gel, the bands for all backbone versions could be found at the expected sizes.

---

##### Backbone 3

To obtain a comparison regarding expression efficiency, **three different backbone 3 were created**. The first and second version each contain one expression cassette that varies only in terms of the used promoter. The third expression cassette entails four different expression cassettes.

The backbone 3 were obtained by Golden Gate cloning. All backbone three versions contain a nourseothricin resistance to enable a selection of the E.coli clones including the plasmid after the Golden Gate reaction was transformed into *E.coli*.

**BB3-pGAP-MaSp-RPL2Att**

The **first created backbone 3** is the plasmid BB3-pGAP-MaSp-RPL2Att (Fig.8). It contains the promoter “pGAP”, the terminator “RPL2Att” and the MaSp insert including the secretion signal. This plasmid was synthesized by doing a Golden Gate reaction including the Backbone 1 with the MaSp coding sequence and the secretion signal for *Pichia pastoris*, the “pGAP” promoter and the “RPL2Att terminator. The restriction enzyme BbsI was used to create the desired sticky ends. For the empty backbone the backbone 3 version, building the fusion sites 1 and 4, when digested with BbsI, was chosen.

![](https://static.igem.wiki/teams/4423/wiki/engineering/fig8.png)

Fig.8: BB3\_pGAP\_MaSp\_RPL2Att plasmid

After an incubation period of 18 hours at 37 degrees Celsius, colony growth could only be witnessed on the nourseothricin-plates containing the complete BB3-pGAP-MaSp-RPL2Att plasmid but not on the control plates only containing the empty and, after the Golden Gate, linearized backbone 3. As further proof for the successful cloning a control digest was performed using the restriction enzyme BglI. After loading the digest on a 1% agarose gel, the bands could be found at the expected sizes.

**BB3-pTEF-MaSp-RPL2Att**

The first created backbone 3 is the plasmid BB3-pTEF-MaSp-RPL2Att (Fig. 9). It contains the promoter “pTEF”, the terminator “RPL2Att” and the MaSp insert including the secretion signal. This plasmid was synthesized by doing a Golden Gate reaction including the Backbone 1 with the MaSp coding sequence and the secretion signal for *Pichia pastoris*, the “pTEF” promoter and the “RPL2Att terminator. The restriction enzyme BbsI was used to create the desired sticky ends. For the empty backbone the backbone 3 version, building the fusion sites 1 and 4, when digested with BbsI, was chosen.

![](https://static.igem.wiki/teams/4423/wiki/engineering/fig9.png)

Fig.9: BB3\_pTEF\_MaSp\_ROL2Att plasmid

After an incubation period of 18 hours at 37 degrees Celsius, colony growth could only be witnessed on the nourseothricin-plates containing the complete BB3-pTEF-MaSp-RPL2Att plasmid but not on the control plates only containing the empty and, after the Golden Gate, linearized backbone 3. As further proof for the successful cloning a control digest was performed using the restriction enzyme BglI. After loading the digest on a 1% agarose gel, the bands could be found at the expected sizes.

**BB3-4xMaSp**

The third plasmid version was created out of the four previously described backbone 2 versions. These backbone 2 versions were used in a Golden Gate reaction with BsaI as restriction enzyme, also including an empty backbone 3 containing the fusion sites A and E after digest with BsaI.

![](https://static.igem.wiki/teams/4423/wiki/engineering/fig10.png)

Fig.10: BB3\_4xMaSp plasmid

After an incubation period of 18 hours at 37°C, extensive colony growth could be witnessed on the nourseothricin-plates, whereas only few colonies appeared on the plates only containing the empty and, after the Golden Gate, linearized backbone 3. A first control digest with the enzyme BglI was not entirely rewarding since it led to several not very distinctive bands. After some consideration a different restriction enzyme, XhoI, was chosen for a second digest. The second digest was more successful, resulting in bands at the expected sizes on a 1% agarose gel.

---

#### Expression in *Pichia pastoris*

To transform the obtained plasmids into the genomic DNA of *Pichia pastoris*, they were first linearized using the restriction enzyme AscI. The restriction site of AscI is located between the two pRGI parts of the plasmid, later attaching to homologous parts in the genomic DNA, while **inserting the MaSp expression cassette**.

