Part:BBa_K2765021:Experience

After transform it into Saccharomyces cerevisiae, we begin the tests.

We first measured the ROS production of yeast cells cultured in non-screening or screening media by Fluorescent microplate reader. DCFH-DA, which can be oxidized by ROS to become a strong green fluorescent substance DCF (dichlorofluorescein) that cannot penetrate the cell membrane, was mixed with yeast cells. Fluorescent microplate reader analysis showed that overexpression of yno1 caused significant ROS production as revealed by green fluorescent substance DCF.

Fig.7 accumulation of ROS through overexpress gene ndi1 & yno1

In order to make sure the expression of yno1 is totally under control, we overexpress yno1 by replace its promoter to gal through Homologous reorganization.

Fig.8 process of replacing promoter of yno1

Fig.9 target genes

1. The size of: Leucine-deficient screening marker (1000bp)

2. The size of upstream homology arm (500bp)

4. The size of Downstream homology arm+gal1p (1000bp)

We sequenced the constructed gene expression vector and obtained the correct sequencing results.

Fig.10 sequencing results

After yeast transformation, we screened and identified yeast strains that were transferred to the target gene using SD-Leu-deficient medium.

http://2018.igem.org/File:T--BIT-China--iGEM2018-yno1_form.png

We used a suitable concentration of galactose to induce overexpression of the target gene, resulting in an increase in ROS accumulation compared to the control group.

After that, we transform the plasmid pESC-TEF1p-roGFP2-Orp1-CYC1t, a shuttle vector marked with trp1, cellular redox status were monitored by measuring the fluorescence ratio at 488 nm(reduced state) and 405 nm(oxidized state). Cells were cultured in SD medium supplemented with 1% galactose at 30 ℃.

Fig.11 Experimental result

When galactose was added to a culture, GAL1-GAL10 promotor was induced, overexpression of yno1 gene increased cells ROS level, the fluorescence ratio at 488 nm and 405 nm was declined, which means there are more ROS in our modified strain.

When galactose was added to a culture, GAL1-GAL10 promotor was induced, overexpression of yno1 gene increased cells ROS level, the fluorescence ratio at 488 nm and 405 nm was declined, which means there are more ROS in our modified strain.

References：

Mark Rinnerthalera, Yno1p/Aim14p, a NADPH-oxidase ortholog, controls extramitochondrial reactive oxygen species generation, apoptosis, and actin cable formation in yeast ，2012

朱凯川 酵母转录因子 Gal4 研究进展 2011.10.16

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