Difference between revisions of "Part:BBa K1907007"
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| − | We have created a device that uses oxidative stress as an inducer for reporter signal production by taking the promoter region (260bp) from the TSA1 gene and ligating it with the protein-coding sequence of Venus yellow fluorescent protein. The length of promoter region is chosen so that it contains identified Skn7 and Yap1 binding sites and doesn’t overlap with the next gene in the genome. (He et al., 2005, SGD ID:S000004490) Skn7 and Yap1 are the main transcription factors activated in oxidative stress and are thus responsible for TSA1 promoter activation. The gene sequence for the TSA1 promoter region is taken from strain S288C of Saccharomyces cerevisiae, obtained from the Saccharomyces Genome Database. | + | We have created a device that uses oxidative stress as an inducer for reporter signal production by taking the promoter region (260bp) from the TSA1 gene and ligating it with the protein-coding sequence of Venus yellow fluorescent protein. The length of promoter region is chosen so that it contains identified Skn7 and Yap1 binding sites and doesn’t overlap with the next gene in the genome. (He et al., 2005, SGD ID:S000004490) Skn7 and Yap1 are the main transcription factors activated in oxidative stress and are thus responsible for TSA1 promoter activation. The gene sequence for the TSA1 promoter region is taken from strain S288C of <i>Saccharomyces cerevisiae</i>, obtained from the Saccharomyces Genome Database. |
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<p style="margin-left: 40px">https://static.igem.org/mediawiki/parts/6/6e/TSA1_3.png</p> | <p style="margin-left: 40px">https://static.igem.org/mediawiki/parts/6/6e/TSA1_3.png</p> | ||
<p style="margin-left: 40px">Figure 3. Fluorescence values as a function of different hydrogen peroxide concentrations. GPD1 is a negative control to assess background effects of hydrogen peroxide on fluorescence.</p> | <p style="margin-left: 40px">Figure 3. Fluorescence values as a function of different hydrogen peroxide concentrations. GPD1 is a negative control to assess background effects of hydrogen peroxide on fluorescence.</p> | ||
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Notably, the fluorescence seems to increase with hydrogen peroxide induction also in the control (GPD1 promoter). This could be due to the fact that because hydrogen peroxide slows culture growth, a relatively bigger portion of the cells in the culture are older, meaning that they have have accumulated more fluorescence. Because a defined number of cells are measured to obtain the fluorescence distribution, slower culture growth can show up as this kind of a bias. To account for this, fluorescence values were divided by the respective uninduced values of each promoter. The fluorescence values were normalized to uninduced values to gain an understanding of the resulting increase in fluorescence; the results for this are presented in figure 5.</p> | Notably, the fluorescence seems to increase with hydrogen peroxide induction also in the control (GPD1 promoter). This could be due to the fact that because hydrogen peroxide slows culture growth, a relatively bigger portion of the cells in the culture are older, meaning that they have have accumulated more fluorescence. Because a defined number of cells are measured to obtain the fluorescence distribution, slower culture growth can show up as this kind of a bias. To account for this, fluorescence values were divided by the respective uninduced values of each promoter. The fluorescence values were normalized to uninduced values to gain an understanding of the resulting increase in fluorescence; the results for this are presented in figure 5.</p> | ||
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<p><b>References</b></p><p> | <p><b>References</b></p><p> | ||
| − | He, X.J. and Fassler, J.S., 2005. Identification of novel Yap1p and Skn7p binding sites involved in the oxidative stress response of Saccharomyces cerevisiae. Molecular microbiology, 58(5), pp.1454-1467. | + | He, X.J. and Fassler, J.S., 2005. Identification of novel Yap1p and Skn7p binding sites involved in the oxidative stress response of <i>Saccharomyces cerevisiae</i>. Molecular microbiology, 58(5), pp.1454-1467. |
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Revision as of 20:05, 19 October 2016
TSA1 promoter + Venus YFP
Introduction
TSA1 is a gene for thioredoxin peroxidase - a protein that acts as both a ribosome-associated and a free cytoplasmic antioxidant. It self-associates to form a high-molecular weight chaperone complex under oxidative stress. As TSA1 is produced under oxidative stress, its promoter is activated in these conditions. (Saccharomyces genome database ID: S000004490)
We have created a device that uses oxidative stress as an inducer for reporter signal production by taking the promoter region (260bp) from the TSA1 gene and ligating it with the protein-coding sequence of Venus yellow fluorescent protein. The length of promoter region is chosen so that it contains identified Skn7 and Yap1 binding sites and doesn’t overlap with the next gene in the genome. (He et al., 2005, SGD ID:S000004490) Skn7 and Yap1 are the main transcription factors activated in oxidative stress and are thus responsible for TSA1 promoter activation. The gene sequence for the TSA1 promoter region is taken from strain S288C of Saccharomyces cerevisiae, obtained from the Saccharomyces Genome Database.