The transformation was done by electroporation into electrocompetent *Pichia pastoris* (strain CBS2612). The transformed *Pichia pastoris* was plated on YPD-Agar plates containing nourseothricin and incubated for 72 hours at 28°C. After the incubation period, a large number of colonies could be noticed on the plates containing the plasmids compared to only a few colonies on the negative control, transformed only with sterile water.

Four colonies of each transformed *Pichia pastoris* were chosen and plated on new YPD-Agar plates containing nourseothricin, using the fractional smear method. After an incubation period of 72 hours at 28°C, this step was repeated with colonies of the new plates, ensuring the purity of the clone.

A preculture of 10mL of liquid YPG-medium containing nourseothricin was inoculated with two clones for each plasmid transformed in *Pichia pastoris* and incubated for at 28°C for 24 hours shaking at 180 RPM. After the incubation period a main culture of 50mL liquid BM-Medium with a glucose concentration of 2% was set to an OD600 of 0,1 and incubated for 48 hours. After 12, 24, 36, 48 hours, 2mL samples were taken and the glucose concentration was set to 1%.

1mL of the obtained samples was centrifuged at 16,000xg for 5min. The supernatant was used as a sample for SDS-Page, confirming the expression of the main strain spider silk protein. The other 1mL was used to monitor the cell growth by OD600 measurement.

The proteins encoded by the backbone 3 plasmids are expected to be secreted into the surrounding media, due to the secretion factor added to the backbone 1 plasmid. Even though the secreted protein has a molecular weight of approximately 28kDa, the main strain spider silk **is suggested to build homodimers** formed by two main strain spider silk monomers and therefore resulting into band sizes of approximately 56kDa [**1**].

In a first trial protein samples, gained from the Pichia pastoris clones using the pGAP and the pTEF promoter after 12, 24 and 36 hours were loaded on an SDS-PAGE gel (Fig.11). A **distinctive band** can be seen in all samples but not the wild type control at a size of approximately 56kDa which would be appropriate for the homodimers suggested to be built by the main strain spider silk protein.

![](https://static.igem.wiki/teams/4423/wiki/results/fig4.png)

Fig.11: SDS-Page of the main strain spider silk protein

---

## References

* [1] Bin Liu, Tao Wang, Liyan Xiao, Guilan Zhang, Guangshen Li, Jingzhi Luo & Xiaobing Liu (2018) A directed self-assembly quasi-spider silk protein expressed in Pichia pastoris, Biotechnology & Biotechnological Equipment, 32:2, 451-461, DOI: 10.1080/13102818.2017.1327823

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/4423/wiki/images-for-pages/experiments.png)

Designed in **Freepik**

## Experiments

In our project, we worked in smaller groups on the following goals: [cloning](#cloning) of inserts for **spider silk**, **gelatin** and **PHB** into backbones for *Pichia pastoris* and, in parallel, we were making **[brick mixtures](#bricks)** and testing these materials to analyze their properties.

---

## Cloning Protocols

{% include 'pages/methods-2.html' %}

#### Protocols for Cloning

All of our protocols and methods regarding cloning are available for download here.

* [Determination of Concentration via NanoDrop](https://static.igem.wiki/teams/4423/wiki/methods/concentration-determination.pdf)
* [Enzyme Digest](https://static.igem.wiki/teams/4423/wiki/methods/digest.pdf)
* [*E.coli* transformation](https://static.igem.wiki/teams/4423/wiki/methods/ecoli.pdf)
* [Gel electrophoresis (Agarose gels)](https://static.igem.wiki/teams/4423/wiki/methods/gel-electrophoresis.pdf)
* [Golden Gate Cloning](https://static.igem.wiki/teams/4423/wiki/methods/ggc.pdf)
* [Mini-Prep](https://static.igem.wiki/teams/4423/wiki/methods/mini.pdf)
* [Optical Density Determination](https://static.igem.wiki/teams/4423/wiki/methods/od.pdf)
* [PCR/Gel Cleanup](https://static.igem.wiki/teams/4423/wiki/methods/pcr-gel-cleanup.pdf)
* [PCR](https://static.igem.wiki/teams/4423/wiki/methods/pcr.pdf)
* [Cultivation of *P. pastoris*](https://static.igem.wiki/teams/4423/wiki/methods/pcultivation.pdf)
* [Transformation of *P. pastoris*](https://static.igem.wiki/teams/4423/wiki/methods/pptrafo.pdf)
* [SDS-PAGE with Coomassie Staining](https://static.igem.wiki/teams/4423/wiki/methods/sds.pdf)
* [Sequencing (Microsynth)](https://static.igem.wiki/teams/4423/wiki/methods/sequencing.pdf)

