Difference between revisions of "Part:BBa K2765021:Experience"
| Line 3: | Line 3: | ||
After transform it into<i> Saccharomyces cerevisiae</i>, we begin the tests. | After transform it into<i> Saccharomyces cerevisiae</i>, 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. | + | 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 <i>yno1</i> caused significant ROS production as revealed by green fluorescent substance DCF. |
| − | + | [[Image: T--BIT-China--iGEM2018-Partsregulator-7.png |center|400px|]] | |
| − | + | ||
| − | + | ||
In order to make sure the expression of <i>yno1</i> is totally under control, we overexpress yno1 by replace its promoter to gal through Homologous reorganization. | In order to make sure the expression of <i>yno1</i> is totally under control, we overexpress yno1 by replace its promoter to gal through Homologous reorganization. | ||
| − | + | [[Image: T--BIT-China--iGEM2018-Partsregulator-8.png |center|400px|]] | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| + | [[Image: T--BIT-China--iGEM2018-Partsregulator-9.png |center|400px|]] | ||
We sequenced the constructed gene expression vector and obtained the correct sequencing results. | We sequenced the constructed gene expression vector and obtained the correct sequencing results. | ||
| − | + | [[Image: T--BIT-China--iGEM2018-Partsregulator-10.png |center|400px|]] | |
| − | + | ||
| − | + | ||
After yeast transformation, we screened and identified yeast strains that were transferred to the target gene using SD-Leu-deficient medium. | After yeast transformation, we screened and identified yeast strains that were transferred to the target gene using SD-Leu-deficient medium. | ||
| − | + | [[Image: T--BIT-China--iGEM2018-Partsregulator-form.png |center|400px|]] | |
| Line 42: | Line 27: | ||
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 ℃. | 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 ℃. | ||
| − | + | [[Image: T--BIT-China--iGEM2018-Partsregulator-11.png |center|400px|]] | |
Fig.11 Experimental result | Fig.11 Experimental result | ||
Revision as of 22:45, 17 October 2018
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.
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.
We sequenced the constructed gene expression vector and obtained the correct sequencing results.
After yeast transformation, we screened and identified yeast strains that were transferred to the target gene using SD-Leu-deficient medium.
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
Applications of BBa_K2765021
User Reviews
UNIQ01669c5f0c186c68-partinfo-00000000-QINU UNIQ01669c5f0c186c68-partinfo-00000001-QINU