iGEM 2019 — Team ShanghaiFLS_China

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# Team:ShanghaiFLS China/Notebook

ShanghaiFLS\_China: Notebook

# [Team:ShanghaiFLS\_China](https://2019.igem.org/Team%3AShanghaiFLS_China)

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## Notebook

We documented our work in detail during the whole experiment. This notebook proved very useful when we were trying to find results from several weeks ago, to check if a construct was correct, *et cetera*.

### Notebook

This browser does not support PDFs. Please download the PDF to view it: [Download PDF](https://static.igem.org/mediawiki/2019/0/02/T--ShanghaiFLS_China--Notebook.pdf).

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* igem2019\_sfls
* igem\_shanghaifls\_china

Copyright © iGEM 2019 Team ShanghaiFLS\_China. All rights reserved. Email: SFLS\_x\_iGEM@outlook.com


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# Team:ShanghaiFLS China/Experiments

ShanghaiFLS\_China: Experiments

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* [Judging Form](https://igem.org/2019_Judging_Form?id=3239)

## Experiments

The YNM media used in the 108 h incubation consists of 1.34% yeast nitrogenous base with 0.5% methanol, 50 µg/mL histidine and 40 µg/mL biotin. The total volume in each shake flask was 50 mL. 0.5% methanol was added to the media every 24 hours to maintain the methanol concentration.

The YNM media used in the short-term methanol induction experiment consists of 1.34% yeast nitrogenous base with 0.75% methanol, 50 µg/mL histidine and 40 µg/mL biotin. The total volume in each shake flask was 50 mL.

The 108 h incubation in shake flasks approximates incubations in fermenters right from the start (when yeasts just got exposed to methanol media), whereas the short-term methanol induction experiment illustrates the instantaneous response of the yeast cells to methanol induction once they are accustomed to the methanol media.

To better simulate industrial fermenter incubation, we calculated the total production of each reaction system (represented as each shake flask) and constructed a mathematical model to calculate the growth rates, the oxygen consumption rates and the heat generation of each reaction system. We did this because such data cannot be directly measured in a shake flask. For more details, see demonstration.

For each reaction system, 2~3 parallels were performed. We did the 108 h incubation twice and the results from the two rounds corroborate each other. The following data stems from the second round of 108h incubation as we collected more data in this round.

When collecting data, we considered that the activity of *PAOX1* should not be reflected by the unit cell fluorescence intensity alone, as *PAOX1*AOX1. While unit cell GFP production allows us to measure the expression of GFP, the expression of *AOX1* shall be represented by cell growth as yeasts in which more *AOX1* is expressed should also be more capable of metabolizing methanol, *i.e.* should be able to thrive more in methanol media.

Our measured data are illustrated in the following diagrams:

![](https://static.igem.org/mediawiki/2019/f/f3/T--ShanghaiFLS_China--protocols1.png)
![](https://static.igem.org/mediawiki/2019/c/c3/T--ShanghaiFLS_China--protocols2.png)

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* igem2019\_sfls
* igem\_shanghaifls\_china

Copyright © iGEM 2019 Team ShanghaiFLS\_China. All rights reserved. Email: SFLS\_x\_iGEM@outlook.com

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:ShanghaiFLS China/Results

ShanghaiFLS\_China: Results

# [Team:ShanghaiFLS\_China](https://2019.igem.org/Team%3AShanghaiFLS_China)

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* [Judging Form](https://igem.org/2019_Judging_Form?id=3239)

## Results

We cloned our plasmids *pGMP1* (carries *PMIT1*-*PRM1* cassette) and *pAPM1* from the *pAPP1*(carries *PPRM1*-*PRM1* cassette) and *pGMM1*(carries *PMIT1*-*MIT1* cassette) plasmids used in the Wang et al., 2016 study, given to us as a gift from the ECUST school of Bioreactor Engineering. *(Thank you!)* The following diagram illustrates our construction process:

![](https://static.igem.org/mediawiki/2019/5/59/T--ShanghaiFLS_China--Fig.456.png)

### References

Mateus, C., & Avery, S. V. (2000). Destabilized green fluorescent protein for monitoring dynamic changes in yeast gene expression with flow cytometry. *Yeast*, 16(14), 1313–1323. https://doi.org/10.1002/1097-0061(200010)16:14<1313::AID-YEA626>3.0.CO;2-O
Wang, X., Wang, Q., Wang, J., Zhou, M., Shi, L., Zhou, X., … Shen, W. (2016). Mit1 Transcription Factor Mediates Methanol Signaling and Regulates the Alcohol Oxidase 1 (AOX1) Promoter in Pichia pastoris. *Journal of Biological Chemistry*, 291(12), 6245–6261. https://doi.org/10.1074/jbc.m115.692053

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* igem2019\_sfls
* igem\_shanghaifls\_china

Copyright © iGEM 2019 Team ShanghaiFLS\_China. All rights reserved. Email: SFLS\_x\_iGEM@outlook.com