---

## Brick Experiments

**Manufacturing Pichitecture Bricks**

**Goal:** Making bricks of varying size and different portioning of components to test their properties after drying.

| Component | Purchased |
| --- | --- |
| River Sand | MERCK. CAS: 14808-60-7 |
| Desert Sand | Wüstenland |
| Lignin | Sigma-Aldrich. CAS:8068-05-1 |
| Gelatine | Dr. Oetker (gemahlen) |
| Agar-Agar | MERCK. CAS: 9002-18-0 |
| Cyanobacteria from liquid culture | Synechocystis PCC 6803 |
| BG-11 medium | [Recipe](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27bg-11%27%29%7D%7D) |
| CaCO3 | MERCK. CAS: 471-34-1 |

**Procedure:** Each brick is made from three components: **binder**, **liquid**, **structure** (solid material). The overall ratio was set as: **30% binder + liquid** and **70% solid structure**

**Step by Step Protocol:**

| Step # | Instruction |
| --- | --- |
| 1 | Add 0.1M CaCO3 |
| 2 | Weigh the binder in a flask |
| 3 | Add liquid |
| 4 | Put the flask in a water bath (50°C) until binder is melted |
| 5 | Add the solid component |
| 6 | Fold the solid component into the mixture. Avoid air bubbles. |
| 7 | Use a spoon to transfer the mixture into the molds |
| 8 | Let dry and store at room temperature |

The Mixture Recipes

##### Brick Mixtures

Mixture 1

| # | Solid | Binder | Liquid | CaCO3 |
| --- | --- | --- | --- | --- |
| 1a | 70% River Sand | 3% Gelatine | 27% BG11 | 0.1M CaCO3 |
| 1b | 70% River Sand | 3% Gelatine | 27% BG11 | - |
| 1c | 70% River Sand | 3% Gelatine | 27% BG11 + Cyanobacteria | 0.1M CaCO3 |
| 2a | 63% River Sand + 7% Lignin | 3% Gelatine | 27% BG11 | 0.1M CaCO3 |
| 2b | 63% River Sand + 7% Lignin | 3% Gelatine | 27% BG11 | - |
| 2c | 63% River Sand + 7% Lignin | 3% Gelatine | 27% BG11 + Cyanobacteria | 0.1M CaCO3 |
| 3a | 70% River Sand | 3% Agar-Agar | 27% BG11 | 0.1M CaCO3 |
| 3b | 70% River Sand | 3% Agar-Agar | 27% BG11 | - |
| 3c | 70% River Sand | 3% Agar-Agar | 27% BG11+Cyanobacteria | 0.1M CaCO3 |

Mixture 2

| # | Solid | Binder | Liquid | CaCO3 |
| --- | --- | --- | --- | --- |
| 1a | 70% Desert Sand | 3% Gelatine | 27% BG11 | 0.1M CaCO3 |
| 1b | 70% Desert Sand | 3% Gelatine | 27% BG11 | - |
| 1c | 70% Desert Sand | 3% Gelatine | 27% BG11 + Cyanobacteria | 0.1M CaCO3 |
| 2a | 66.5% Desert Sand + 3.5% Lignin | 3% Gelatine | 27% BG11 | 0.1M CaCO3 |
| 2b | 66.5% Desert Sand + 3.5% Lignin | 3% Gelatine | 27% BG11 | - |
| 2c | 66.5% Desert Sand + 3.5% Lignin | 3% Gelatine | 27% BG11 + Cyanobacteria | 0.1M CaCO3 |
| 3a | 66.5% Desert Sand + 3.5% Lignin | 3% Gelatine | 27% H2O | 0.1M CaCO3 |
| 3b | 66.5% Desert Sand + 3.5% Lignin | 3% Gelatine | 27% H2O | - |