The device has been proved to be functional when fluorescence production is studied as a response to oxidative stress caused by hydrogen peroxide. The TSA1 promoter has a relatively high base level of expression, but expression levels are further driven up when induced.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal SpeI site found at 290
Illegal SpeI site found at 364 - 12INCOMPATIBLE WITH RFC[12]Illegal SpeI site found at 290
Illegal SpeI site found at 364 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 453
- 23INCOMPATIBLE WITH RFC[23]Illegal SpeI site found at 290
Illegal SpeI site found at 364 - 25INCOMPATIBLE WITH RFC[25]Illegal SpeI site found at 290
Illegal SpeI site found at 364
Illegal NgoMIV site found at 613
Illegal NgoMIV site found at 623 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 158
Previous use
We studied function of this device in the pRS415 plasmid backbone in S. cerevisiae strain SS328-leu. The device was inserted to the backbone so that it replaced multiple cloning site and the promoter site, and was directly followed by the cyc1 terminator.
Expression
Yeast containing the plasmids was precultured overnight at + 30 C with shaking in selective SD medium. The following morning, the cultures were refreshed with new medium to an OD of 0.2, and growth was continued until induction could be done at an OD of 0.5 by adding hydrogen peroxide. Hydrogen peroxide concentrations of 0-1mM allowed cell growth and YFP expression. A promoter expression profile was obtained by growing the induced cells in a microplate reader(Cytation3, BioTek) with vertical shaking at + 30 C; yellow fluorescence and cell density were measured at regular intervals. As the yeast easily formed clumps that interfere with the measurement, data over a longer time interval becomes inaccurate. The obtained expression profiles can be found in Figures 1 and 2. Because hydrogen peroxide has a negative effect on cell growth, plotting fluorescence as a function of cell density (OD600) gives more accurate information of whether the cells are producing more fluorescence when induced.
Figure 1. Yellow fluorescence produced under the TSA1 promoter as a function of time in different hydrogen peroxide concentrations.
Figure 2. Yellow fluorescence produced under the TSA1 promoter as a function of cell density (OD600) in different hydrogen peroxide concentrations.
These results were confirmed with flow cytometry analysis; cells were induced in the same way, and measured with a flow cytometer (FACSAria III) after 2 h and 4 h induction. The obtained fluorescence values from 2 hour induction are presented in figure 3.
Figure 3. Fluorescence values as a function of different hydrogen peroxide concentrations. GPD1 is a negative control to assess background effects of hydrogen peroxide on fluorescence.
Notably, the fluorescence seems to increase with hydrogen peroxide induction also in the control (GPD1 promoter). This could be due to the fact that because hydrogen peroxide slows culture growth, a relatively bigger portion of the cells in the culture are older, meaning that they have have accumulated more fluorescence. Because a defined number of cells are measured to obtain the fluorescence distribution, slower culture growth can show up as this kind of a bias. To account for this, fluorescence values were divided by the respective uninduced values of each promoter. The fluorescence values were normalized to uninduced values to gain an understanding of the resulting increase in fluorescence; the results for this are presented in figure 5.
Figure 4. Normalized fluorescence values as a function of different hydrogen peroxide concentrations.GPD1 is a negative control to assess background effects of hydrogen peroxide on fluorescence.
References
He, X.J. and Fassler, J.S., 2005. Identification of novel Yap1p and Skn7p binding sites involved in the oxidative stress response of Saccharomyces cerevisiae. Molecular microbiology, 58(5), pp.1454-1467.