Mixture 3

| # | Solid | Binder | Liquid | CaCO3 |
| --- | --- | --- | --- | --- |
| 1a | 70% River Sand | 1.5% Gelatine | 28.5% H2O | 0.1M CaCO3 |
| 1b | 70% River Sand | 1.5% Gelatine | 28.5% H2O | - |
| 1c | 70% Desert Sand | 1.5% Gelatine | 28.5% H2O | 0.1M CaCO3 |
| 1d | 70% Desert Sand | 3% Gelatine | 27% H2O | - |
| 2a | 70% River Sand | 3% Gelatine | 27% H2O | 0.1M CaCO3 |
| 2b | 70% River Sand | 3% Gelatine | 27% H2O | - |
| 2c | 70% Desert Sand | 3% Gelatine | 27% H2O | 0.1M CaCO3 |
| 2d | 70% Desert Sand | 3% Gelatine | 27% H2O | - |

Mixture 4 - BioGlue (provided in collaboration with the BOKU-IFA Project BioGlue

| # | Solid | Binder | Liquid | CaCO3 |
| --- | --- | --- | --- | --- |
| 1a | 70% River Sand | 30% Bio-Glue | - | - |
| 1b | 70% River Sand | 15% BioGlue + 1.5% Gelatine | 13.5% H2O | - |
| 1c | 70% River Sand | 3% Gelatine | 21.6% H2O + 5.4% unpolymerized lignin | - |
| 2a | 70% Desert Sand | 30% BioGlue | - | - |
| 2b | 66.5% Desert Sand + 3.5% BioGlue | 15% BioGlue + 1.5% Gelatine | 13.5% H2O | - |
| 2c | 70% Desert Sand | 3% Gelatine | 21.6% H2O + 5.4% unpolymerized lignin | - |

**Testing the Bricks**

A short description of the testing methods can be found [here](https://static.igem.wiki/teams/4423/wiki/methods/mat-test.pdf).

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![](https://static.igem.wiki/teams/4423/wiki/images-for-pages/results.png)

Designed in **Freepik**

## Our results

Our project encompassed two main approaches: the [cloning of coding sequences](#cloning) for our chosen biopolymers into plasmids for *Pichia pastoris*, and, simultaneously, making a variety of [brick mixtures](#brick) of different sizes and components, and testing them. Throughout our work, we adjusted our experiments based on troubleshooting according the engineering cycle and after gaining insights during our human practices analyses.

---

#### The Cloning

We had varying success with out cloning, and will show our progress in the following order: [spider silk](#spisi), [PHB](#phb) and [gelatin](#gelatin).

##### Main Strain Spider Silk

**Insert**

Since the insert can not be synthesized due to a high number of repeating sequences, it was built together by PCR. After a PCR-product in the desired size was retrieved, it was sent to the company “Microsynth” for sequencing.

Three further PCR-products, were created out of the first PCR-product, together creating the complete main strain spider silk sequence. The sequences of these PCR-products were also confirmed by sending them for sequencing to the company “Microsynth”.

**Backbone 1**

After receiving the correct sequences for the three PCR-products, the DNA-parts were cloned into an empty backbone 1 by Golden Gate. A first indication for a successful cloning is given by the comparison of the incubated *E.coli* plates of the negative control with the sample. Since no colonies could be observed on the negative control a successful cloning appeared to be likely.

A further sign for the correct plasmid was given by the control digest, showing bands at the correct sizes for most of the Mini-Prep samples. These samples were again sent to the company “Microsynth” for sequencing.

**Backbone 2**

**Four backbone 2** versions were created by Golden Gate cloning of the backbone 1 plasmid, proven to obtain the correct sequence. Since the sequence of the insert was already confirmed by sequencing the backbone 1, only the comparison of the *E.coli* plates and the restriction digest of the Mini-Preps were needed as a evidence for a successful cloning.

**Backbone 3**

Three different versions of the backbone 3 were obtained, two containing one expression cassette each (Fig. 1 & 2), only differing from each other in the used promoters to create a direct comparison of the efficiency of these promoters. The third created backbone 3 version entails four different expression cassettes (Fig. 3). Similar to the backbone 2 versions, the successful cloning was proven by the comparison of the *E.coli* plates and the restriction digest of the Mini-Preps.

![](https://static.igem.wiki/teams/4423/wiki/results/fig1.png)

Fig.1: BB3-MaSp-RPL2Att Plasmid

![](https://static.igem.wiki/teams/4423/wiki/results/fig2.png)

Fig.2: BB3-pTEF-MaSp-RPL2Att Plasmid

![](https://static.igem.wiki/teams/4423/wiki/results/fig3.png)

Fig.3: BB3-4xMaSp Plasmid

**Protein Expression in *P.Pastoris***

The proteins encoded by the backbone 3 plasmids are expected to be secreted into the surrounding media, due to the secretion factor added to the backbone 1 plasmid. Even though the secreted protein has a molecular weight of approximately 28kDa, the main strain spider silk **is suggested to build homodimers** formed by two main strain spider silk monomers and therefore resulting into band sizes of approximately 56kDa [1].

In a first trial protein samples, gained from the Pichia pastoris clones using the pGAP and the pTEF promoter after 12, 24 and 36 hours were loaded on an SDS-PAGE gel (Fig.4). A **distinctive band** can be seen in all samples but not the wild type control at a size of approximately 56kDa which would be appropriate for the homodimers suggested to be built by the main strain spider silk protein.

![](https://static.igem.wiki/teams/4423/wiki/results/fig4.png)

Fig.4: SDS-Page of the main strain spider silk protein

Further SDS-PAGE gels will be shown at the Jamboree.

##### PHB

**Backbone 1**

To gain PHB, four different proteins need to be present in the final plasmid.

In a first step the synthesized inserts, each coding for a one of the four proteins needed for the PHB synthesis, were cloned into a backbone 1 by Golden Gate.

To ensure the successful cloning, the samples were sent to the company “Microsynth” for sequencing after first indications for a successful cloning were obtained by comparing the E.coli plates to the negative control and restriction digest.

**Backbone 2**

After the sequences were verified, the backbone 1 versions were cloned into backbone 2, adding various promoters and terminators to create four separate expression cassettes.

Since the sequences of the inserts were already confirmed in the backbone 1 versions, only a comparison of the *E.coli* plates to the negative control and a restriction digest were needed to prove the cloning success.

**Backbone 3**

All four backbone 2 versions were combined into one backbone 3 plasmid, to ensure all four proteins are produced by *Pichia pastoris* (Fig.5).

The successful cloning was proven by the comparison of the *E.coli* plates to the negative control and a restriction digest of the Mini-Preps, similar to the confirmation of the cloning success of the backbone 2 versions.

![](https://static.igem.wiki/teams/4423/wiki/results/fig5.png)

Fig.5: BB3-PHB plasmid

##### Gelatin

Already early on in the project, gelatin presented a challenge.

Over the passing weeks, we performed Golden Gate Assembly to clone our BB1 plasmid with the sequence that codes for gelatine and the alpha mating factor as an insert. After that, the plasmids were transformed into Ecoli which were then plated on LB-Kana agar plates. The plasmids were then later extracted.

![](https://static.igem.wiki/teams/4423/wiki/model/seq-gel.png)

Fig.:6 Sequencing results for gelatin plasmid.

To confirm that we did indeed have a correct plasmid we performed a digest with BglI and ran it on a 1% agarose gel. In one of our two samples, the bands were wrong, but the other one looked good, so we sent it to sequencing. The sequencing results confirmed that we have a correct plasmid.

We then took our BB1 plasmid and performed a Golden Gate Assembly into backbone 3, together with the promoter and the terminator. The assembled plasmid was transformed into Ecoli and plated on LB-Nat agar plates. After isolating the plasmids
we performed a digest with PstI, but we don’t have the results for that yet.

---

#### Our Bricks

![](https://static.igem.wiki/teams/4423/wiki/brick-results/brick1.jpg)

Fig.7: 1x1x1 bricks

Mixture 1

| # | Solid | Binder | Liquid |
| --- | --- | --- | --- |
| 1a | 70% River Sand | 3% Gelatine | 27% BG11 |
| 1b | 70% River Sand | 3% Gelatine | 27% BG11 |
| 1c | 70% River Sand | 3% Gelatine | 27% BG11 + Cyanobacteria |
| 2a | 63% River Sand + 7% Lignin | 3% Gelatine | 27% BG11 |
| 2b | 63% River Sand + 7% Lignin | 3% Gelatine | 27% BG11 + Cyanobacteria |
| 2c | 63% River Sand + 7% Lignin | 3% Gelatine | 27% BG11 + Cyanobacteria |
| 3a | 70% River Sand | 3% Agar-Agar | 27% BG11 |
| 3b | 70% River Sand | 3% Agar-Agar | 27% BG11 with CaCO3 |
| 3c | 70% River Sand | 3% Agar-Agar | 27% Syn. PCC6803 in BG11 |

We divided the mixtures of our bricks into three components: solid, binder and liquid.

For our first brick experiment we used river sand as a solid and also mixed it with lignin. As a binder we used gelatin and Agar-Agar, and the liquid was a) only BG11 medium b) BG11 medium with CaCO3 and 3) our *Synechocystis* culture. The function of 3) was not to let the *Synechocystis* precipitate CaCO3 but solely to see how the cell material is interacting with the mixture. We made five cubes of each combination and two sets of the experiment: we let them dry at RT and 4°C.

The full, detailed results of our first brick tests you can find [here](https://static.igem.wiki/teams/4423/wiki/brick-results/brick-test-1.pdf).

![](https://static.igem.wiki/teams/4423/wiki/brick-results/brick2.png)

Fig.8: The first brick experiment.

**Brick experiment 2: 19.08.2022**

Mixture 2 bricks.

| # | Solid | Binder | Liquid | CaCO3 |
| --- | --- | --- | --- | --- |
| 1a | 70% Desert Sand | 3% Gelatine | 27% BG11 | 0.1M CaCO3 |
| 1b | 70% Desert Sand | 3% Gelatine | 27% BG11 | - |
| 1c | 70% Desert Sand | 3% Gelatine | 27% BG11 + Cyanobacteria | 0.1M CaCO3 |
| 2a | 66.5% Desert Sand + 3.5% Lignin | 3% Gelatine | 27% BG11 | 0.1M CaCO3 |
| 2b | 66.5% Desert Sand + 3.5% Lignin | 3% Gelatine | 27% BG11 | - |
| 2c | 66.5% Desert Sand + 3.5% Lignin | 3% Gelatine | 27% BG11 + Cyanobacteria | 0.1M CaCO3 |
| 3a | 66.5% Desert Sand + 3.5% Lignin | 3% Gelatine | 27% H2O | 0.1M CaCO3 |
| 3b | 66.5% Desert Sand + 3.5% Lignin | 3% Gelatine | 27% H2O | - |

For our second mixtures, we tested how **desert sand** instead of **river sand** as a solid component would influence the mixture. In the building industry, river sand and sea sand are the most frequently used sand types. Desert Sand, though easily available in huge quantities, is not suitable; the reason for this is that its surface is much more rounded, which hinders the sand particles to stable each other, leading to crumbling, especially when the sand gets wet. We hoped that in the combination with lignin, the properties of desert sand might become more usable.

* Desert sand was used in 1), to find out how desert sand works in our mixture.
* for 2) we used Desert Sand and Lignin, the amount of lignin is decreased in comparison to the first experiment.
* 3) H2O instead of BG11 was used, to see how the brick would behave if no cyanobacteria at all would be integrated into our final product.

![](https://static.igem.wiki/teams/4423/wiki/brick-results/brick3.png)

Fig.9: The second bricks experiment.

We found out that over all, the material made by the second mixture appears to be less dense, compared to the river sand mixture, the:

* The 1) brick was as expected losing its structural integrity at the edges, and was especially crumbly when in contact with water.
* The 2)had similar properties to the river sand + lignin mixture, we therefore decided to do further experiments with lignin.
* The 3) mixture with H2O as the liquid component, instead of BG11 had similar properties then the 1) (Mixture 2).

Overall no big difference was picked up by our testing methods between the control groups (a,b,c). It is important to note that our material testing protocol is only useful to give a basic understanding about its properties, further test hare highly necessary, and still on-going in **cooperation with one of our sponsors, Wienerberger, as of the date of the wiki freeze**.

Additionally, we found out that it is **important to integrate the sand softly to the liquid**, and avoid creating bubbles, otherwise the stability and density reduces.

The full, detailed results of our second brick tests you can find [here](https://static.igem.wiki/teams/4423/wiki/brick-results/brick-test-2.pdf).

**Brick experiment 3: 25.09.2022**

Mixture 3

| # | Solid | Binder | Liquid | CaCO3 |
| --- | --- | --- | --- | --- |
| 1a | 70% River Sand | 1.5% Gelatine | 28.5% H2O | 0.1M CaCO3 |
| 1b | 70% River Sand | 1.5% Gelatine | 28.5% H2O | - |
| 1c | 70% Desert Sand | 1.5% Gelatine | 28.5% H2O | 0.1M CaCO3 |
| 1d | 70% Desert Sand | 3% Gelatine | 27% H2O | - |
| 2a | 70% River Sand | 3% Gelatine | 27% H2O | 0.1M CaCO3 |
| 2b | 70% River Sand | 3% Gelatine | 27% H2O | - |
| 2c | 70% Desert Sand | 3% Gelatine | 27% H2O | 0.1M CaCO3 |
| 2d | 70% Desert Sand | 3% Gelatine | 27% H2O | - |

The third experiment was done to find out if a smaller amount of gelatin would work equally as efficient, as a smaller amount needed, equals less energy during the gelatin production would be needed per kg material.

In some mixtures we didn't add cyanobacteria, but added CaCO3. This was due to our safety concept and proposed implementation plans; using solely CaCo3 ensures that no microorganism will be introduced through the brick in to the environment.

![](https://static.igem.wiki/teams/4423/wiki/brick-results/brick4.png)

Fig.10: The third brick experiment

The stability of the 2) mixture with the higher amount of gelatine was better. The sides, however, crumbled more.

The full, detailed results of our third brick tests you can find [here](https://static.igem.wiki/teams/4423/wiki/brick-results/brick-test-3.pdf).

**Brick experiment 4: 28.09.2022**

Mixture 4 - BioGlue (provided in collaboration with the BOKU-IFA Project BioGlue

| # | Solid | Binder | Liquid | CaCO3 |
| --- | --- | --- | --- | --- |
| 1a | 70% River Sand | 30% Bio-Glue | - | - |
| 1b | 70% River Sand | 15% BioGlue + 1.5% Gelatine | 13.5% H2O | - |
| 1c | 70% River Sand | 3% Gelatine | 21.6% H2O + 5.4% unpolymerized lignin | - |
| 2a | 70% Desert Sand | 30% BioGlue | - | - |
| 2b | 66.5% Desert Sand + 3.5% BioGlue | 15% BioGlue + 1.5% Gelatine | 13.5% H2O | - |
| 2c | 70% Desert Sand | 3% Gelatine | 21.6% H2O + 5.4% unpolymerized lignin | - |

We had the possibility to work together with the Bio Glue Team and cooperate there BIoGue as a Binder into our bricks. The amounts we decided on using were recommended by the project leader of BioGlue, **Sandra Bischof**. Her glue is **20% polymerized lignin and 80% water**. Because of the polymerisation that has been achieved by an enzyme the lignin should be water resistant, and even more sticky. We are **still waiting for the results**, as the glue needs 2 weeks to completely dry.

Also, we decided to switch to the construction sand, available in a local building store. The reason was that our product should work well with the solid materials that are already used in construction.

![](https://static.igem.wiki/teams/4423/wiki/brick-results/brick5.png)

Fig.11: The fourth brick experiment.

**In Conclusion**

Our goal in our brick experiments was to find a couple of prototypes that we could explore further with more **robust testing** with **Wienerberger**. As of the time of the wiki freeze, we are still in the process of creating new brick mixtures (adding construction rubble for example), and sending them for lab testing to see if they are fit to use for construction. After these tests, we would be able to judge more accurately what influences the properties of our bricks.

---

## References

* [1] Bin Liu, Tao Wang, Liyan Xiao, Guilan Zhang, Guangshen Li, Jingzhi Luo & Xiaobing Liu (2018) A directed self-assembly quasi-spider silk protein expressed in Pichia pastoris, Biotechnology & Biotechnological Equipment, 32:2, 451-461, DOI: 10.1080/13102818.2017.1327823

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